General Zoological Microtechniques

GENERAL
ZOOLOGICAL
MICROTECHNIQUES
/ GENERAL
ZOOLOGICAL
MICROTECHNIQUE8_;
(.::::I:=LJf1!:::::n~~:2 Labo"'.'Y Tuhnician

Department of Zoology, University of California, Berkeley
Baltimore
The Williams & Wilkins Company
Calcutta
Scientific Book Agency
G. • LIbrary
Copyright © 1960
The Williams & Wilkins Company
428 East Preston Street, Baltimore 2, Maryland, U.S.A.
Reprinted March, 1964

Reprinted June, 1965
Indian Edition Published August 1968

By Scientific Book Agency
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Library of Congress Catalog Card Number 60-128J1
Indian edition published by special arrangement with the

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Preface
This book is a product of ten year' of teaching a general course in

microscopic techniques in the Department of Zoology at the University
of California, Berkeley. Since no appropriate text was available, a
laboratory syllabus was prepared to meet the needs of the course. It
was both gratifying and revealing to find that the laboratory directions
for the course were generally useful to more advanced students pursuing
research problems of their own. There are many books dealing with
specialized techniques and compendium of technique methods, but
there is a need for a step-by-step pre entation of the basic procedure
upon which the more specialized techniques are ultimately dependent.
Thus, the proper preservation of the material is universal to all tech-
niques; and subdivision of larger organisms and ti Rues following paraffin
or celloidin embedding is usually preliminary to even the most pe-
cialized staining procedure.
The present book is .an expansion of the earlier yllabus, including the
material which was originally presented in lecture or demonstrations.
The text has been extended to include those procedures and problems
most frequently encountered by more advanced students. It is my hope
that the present volume will serve a double purpose. First, as a general
text for use in rnicrotechnique <,ourses and, second, a a guide to the
worker who has · not had a technique course 01' \\'ho, as is so often the
case, needs a "refresher" when faced with the problem of processing
tissues after a long absence from the techniq laboratory.
The primary problem has been that of Imiting the overage to a
practical area while providing enough diver ity to include most types of
preparations. In general the techniques a d solution included might
be considered "classical," having . h tood the test of use · by many
workers on a diversity of materials. All of these procedures are subject
to a t remendous variety of modifications depending upor" the material,
the objective and the worker. This is but a reflection of the fact that a
v
"
vi PREFACE
good technician, having mastered and understood the basic procedures

can intelligently modify them to fulfill the requirements which are
peculiar to almost any organism or tissue being processed. There is no
"universal technique" which will work equally well for all materials,
nor, unfortunately, for all technicians.
In general, the formulas, stains and techniques are not original with
the author, but, in keeping with text book procedure, no attempt has
been made to give authoritative references. Many of the formulas and
procedures bear the names of their originators and this permits c6n·
tinual acknowledgment. For an exhaustive consideration of different
techniques and references to original works, the student should consult:
Conn, Biological Stains; Cowdry, Laboratory Technique In Biology And
Medicine; Gray, The Microtomist's Formulary 4.nd Guide; and Lee,
The Microtomist's Vade-mecum. Referencef5 to these and other standard
works will be found in the Appendix.
Much of the material is presented in the form of exercises for the con-
venience of both student and instructor. In each instance the prelimi-
nary discussion should be considered before undertaking the exercise
itself. It should be noted that a single exercise may require more than a
single period or day of work. Furthermore, a number of exercises are
usually carried on simultaneously to utilize the available time to greatest
advantage. It is not expected that the average semester will permit a
con ideration of all of the preparations described, but should allow for a
sampling of different types of procedures. The actual work undertaken
will vary with the availability of materials, equipment, time, and the
particular emphasis of the course. The greatest part of the semester will
probably be devoted to the preparation and processing of paraffin
sections, since this technique is the most generally used and requires
considerable practice to penect. In some instances it may be practical
to provide certain tissues after preliminary preparation by the instructor
(fixation of some tissues; tissue blocks already embedded or even sec-
tioned in celloidin, etc.). Suggestions for scheduling the work are pre-
. ented in the Appendix.
I wish to thank my associates, both faculty and graduate students,
in the Department of Zoology at the University of California, Berkeley,
who directly or indirectly contributed to my general knowledge, ex-
perience and understanding of the problems of technique. I also wish to
express my gratitude to the many teaching assistants, and, further, to
the more than two thousand students in the general technique course,
many of whom offered many helpful suggestions over the years. Finally
I thank my husband, Robert R. Lechleitner, without whose encourage·
ment and help this book would never have been comI?leted.
Contents
PREFACE . . . . . ..... . . . . . . . . . . . . .. .... . . . . . . . . . . . . . . • • . . . v

CHAPTER 1, The Compound Microscope . ...... . ........ .
The Microscope and its Accessories .... .... . .. ... . . . 1
Principles of Image Formation . .. ................. . 6
Setting up the Microscope ........................ . 8
Exercise 1, The Compound Microscope . . ... . .. . .. . . . 14
A. The Field of View and the Microscope Adjust-
ments . ................... . ...... .. . , .. . 14
B. Color Image and Refractive Image .... . .. ..... . 14
CHAPTER 2, P review of Methods . . .. . ................ .. . 16
Types of Preparations . . ....... .. ......... . ....... . 17
Exercise 2 ..... . , .............. . ................ . 21
A. Supravital Staining . ... . ...... , .... . .... .. .. . 21
B. Methyl Green Fixing-Staining 22
C. Demonstration Slides . ....... . ... .. ... .. . . . . 22
CHAPTER 3, Records and Labels . . .. . ..... ... .... ..... . 24
The Working-Label. ... .. . ...... . ... .... ..... ... . . 25
Cleaning, Labeling and Storing the Finished Slide . . . . 26
CHAPTER 4, Fixatives ........ . .. . ...... . ...... .. ...... . 28
Acetic Acid .......... . . .. ... ... ....... .... .... .. . 29
Formalin . . ...... . ....... . . . .. . . . ... . . . .... ... .. . 30
Alcohol .............. ....... ... .. ............ .. . 31
Picric Acid ...... . .. ......... .. . . ........ . ...... . 32
Mercuric Chloride .... . ..... .. . ..... . ... ... . . .. . . . 33
Potassium Biohromate . .. . .. ... .. ..... . . ... ... . .. . 36
Chromic Acid ........ .. ..... ..... .... . . . . . .. . ... . 36
Osmic Acid ...................... . .. . ......... . 37
CHAPTER 5, Fixation . . . . . . . . ..... .. .................. . 39
Anesthetizing and Killing .. .. .. . ..... .. ...... ..... . 41
Fixation of Invettebrates .. ... ... ...... .... ." . . . . . . 42
vii
VlIl CONTENTS
Fixation of Larvae and Embryos ... . ....... .. .... . . 46

Fixation of Tissue Blocks . ..... . . .. . .............. . 47
A. General Procedures 47
B . Suggestions For Specific Tissues . ...... . .... . . 49
Exercise 3, Fixation of I~vertebrates for Smears and
Whole Mounts ..... . ......... . ... . ........ . 51
A. Protozoa Smears . ....... . .... . ...... . .. .. . . 52
B . Sponge Spicules . . ...... . . . ... . ............ . 52
C. Small Hydromedusae . ........ . ............ . 52
D . H ydroids . ........ .. ...... . . . . . . .. .. .. . . . . 53
E . Platyhelminthes ...................... . 53
F. Nematodes for Unstained Glycerine Jelly
Mounts .. . ....... . ..... . . . ........ . . . . . 53
G. Sea Cucumbers for Body Wall Mounts or for
Corrosion Preparations/. ................ . 54
H . Arthropods . . .. . .... .. .... . .... . .... .. . . . . 54
Exercise 4, Fixation of Bird or Reptile Embryos .. . .. . 54
Exercise 5, Fixation of Mesenteric Spreads ...... . . .. . 56
Exercise 6, Fixation of Animal Tissues . .. , . . ...... . 57
Exercise 7, Fixation of Plant Tissues ............ . .. . 57
A. Materials for Whole Mounts ...... . .. ... .. . . 58
B. Materi als for Sections . . . .. ... ....... . '.' .... . 58
C. Onion Root Tips . . ......... . ..... .. . . . . ... . 59
CHAPTER 6, Unstained Preparations ............... . .. . 60
The Slide Ringer ..... . ..... . . . . .. . .. . .. . .. . ..... . 61
A. Preparation of a Cell . .................... . . 61
B. S ealing Procedure . ...... . .. . ............. . . 62
Exercise 8, Dry Mounts ............... . .. ... . .... . 63
A. TransptJrent Mounts . . . ....... . . . ........ . . . 63
B . Opaque Mounts . . . .... . ............... . ... . 64
Exercise 9, Glycerine Mounts . .. ... . . . .. . ........ . . 66
Exercise 10, Glycerine Jelly Mounts .... .. .... .. . . . . 68
A. Mounting in Glycerine Jelly . ... ............ . 69
B. Alternate Method for Small Objects . .... . .. . .. . 70
Exercise 11, Berlese's Gum-Chloral Mounts . ....... . . 71
Exercise 12, Resin Mounts of Unstained Material . . . . 72
A. Soft Tissues .............. . . . ........... . . . ' 72
B. Dry Materials . ... .... ... . .. . .... . ........ . 73
Exercise 13, Corrosion Techniques .... . .... . .... .. . . 74
\ Exercise 14, Ground Sections of Bone or Tooth ...... . 75
CHAPTER 7, Stains . . . . . ..... . . . ..... : ..... . .. ... .. . . . 77
The Natural Dyes and Stains ...... . ......... . .... . 78
CONTENTS ix
The Coal Tar Dyes and Stains . . . . . . . . . . . . . . . . . . . .. 82

CHAPTER 8, Stained Whole Mounts . . . . . . . . . . . . . . . . . . . .. 91
Exercise 15, Grenacher's Alum Carmine . . . . . . . . . . . .. 92
Exercise 16, Grenacher's Borax Carmine . . . . . . . . . . . . . 94
Exercise 17, Whole Mounts of Minute Objects . . ..... 96
Exercise 18, Mesenteric Spread in Mallory's Triple
Stain ...... .. .............. . ...... . ...... , 99
Exercise 19, Alizarin Red S for Bone .......... . . .... 100
Exercise 20, Toluidine Blue for Cartilage ............ 101
CHAPTER 9, Processing Materials on Slides ... . .. . .... . 103
Cleaning Slides and Cover Glasses . . . .. . ........... 103
Containers and General Procedures. . . . . . . . . . . . . . . .. 104
Mounting the Cover Glass. . . . . . . . . . . . . . . . . . . . . . . .. 106
CHAPTER 10, Smears. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 108
Exercise 21, Protozoa, Fecal and Other Wet Smears in
Iron Hematoxylin .......... . .. . . .... . ..... , 108
Exercise 22, Blood Smears . . . . . . . . . . . . . . . . . . . . . . . .. 110
A. Thin Smears . . .. . . .. . .. . ... . ... . . .. ....... 111
B. Thick Smears . ... .. .. ........... , ........ 113
C. Cover Glass Preparations . ........... , . .... . 113
Exercise 23, Sperm, Vaginal and Tissue Smears . . . . .. 114
A. Preparation of Smears . .............. . .. . .. , 114
B. Fixation M ethods . . . . . . . . . . . . . . . . . . . . . . . . .. 115
C. Staining Dry S mears. . . . . . . . . . . . . . . . . . . . . .. 115
D . Staining Wet Smears . . . . . . . . . . . . . . . . . . . . . .. 116
CHAPTER 11, The Paraffin Method, I. Infiltration and
Emhedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 117
General Steps . . . . . . . . . . . . . . . . . . . . . . .. '" . . . . . . . . .. 117
Paraffin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 119
Exercise 24, Paraffin Processing of Tissue Blocks. . . .. 122
Exercise 25, Alternate Paraffin Methods. . . . . . . . . . . .. 134
A. Dioxane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 134
B . T ertiary Butyl Alcohol . ..... . ..... . .. .. ..... 135
Exercise 26, Processing Minute Organisms in Paraffin. 136
A. Centrifugation or Gravity Settling . . . . . . . . . . . . . 136
B. Embedding in a Sac of Animal Tissue . . . . . . .. 138
C. Embedding in Agar-agar . . ... . .............. 140
CHAPTER 12, The Paraffin Method, II. Sectioning ...... 141
The Cutting Blade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Exercise 27, The Rotary Microtome .... . ........... 1~7
Exercise 28, Paraffin Sectioning ......... .. .... ... . . 151
x CONTENTS
CHAPTER 13, The Paraffin Method, III. Affixin g a nd

Processing the Section~ ......... .. ..... ... .... 163
Affixatives ...... .. ......... ' . . . . . . . . . . . . . . . . . . . . .. 163
Sli.de Warmers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 164
Exercise 29, Affixing Paraffin Sections ......... .. ... . 166
Alternate and Supplementary Methods ....... .. .. .. , 170
Exercise 30, Affixing Serial Sections . . . . . . . . . . . . . . . .. 171
Processing Paraffin Sections . . . . . . . . . . . . . . . . . . . . . . .. 176
Setting up the Solutions. . . . . . . . ... . .......... 178
Exercise 31, Heidenhain's Iron Hematoxylin . ..... ... 179
Exercise 32, Ehrlich's Acid Hematoxylin . . . . . . . . . . . . 181
Exercise 33, Harris' Alum Hematoxylin. . . . . . . . . . . .. 183
Exercise 34, Mallory'S Triple Stain. . . . . . . . . . . . . . . .. 185
CHAPTER 14, The Celloidin Method ... ...... . ........ ... 188
Exercise 35, Infiltrating and Embedding in Nitro-
cellulose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 189
Exercise 36, Cutting Celloidin Sections. . . . . . . . . . . . .. 191
Exercise 37, Processing Free Celloidin Sections. . . . . . . 196
A. Mallory'S Triple Stain .. . ........ ....... .... 197
B. Safranin 0 and Fast Green. . . . . . . . . . . . . . . . .. 198
C. Removing the Celloidin before Mounting . . .. ... 198
Exercise 38, Affixing Celloidin Sections ... . ......... , 199
Exercise 39, Double Embedding. . . . . . . . . . . . . . . . . . .. 200
CHAPTER 15, The Freezing Method. . . . . . . . . . . . . . . .. '" 201
Exercise 40, Cutting and Staining Frozen Sections . . .. 201
A. Sectioning . ...... . ........... , . . . . . . . . . . .. 201
B. Sudan II for Fat . . . . . . . . . . . . . . . . . . . . . 204
CHAPTER 16, Miscellaneous Techniques . . . . . .. ........ . 206
Exercise 41, Bulk Staining . . ....................... 206
Exercise 42, Methylene Blue and Eosin . . . . . . . . . . . .. 207
Exercise 43, Feulgen's Nuclear Stain ................ 208
Exercise 44, Flemming's Triple Stain . . . . . . . . . . . . . . .. 209
Exercise 45, Masson's Triple Stain ..... .. .... . ... . . . 210
Exercise 46, Heidenhain's Mallory-Azan ............ ' 210
APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . • . . . . 212
I. Reagents, Formulas and Resins . . . . . . . . . . . . . . . . .. 212
A. General Rules for Preparing and Handling
Solutions . ................ .. . .. '......... 212
. B. Accuracy Requ1:red in Preparing Solutions. . . .. 213
C. Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 213
D . Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 216
II. Disposal of Wastes . . . . . . . . . . . . . . . . . . . . . . . . . . .. 217
CONTENTS xi
III. Equipment Usually Needed by the Individual for

Routine Technique Work ... . ......... . . .. .. , 218
IV. Scheduling of Work .... . . " .... . .. ...... ...... 219
V. Selected References ...... .. .. ...... . ...... . . . . , 221
INDEX .... ... ... .. .. . . . . . . . . . . . . • • . . . . . • .. ... . .. .. . . . . . 223
1
The
Compound
Microscope
The most fundamental requirements for the technician are the ability
to use the light microscope properly and to understand its capacities
and limitations. The processes utilized in preparing material for study
with the microscope are dependent upon the characteristics of the in-
strument as well as the nature of the material to be examined. Mi-
croscopic technique is not an end in itself but a preliminary step to the
examination of the material. By keeping in mind the requirements of
the microscope system, many of the procedures used in various tech-
niques are more readily appreciated. From the practical standpoint,
the proper use of the microscope permits the technician to obtain an
adequate picture of the material during processing when the slide must
be examined under adverse optical conditions in water or alcohol. The
greatest difficulty that many technicians encounter is the inability to
see and judge the material during and after its preparation.
The following discussion of the microscope is far from complete, but
attempts to provide a working knowledge of the instrument. For fur-
ther technical information regarding optical systems the student should
consult standard physics texts and the manufacturer's manuals.
The Microscope and Its Accessories
The microscope lenses are supported at either end of a tube which
may be moved in a vertical direction above a stationary stage on which
the material is placed. The center of the stage is perforated by an open-
1
2 GENERAL ZOOLOGICAL MlCROTECHNIQUES
ing which permits light transmission through the specimen and into
the lens system. There are two sets of lenses : an ocular at the top of
the tube, next to the eye, and an objective at the bottom of the tube,
next to the material. The microscope may be monocular or binocular.
Oculars are available in various powers, the 5x, 8x and lOx being the
most useful.
The microscope is usually provided with three objectives arranged
on a rotating nosepiece which permits anyone of them to be brought
into alignment with the tube and ocular. The objectives usually in-
clude: a "low power" (50 to 32 mm.) ; "intermediate power" (usually
16 mm.) ; and "high-dry" (8 to 4 mm.) lens systems. An oil immersion
obj ective (about 1.4 mm.) may be present in addition to the above, or
may replace the low power objective. The oil immersion objective
should not be used with unfinished or wet preparations. For technique
purposes, a 8x or lOx ocular in combination with the 48-, 16- and
4-mm. objectives is satisfactory. The low power objective permits ex-
amination of thick, whole mount material. The 16- and 4-mm. objec-
tives are necessary for the examination of smears or thin sections.
The following points should be kept in mind regarding the objec-
tives: (1) the size of the lens decreases from lower to higher magnifica-
tion objectives; (2) the fi eld of view or visible area of the material
decreases from lower to higher magnifications; and (3) the clearanct!
distance decreases from lower to higher magnification objectives. Clear-
ance distance refers to the distance from the front lens of the objective
to the obj ect, when the obj ect is in focus. The 48-mm. lens has a clear-
ance distance of about 30 mm.; the 16-mm. lens a clearance of about
6 mm., and the 4-mm. lens a clearance distance of less than 1 mm.
Many microscopes have a fixed tube length; that is, the distance be-
tween the bases of the oC.lllar and objectives cannot be varied. Others
have an adjustab le draw tube below the ocular, and a line or scale will
be found on the tube indicating tube length. Most lens systems are cor-
rected for a tube length of 160 mm. It is well to determine the proper
tube length for your microscope by referring to the manufacturer's
specifications. An incorrect tube length will interfere with definition of
detail, especially at high magnifications. Tube length affects the amount
of magnification obtained with a given ocular-objective combination.
The tube length may be varied to correct for cover glasses which are
too thick or too thin. (This is described under the section dealing with
the 4-mm. objective.)
The mirror, below the stage, is used to direct the light from its source
upwards through the aperture in the stage. The mirror usually has two
THE COMPOUND MICROSCOPE 3
surfaces, one plane and the other concave. The plane mirror simply
redirects the light without altering its form, and is designed for use
with the substage condenser. The concave mirror directs the light up-
wards and serves as a crude condenser converging the light rays into a
cone. It does not provide adequate illumination for higher magnifica-
tion systems and should never be used with the substage condenser.
The substage condenser includes a set of lenses mounted between the
mirror and the microscope stage. The condenser may be moved in u
vertical direction by means of a ratchet mechanism, and serves as a
critical light-focusing system. The column of light entering the lens
system from the plane mirror is bent inward to form a cone the rays
of which bisect above the upper lens of the condenser. The point of bi-
secting light rays should be focused upon the material being examined
(fig. 1, D). If the concave mirror is used with the substage condenser,
the light rays bisect within the upper lens of the condenser and it be-
comes impossible to focus them on the material.
The substage iris diaphragm, which li es below the condenser, is an
aperture diaphragm used to control the diameter of the light column
entering the substage condenser. The angle of the light rays emerging
from the material is directly proportional to the size of the light col-
umn entering the condenser. The wider the column the greater the angle
of the emerging light (fig. I, D and F) .
The microscope lamp utilized in the technique laboratory may vary
from complex models with lenses, filters and an iris diaphragm to sim-
ple table lamps. Since the formation of the image of the object is de-
pendent upon the light which is transmitted through the material and
picked up by the lens system and the eye, it is apparent that the light
source is an important part of the total system. The character of the
image obtained is as dependent upon proper illumination as it is upon
a good objective. Although the best possible microscope lamp is to be
recommended it is not always practical. It is important to remember
that the proper adjustment of the crudest lighting system will improve
the Image tremendously. On the other hand, the incorrect use of the
most expensive lamp results in a poor image. A relatively simple lamp,
properly used, will provide adequate illumination for most technique
purposes. A partially enclosed light, sometimes referred to as a "tor-
pedo lamp," is to be preferred over a more open light such as a goose-
necked lamp. If the latter must be used it should be inclined as close
to the table as possible so that the light will not strike the top of the
microscope stage. When a simple lamp is used without a ground glass
it should be provided with a frosted, blue "daylight" bulb.
4 GENERAL ZOOLOGICAL MICROTECHNIQUES
@ A
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c E
~XCESSIVE
,/ ,l LIGHT
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MIRROR- ______ _
F
B o
o G
H
FIG. 1. The fields of view,-e.nd adjustments of the substage condenser, substage
iris diaphragm and lamp diaphragm for critical illumination. A, C and E show the
fields of view observed with the situations illustrated in figures B, D and F, respec-
tively. B, D and F show the adjustments of the substage condenser and substage iris
diaphragm. In B the substage condenser is too low and the light rays bisect below
the material being observed . If the excessive light is eliminated by closing the sub.
stage iris, the image will still be impaired since the lens is not being provided with
light at the greatest angle which it can utilize. (The set-up approximates the situa-
tion for the high-dry objective.) D shows the correct set-up; the condenser has
been raised so that the light rays bisect at the level of the material being observed.
This is indicated optically by the fact that the image of an object, such as a needle,
held at the light source is projected into the field of view as indicated by 1 in figure
C. Note that the lens is now provided with light at a greater angle than was utilized
with the set-up shown in B . This will greatly improve resolution. F shows the cor-
rect set-up' for intermediate power. Note that although the field of view (E) is
The theoretically ideal illumination source is a point of light, but

this is difficult to achieve or to utilize. One compromise is Kohler illu-
mination, utilizing an aspheric lens in front of the actual light source
as a source of illumination. This is very effective since it provides a
large area of light of uniform intensity derived from a relatively small
source. When using this type of lamp the image of the light filament is
focused on the substage iris by adjusting the focusing device in the
lamp. The image of the aspheric lens is then projected into tIN:.. field of
view by adjusting the level of the substage condenser. Since )he lens
involved is very expensive, such a lamp may not be available. I
A second compromise involves the use of a pair of planoconvex lenses

in front of the light, with a ground glass in front of these lenses. Usu-
ally a blue "daylight" ground glass filter is used. This glass serves as
a projected light source and is focused on the object by means of the
substage condenser. This type of lamp is relatively inexpensive and
is more frequently available than one with an aspheric lens. When a
simple light must be used the surface of the blue frosted bulb should
be focused on the material as though it were the ground glass filter of
a lamp. The procedure for focusing the light at the level of the field of
view will be described later.
The lamp iris diaphragm, or field diaphragm, provided on the face
of some lamps, should be adjusted according to the ocular-objective
combination being used. Only the field of view should be illuminated.
Since this area is reduced as higher magnification systems are used, the
lamp diaphragm must be closed accordingly (fig. 1, G and H). With
the lOx ocular and the 16-mm. objective, the correct lamp aperture is
about 75 mm., whereas with the 1.4-mm. oil immersion obj ective it is
only about 10 mm., with the lamp about 8 inches away from the mi-
croscope. The lamp diaphragm reduces excessive light and helps to
eliminate "glare." This phenomenon results from th e lateral movement
of light through the material and imparts a hazy or fuzzy appearance
larger, the angle of the light which is utilized by the lens is less than in D and the
substage iris has been closed down just enough to eliminate the light which is not
being utilized . The area of the material which is illuminated is controlled by the size
of the lamp aperture and is independ ent of the angle of the light rays which is con-
trolled by the substage iris diaphragm . This is discussed in the text. E shows the
method of focusing the light source on the material by closing the lamp diaphragm
and raising (or lowering) the substage condenser until the image of the diaphragm
(!) is superimposed on the image of the material. The diaphragm is then opened
until the image of the diaphragm coincides with the edge of the field of view (8) .
Figures G and H show the relative openings of the lamp diaphragm for high-dry
(G) and intermediate (H) power objectives.
to the image. The field diaphragm does not affect the angle of light as
does the aperture diaphragm.
Some lamps are provided with a rotating shield, having a series of
holes of various diameters for use with different objectives. Simple
lamps, especially those of the "torpedo" type, may be provided with
a cardboard or metal shield having appropriate holes punched out of
the centers. The original aperture of these lamps will fill the field of
view with the 16-mm. objective, if placed at the proper distance. The
improvised diaphragms may be held in place by a metal or cardboard
frame attached to the lamp housing. If the lamp has an overhanging
shield, the diaphragms may be held in place by a magnet glued to the
diaphragm. The aperture in such shields should be carefully centered
over the light source. Such simple arrangements greatly improve the
illumination of the material.
It should be noted that neither the field nor aperture diaphragms
affect the intensity of the light. Intensity varies with the wattage of
the bulb, the voltage used, and the distance of the illuminator from the
object illuminated In the case of a simple bulb or light from a ground
glass filter in front of a lens, the intensity of the light is inversely pro-
portional to the square of the distance from the illuminator to the
object. Voltage may be controlled by use of a rheostat. Intensity may
also be varied by the use of special filters.
Principles of Image Formation
With the compound microscope an image is form ed as a result of
light rays passing through the material being examined (transmitted
light) into the lens system of the microscope and reaching the eye of
the observer. During this process the light rays are shifted in such a
way as to form an inverteg image of the object. The simple microscope,
or magnifying glass, does not invert the image.
In order to discuss the basic principles involved in light microscopy
it is necessary to define several terms.
Magnification. The enlargement of the image of an object. In effect
the object is brought closer to the eye.
Resolution. The ability to distinguish two closely associated objects
as being distinct from one another.
Color Image. The image formed by the differential absorption of
certain wave lengths of light by a pigmented or dyed object.
Refractive Image. An image resulting from differences in the re-
fractive index of a transparent object and the medium which surrounds
it.
Refractive Index. The relative velocity of light passing through a

transparent object or medium as compared with the velocity of light
through a vacuum. .
Velocity of light in vacuum .. .
\ ' eI oClty
' f r h' d' = refractive mdex (R.I.) of medIUm
0 Ig t III me lUm
It should be kept in mind that although mo t images are a combina-

tion of a color image and a refractive image, these two phenomena are
actually distinct from one another. A clean, smooth piece of colorless
glass may be observed in air because the refractive index of the glass
(R.I., about 1.60) is different from the refractive index of air (R.I.,
1.00). If the glass is placed in a solution having an identical refractive
index the glass will disappear optically. If, however, the glass is col-
ored, both a color image and a refractive image are observable in air.
Tf a colored glass is placed in a colorless solution having a refractive
index identical with that of the glass, the area occupied by the glass
will be yisihle as a color image. If only a portion of the glass is colored,
only the colored area will be visible.
The observation of a refractive image is dependent upon the trans-
mission of parallel light rays through the material from one direction.
Although the microscope system appears ideal for transmitting par-
allel light through the material, such li ghting conditions are not favor-
able for good resolution and, therefore, will not permit the observation
of finer details even though the image is enlarged. Magnification in it-
self accomplishes little, since increasing the size of the image may not
reveal any new detail .of structure. Magnification must be combined
with a system which will provide resolution.
Resolution is favored by the use of a cone of light and such a system
is compatible with the observation of a color image. Consequently,
most microscopy is based upon utilization of the color image produced
by natural pigmentation or, more usually, by staining, rather than on
a refractive image produced by differences in the refractive indices of
yarious portions of the material and the medium which surrounds it.
It should be remembered that certain structures may not take a stain
and must be viewed on the basis of a refractive image even though the
finer details may not be observed. Furthermore, the observation of
living cells is almost entirely dependent upon the differences in the re-
fractive index of different portions of the cells and the medium in which
they are observed.
The resolving power of an objective is dependent upon what portion
of the light cone the objective can utilize or upick up" as light is trans-
mitted through the material. The inherent capacity of an objective to

gather in light is indicated by the "numerical aperture" (N.A.) of the
objective. The larger the N.A. the greater the resolving power possible.
Resolution may be improved by using short wave length light and/oJ
by slowing down the light. Both have the effect of bending the light in-
wards so that a greater proportion of the light waves leaving the ma-
terial can be picked up by the objective. Short wave length light may
be obtained by using a blue ("daylight") bulb or a blue filter in front
of a white bulb. The light may be slowed down by mounting the ma-
terial in a medium having a high refractive index, and by increasing
the refractive index of the material itself. Resolution is further im-
proved by the use of immersion oils between the objective and the ma-
terial. The immersion oils have a higher refractive index than does air
and retain a greater amount of light emerging from the material within
the angle that the objective can utilize. Oil immersion systems are usu-
ally employed with higher magnifications, but may also be obtained
to replace some of the "dry" objectives. These relatively low power
oil immersion objectives are useful in photographic work. Water im-
mersion lenses are also available.
It should be noted that a dry lens should not be used in oil nor water,
nor an oil or water immersion lens in air. Each is constructed to be
utilized in a specific medium. The numerical aperture of a given ob-
jective is calculated on the basis of the refractive index of the medium
in which it is to be used.
The fullest capacity of an objective is realized only if it is provided
with light at the maximal angle that it can pick up tfig. 1, B and D).
Therefore, the higher the N.A. of the objective, the greater should be
the angle of light emerging from the material. As indicated earlier, the
angle of light is dependent upon the adjustment of the substage iris
diaphragm. The greater the N.A. of the objective, the wider must be
the opening of the substage iris. Since the N.A. of the higher powered
objectives is greater than that of low powered, the substage iris must
be opened when going fro.m lower to higher powered objectives. Failure
to make this adjustment is one of the most common errors in setting up
the microscope.
Setting Up the Micro8cope
General Rules for Care and Use
1. Carry the microscope with both hands, using one to support the
base.
2. Do not tip it excessively or the oculars may slip out!

3. The lenses of the ocular, objectives and the condenser, as well as
the surfaces of the mirror should be kept clean and free of dust. They
should be cleaned with lens paper. When it is necessary to use a solvent
(water, xylol or chlorofomr;-depending upon the material to be re-
moved) use it sparingly and dry the lens. Alcohol should never be used
'for cleaning lenses, because it will dissolve the cement which holds the
lenses in place; touching the lenses or mirror surfaces with your fingers
may result in corrosion of the glass.
4. Keep the stage covered with a thin glass plate when examining
wet preparations during processing. This protects both the stage and
the lenses of the substage condenser. The plate must be cleaned fre-
quently and should not be allowed to collect enough fluid to flood on to
the stage.
5. Never force an adjustment on the microscope. If the ratchet mech -
anisms do not work readily they should be cleaned and checked.
6. If the fine adjustment does not seem to be working in one direc-
tion it has probably reached the limit of adjustment. Rack up the tube
with the coarse adjustment knob so that the objective is clear of the
slide; turn the fine adjustment knob in the direction opposite to that in
which it has stopped. Refocus on the slide.
7. When using a monocular microscope keep both eyes opcn. If you
have difficulty at first in "blanking out" the view with the eye you.are
not using with the microscope try holding a white card, or your hand,
about 4 inches away from the eye to break the image. If you use a
monocular for long periods it is best to train yuurself to use both eyes
for viewing.
8. When the microscope is not in use it should be protected with
some type of covel' to f:>xclude dust. A paper or plastic bag is quite ef-
fective.
Focusing Procedure
Gross 'vertical movements of the tube and attached lenses are ac-
complished by means of the coarse adjustment knob. Adjustments
made with this knob, while looking through the microscope, should al-
ways be made in an upward direction. When racking down with this
knob the objective should be viewed from the side so that contact be-
tween the lens and the slide is avoided. Because of the difference in
clearance distances, it is never safe to swing directly from one objec-
tive to a second, higher magnification, objective. The tube should be
racked' upwards before a higher powered objective is swung into posi-
tion. The lens is lowered until it is close to the material while watching
from the side with the eye at the level of the stage and then racked up-
wards until the material is brought into focus. Minor focusing adjust-
ments in either direction are made with the fine adjustment knob.
The procedure of racking up the tube when changing objectives
!lhould be followed even though the microscope is parfoeal. A parfocal
system does not guarantee that there is sufficient clearance distance.
When a microscope is parfocal the object may be brought into focus
with one objective and will be in focus when another objective is swung
into place. If, however, the preparation is too thick the lens will be
brought into contact with the slide. In examining thin preparations it
would be expected that the clearance distance would be adequate with
a parfocal system, but extreme care must be exercised to be sure that
the slide is placed tissue-side-up on the stage. An inverted slide will go
undetected with the 16-mm. lens and will not yield sufficient clearance
for the 4-mm. objective because of the thickness of the slide. Slides are
occasionally inverted, especially before a paper label has been attached
and while the material is being processed.
Setting Up the Low Power Objective (50 to 32 mm.)
1. The usual substage condenser does not illuminate a large enough
field to be used with low power objectives. Remove the condenser and'
us~ the concave surface of the mirror. The condenser is usually removed
by racking it all the way down and swinging it to one side.
2. Adjust the concave mirror so that it will direct the light from the
lamp upwards through the material. The intensity of the light and the
area illuminated are controlled by varying the distance of the light from
the substage mirror. The lamp should be placed in a- straight line with
the microscope and the mirror adjusted so that the light is centered in
the field of view.
If a low power condenser is available, or if the one provided for
higher magnifications has a removable upper lens, the condenser and
plane mirror are used for illumination. In this case adjust the micro-
scope as directed for the 16-mm. objective.
Setting Up the intermediate Objective (16 mm.)

1. Place the microscope and lamp in a straight line and about 6 to 8
inches apart. The lamp-to-microscope distance should be adjusted so
that the greatest lamp aperture is sufficient to completely fill the field
of view visible with this objective (with the material in focus).
2. If neces. ary, adjust the tube length (usually 160 mm.).
3. Use the plane mirror and the substage condenser. Open the sub-
stage iris diaphragm.
4. Adjust the light so it falls upon the mirror and not upon the table
or microscope stage.
5. Adjust the mirror so that the light is directed upward through the
material and is centered in the field of view as observed through the
microscope.
6. Focus on the material, following the procedure outlined earlier.
This step must be carried out before the following steps can be properly
completed.
7. Focus the light source on the material by raising or lowering the
substage condenser. When the light source is in focus the light, or an
object such as a needle held at the light, will be visible in the field of
view, superimposed on the image of the material (fig. 1, C). If a ground
glass filter is used in front of the lamp it serves as a projected light
source and should be used as a point of focus. A small x may be pen-
ciled in the center of the ground glass to serve as a focus point. When
a blue, frosted bulb is used as a light source, focus the surface of the
bulb on the material.
8. Adjust the lamp diaphragm (or lamp di stance with this objec-
tive) so that the visible field is just completely illuminated.
9. Remove the ocular and, while looking down the tube, adjust the
substage iris diaphragm so that it is just visible at the edge of the field
of view as observed in the back lens of the objective. In this position
the objective is receiving as much light as it can utilize. If the substage
iris· is opened beyond this point, a fuzzy image results. If the substage
iris is closed down, the resolving power of the system will be impaired.
If a refractive image is required, the microscope is set up in exactly
the same way, but the substage iris is closed down. By simply opening
and shutting the substage iris, therefore, it is possible to shift from a
color image to a refractive image and back again.
If a lamp with an aspheric lens is available the following procedure
should be followed to obtain Kohler illumination. Position the substage
condenser (and substage iris) so they are at the approximate level
necessary to focus the Jight on the material. Usually the upper lens of
the condenser will be close to the lower surface of the slide. Close the
substage iris diaphragm and focus upon its lower surface the image of
the lamp filaments. The face of the iris may be examined with a small
mirror while focusing the lamp filaments by adjusting the position of
the aspheric lens in the lamp. Once the filaments have been focused on
the iris, open the substage iris and adjust the light as outlined above.
12 GENERAL ZOOLOGICAlr .MICBOTECHNIQUES
In order to focus the light at the level of the field of view (step 7), close
down the lamp iris and adjust the condenser so that the image of the
lamp iris is projected into the field of view (fig. 1, E). Then open the
lamp iris so that the field is completely illuminated.
Setting Up the High-dry Objective (8 or -4 mm.)
Since the area visible with this objective is relatively small it is de-
sirable to examine the slide first with the 16-mm. objective, setting up
the microscope as outlined above. The area to be examined is then
centered, the objective is racked up and the high-dry objective swung
into position.
1. Focus on the material ; bring the objective close to the cover glass
while watching from the side and rack up slowly while watching through
the microscope.
2. Check that the light source is still focused and centered on the
material. Adjust the mirror and substage condenser if necessary.
3. Reduce the size of the lamp aperture, if a lamp diaphragm is
available, so that just the fi eld of view is illuminated.
4. Remove the ocular and adjust the substage iris so that it is just vis-
ible at the edge of the field of view in the back lens of the obj ective.
Note that whereas the lamp diaphragm must be closed down, the sub-
stage iris m1Jst be opened wider.
In the case of the high-dry objectives it may be necessary to make
corrections for a cover glass which is too thick or too thin for the lens
system. The objective is constructed so that the cover glass and the
mounting medium is a part of the lens system. In the case of the .4-
rom. lens the deviation allowable is very slight, and if exceeded in either
direction a blurred or foggy image results. For this reason it is impor-
tant that the correct thickness of cover glass be employed and that a
minimum of mounting medium is used. In general the " No. 1" cover
glass is approximately correct in thickness for the high-dry obj ective.
For critical work it is best to measure even these and retain only those
cover glasses which have a thickness of 0.15 to 0.18 mm. The cover
glass thickness for which objectives are corrected ·varies somewhat with
different lens manufacturers. If a cll"ar, sharp image cannot be ob-
tained, even though the light has been properly adjusted, it is prob-
able that the cover glass is not of the correct thickness. This may be
compensated for, within narrow limits, by varying the tube length. If
the cover glass is too thick, the tube length is decreased; if too thin, the
tube length is increased. Some objectives are provided with a correc-
tion collar to compensate for deviations in cover glass thickness. If the
cover glass or preparation is so thick that there is not sufficient clear-

ance distance the material cannot be viewed with the high-dry or oil
immersion objectives.
. Setting Up an Oil Immersion Objective
The oil immersion objective should not be used on unfinished or
freshly prepared slides. Be sure the mounting medium has hardened
completely before examining the slide with an oil immersion system.
For the most critical work a drop of immersion oil should be placed
between the slide and the condenser, as well as between the cover glass
and the objective.
1. Locate the area to be examined, using the high-dry objective.
2. Rack up the tube and swing the oil immersion objective into place.
3. Place a drop of immersion oil on the cover glass surface in the
area to be examined.
4. Watching from the side, bring the objective into contact with the
oil, and as close to the cover glass as possible.
5. Looking through the microscope, bring the material into focus by
racking up the objective very slowly. If the material does not come
into focus by the time the objective breaks free of the oil, readjust the
objective, while watching from the side, and begin again. If you can-
not bring the material into focus, the cover glass may be too thick. Do
not be tempted to rack downward since you may bring the lens into
contact with the cover glass and may damage both lens and preparation.
6. Check that thc light source is still focused on the material. Adjust
the substage condenser if necessary
7. Reduce the size of the lamp aperture so that it just fills the field
of view.
8. Adjust the substage iris so that it is just visible as viewed in the
back lens of the objective with the ocular removed. Usually with an
oil immersion objective, the condenser iris must be opened to its fullest
extent.
Be sure that all traces of oil are removed from the objective when
you finish work for the day. Remove the excess with a piece of dry lens
paper. Then wipe the lens with a piece of lens paper slightly moistened
with xylol, and again with a dry paper. The oil should also be removed
from the condenser lens if oil was used under the slide. The slide itself
should be cleaned with a cloth moistened with xylol. If an uncovered
film has been examined do not try to wipe it clean, since the prepara-
tion will be removed. Such slides may be cleaned by placing them tn
xylol and then draining the slide.
14 GENERAL ZOObOGICAL MICROTECHNIQUES
Exercise 1
The Compound Microscope
The principles of microscopy presented here are more readily ap-
preciated once they have been visualized. The two procedures which fol-
low have been found to give a simple and effective demonstration of
the effects of lighting and the difference between color and refractive
images. The material required can be prepared by the students or can
be supplied in the form of two prepared slides loaned to each individ-
ual for the occasion.
A. The Microscop e Adjustments
Mark a 25- by 75-mm. (1- by 3-inch) sheet of white bond paper
with a series of circles, one within the other. The inner circle should
be as small as possible and the outer circle about 20 mm. in diameter.
Tape the sheet to a microscope slide. The paper will serve as a diffu-
sion plate which will permit observation of the light at the level of the
field of view, and will show the effects of .adjusting the substage con-
denser, substage iris diaphragm, the mirror, and the lamp diaphragm.
The circles aid in demonstrating the area visible with each ocular-ob-
jective combination.
1. Set up the microscope, following the directions given previously for
low, intermediate and high-dry objectives.
a. In each instance note through the microscope the area of the slide
which is visible.
b. Observe from the side the area of the slide which is illuminated
after adjusting the light as directed for each set-up.
2. With the light adjusted for the high-dry objective, raise the tube
and watch the light at the slide level as observed on the paper.
a. Note the effect of ;hanging from the plane to concave surface of
the mirror.
b. Return to the plane mirror and note the effect of opemng and
closing the substage iris diaphragm.
c. Note the effect of opening and closing the lamp diaphragm if one is
available.
d. Note the effect of lowering the substage condenser.
B . Color Image Versus Refractive Image
Prepare a slide of butterfly wing scales as follows. Press a pencil
eraser firmly on a butterfly wing in an area with colored scales. Then
press the eraser firmly on a clean cover glass, placing the scales slightly
u. . ~.-.,
off center to the right. Wipe the eraser clean. Transfer a group of white
(colorless) wmg scales to the same cover glass, placing them next to
the colored ones and to the left of center. Place a small amount of
mounting medium such as thin Canada balsam or a synthetic resin on
the surface of a clean slide. Invert the slide and bring the drop of
mountant into contact with the surface of the cover glass bearing the
wing scales. Turn the slide right-side-up and allow the medium to flow
to the edge of the cover glass. If the medium is too thick the slide may
be warmed slightly to hasten the flow of the medium. If the correct
amount of mountant has been used it will just flow to the edge of t he
cover glass. This type of mount is easier to prepare with a round cover
glass than with a square one.
1. Place the slide on the microscope stage so that the area of colored
wing scales is in the field of view. Focus on the material, first with in-
termediate and then with the high-dry objective, being careful to set
up the microscope properly. Note the effects of opening and closing
the substage iris and the lamp iris.
2. Focus very carefully on the colored wing scales with the high-dry
objective (intermediate power may also be used). Without altering
the focus, except for very minor movements with the fine adjustment,
search the area where the colorless wing scales were placed. Since both
sets of scales were mounted on the under surface of the cover glass,
they should be at the same focus level. Adjust the light for a refractive
image by closing down the substage iris diaphragm. When you think
you have located a colorless wing scale, open the diaphragm. If it is
a pigmented scale it will still be visible. If it is a colorless scale it will
essentially "disappear" as the substage iris is opened.
3. Place some colorless wing scales on the end of the same slide and
examine them in air. Are they more readily observed in air or in the
mountant? What do your observations indicate regarding the relative
refractive indices of the wing scales, air and the mounting medium used
on the slide?
A similar demonstration which is perhaps even more effective is to
mount on the same slide a stained and unstained section from the same
block of tissue. This type of slide may be used in place of the above or
may be set up on a demonstration microscope.
2
Preview
of
Methods
In view of the characteristics of the light microscope and the princi-

ples of image formation, it is evident that materials to be observed with
such a system must meet certain specifications. They must be relatively
thin if they are to be studied at high magnifications. They should be
colored if maximal resolution is to be obtained. They must be transpar-
ent if they are to be observed with transmitted light.
Some organisms, such as protozoa, are minute enough to be studied
without subdivision, but in most instances some reduction in thickness
is necessary. This may involve dissection and/ or subdivision by smear-
ing, corrosion, maceration, teasing, or by cutting the material into thin
slices. Thinness reduces the problem of transmitting light through the
material. Furthermore, thianess simplifies the image obtained by reduc-
ing the material to a single layer of cells or portions of cells if appropri-
ately subdivided.
Most biological material is not naturally pigmented and a color image
is obtained by treating the material with certain dyes or stains. Some
stains have a special advantage in that they have an affinity for certain
types of structures.
Transparency of the material is achieved by mounting it in a medium
(usually a gum or resin) having a similar or identical refractive index.
Resolution is improved if both the material and the mounting medium
have a relatively high refractive index.
The preparation of material for microscopic examination involves four
major aspects. (1) Preservation of a special type referred to as "fixa-
16
PREVIEW OF METHODS 17
tion," which stabilizes the material in as near a lifelike condition as

possible and prepares it for further processing. (2) Subdivision of large
materiaL (3) Staining, which may proceed but usually follows subdivi-
sion and may involve the use of one or several dyes. (4) Mounting, under
conditions favorable for permanence and observation. These processes
must be integrated and involve a number of intermediate procedures
(such as washing to remove the fixative, dehydration, etc.). The pro-
cedures vary greatly in complexity and will be considered in detail later.
It is important to remember that the finished preparation presents only
a partial and altered picture of the material as it was in the living state.
Types of Preparations
The following discussion presents a general picture of the diversity of
microscopic techniques along with some consideration of their uses and
limitations.
Living material which is small enough, or can be isolated, may be
studied for a limited time with the microscope. Protozoa, invertebrate
embryos and larvae, small invertebrates such as flatworms, nematodes,
rotifers, gastrotrichs, etc., and vertebrate and invertebrate· blood, sper-
matozoa, oocytes and isolated tissue cells are all favorable objects for
such examination. Aquatic forms are examined in fresh or sea water de-
pending upon their origin . Parasitic forms and tissue cells are examined
in physiological saline or Ringer's solution. The material is usually ex-
amined unstained or may be treated with one of a group of dyes which
will stain living protoplasm. These are the vital or supravital dyes. The
two most frequently used are Janus green, which is specific for mito-
chondria, and neutral red, the color of which varies with the pH of the
material stained.
Dry mounts may be prepared of structures which are already dry or
may be dried without distortion. Such .objects may be mounted in air.
If they are thin enough they are studied by transmitted light. If they
are opaque, they are observed by reflected light (illuminated from
above). In most instances it is desirable to use a mounting medium
which will reduce the opaqueness of the material, even though it is dry.
The shells of foraminiferans, spicules of sponges, skeletal plates of sea
cucumbers, wing scales and wings of certain insects, hairs, feathers, and
ground bone or tooth sections may all be prepared as dry mounts.
Wet whole mounts are limited to organisms or parts of organisms
which are small enough to be mounted entire and studied by trans-
mitted light. Unlike the dry mounts the material is not desiccated, since
this leads to distortion of soft tissues and must be carefully avoided. In-
stead, the material is dehydrated by replacing the water with other

solvents, most commonly ethyl alcohol. The alcohol is then replaced with
another solvent which is miscible with the mounting medium as well as
the alcohol. Since the dealcoholizing agent has a high refractive index
and makes the material transparent, it is referred to as a "clearing
agent." Finally, the material is placed in a mounting medium which re-
places the clearing agent and the obj ect is mounted on the slide under a
cover glass. Such preparations are usually stained, before or during de-
hydration. Protozoa, small insects and other arthropods, embryos, larvae
and many parasites are prepared as stained, wet whole mounts. With the
exception of very minute forms, such preparations can usually be stud-
ied with low and intermediate powers only, depending upon the size of
the material. A more detailed study requires further subdivision.
If a stained preparation is not required by the study, or IS impractical
because of the nature of the material, the object may be mounted un-
stained in glycerine or glycerine jelly. These media have sufficiently
high refractive indices to render the material relatively transparent so
that it can be studied by transmitted light. On the other hand, the re-
fractive indices of medium and material are sufficiently different to per-
mit the observation of a refractive image. Helminth eggs, small nema-
todes and insects are frequently mounted in this way.
Smears are prepared by spreading a thin film of material on a slide
or cover glass. Materials commonly prepared by smear techniques are
protozoa, blood, lymph, spermatozoa, contents of the digestive tract,
feces, vaginal epithelium, and any tissue which is soft enough so that in-
dividual cells may be separated from the general tissue mass by pulling
it over a slide. Blood and vaginal smears may be permitted to dry when
they are utilized for the identification of cell types, rather than for a
study of cellular detail. T.estis and epididymis smears which are pre-
pared to determine the presence or absence of spermatozoa, may also be
prepared as dry smears. Distortion is minimized if they are dried as
rapidly as possible. When any detail of structure is desired a wet prep-
aration should be made. Once the material has been spread on the slide,
the processing of a smear is essentially like that of a whole mount prep-
aration.
Squashes are prepared by placing the material in a fluid between slide
and cover glass and tapping on the cover glass until the desired degree
of separation has occurred. This type of preparation is used a.lmost ex-
clusively for the study of chromosomes such as those of Drosophila sali-
vary gland cells. The. material is usually squashed in a fixing-staining
solution. It may be examined in the initial solution or prepared as a

permanent mount by dehydrating, clearing and mounting.
I solation techniques permit the subdivision of tissues into structural
units suitable for examination. These techniques involve an actual
breakdown of the tissues by teasing, maceration or corrosion. Teasing
involves the dissection of fibrous tissues, such as tendons, by separating
the tissue mass with dissecting needles. This procedure may be carried
out without preliminary softening or may follow maceration. Macera-
tion involves a chemical breakdown of intercellular substances ("ce-
ment") so that the individual cells are shaken apart or isolated by teas-
ing. Corrosion techniques are utilized in the preparation of hard secreted
objects (calcareous ~r silicious) and involve the chemical distruction of
soft tissues by reagents which will not destroy the deposits.
The main advantage of isolation techniques is that they allow a study
of complete units of structure; individual cell units in the case of teased
or macerated preparations; individual spicules, plates, etc., in the case of
corrosion tech niques. Such preparations, however, do not permit a study
of cell OJ' tissue interrelationships. Once the material has been isolated
it is handl ed as a wet whole mount in the case of soft tissues, or as a dry
mount in the case of secreted structures.
Sectioning pro\'icles a method of subdivi son which retains the struc-
tural unit in its natural position so that its interre lationship to other
structUl'al units and the whole may be observed. The organism or tissue
block is cut into thin slices or sections of a known and uniform thickness
with a precision instrument called a microtome (fig. 2). Tissues in either
a fresh or fixed state ilre not sufficiently rigid to be cut into sections of
the necessary thinness without distortion. The tissue must be supported
during sectioning, either by freezing or by infiltrating the cells with
some supporting material such as paraffin or celloidin.
Freezing is usually employed when time is essential or when it is
necessary to retain substances, such as fat, which are destroyed by the
reagents used in paraffin or celloidin embedding.
Paraffin is the most widely used embedding material, being effective
for most tissues and providing a relatively rapid procedure compared to
celloidin embedding. Whenever possible, serial sections should be pre-
pared with this technique. Since paraffin is not miscible with water the
fixed tissue is dehydrated , usually with alcohol. This is then removed
with toluol, ch loroform , or some other reagent which is ~iscible with
both alcohol and paraffin. The dealcoholizing agent is then replaced
with paraffin kept just above melting point. The paraffin mass with the
FIG. 2. The rotary microtome (AO model 820 ; photograph courtesy of the Ameri-
can Optical Company). The lid (1) of the microtome has been raised to expose the
advance mechanism (f), which consists of a cone bearing against an inclined plane .
Each time the advance wheel (3) is turned one revolution, the tissue block (~) held
in the block clamp (5) is advanced forward and downward over a stationary knife
(6). The distance of advancement is determined by the setting of a micron scale
which is not visible in the photograph. See Chapter 12 for a description of the action
and use of the rotary microtome.
infiltrated tissue block is then hardened by cooling, the block is trimmed,

attached to a peg, and the material sectioned. The sections are placed on
water on microscope slides and warmed slightly to completely flatten
the tissue. Upon drying the material is attached or "affixed" to the slides.
The paraffin is then removed with a solvent, the solvent removed with
alcohol, and the material is hydrated by running the slides down a
graded series of alcohols into water. The sections are then, stained and
subsequently dehydrated, cleared, and a drop of mounting medium and
the cover glass applied.
Celloidin offers a maximum of tissue support, but the procedure is
relatively slow and is not favorable for the preparation of serial sections.
Its use is indicated when the tissue blocks are large and/ or dense or tend
to over-harden or distort when heated to temp.eratures (about 58° C.)
necessary for paraffin infiltration. The fixed material is dehydrated

through a series of alcohols, and then transferred to anhydrous ether-
alcohol which is a solvent for the celloidin. ·From the solvent it is proc-
essed through three solutions of celloidin of increasing concentrations.
At room temperature, each solution may require days to weeks to infil-
trate the tissue block. The celloidin mass with the infiltrated block is
then hardened by evaporation of most of the solvent and replacement
of t he remainder with chloroform. Sections are cut and the material
may be attached to slides and the celloidin removed. Such slides are then
handled as are paraffin sections after removal of the paraffin. Alter-
nately , the celloidin may be left in the sections which are processed
through the solutions and mounted with the celloidin intact, or the
celloidin may be remo\'(:! d just before clearing and mounting.
Exercise 2
A. Supravital Staining
Although our primary concern is with the preparation of permanent
mounts, the technician should appreciate the value of examining mate-
rial in the living state whenever possible. The following exercise permits
the examination of living protozoa and demonstrates the effects of supra-
vital staining.
Termite protozoa are recommended since they are relatively large and
are obtainable in many areas. Other protozoa may be substituted.
1. Flood a clean slide with neutral red solution (1: 1000 in absolute
alcohol) or with a combination of neutral red and Janus green (add 1
or 2 drops of saturated aqueous Janus green to 10 ce. of the neutral red
solution). Drain off the excess stain and let the film dry completely.
2. With your forceps, grasp a termite by the head or thorax and place
it on its back in the center of the slide. With a needle, catch the last 2 or
3 abdominal segments and pull out the digestive tract.
3. Place a drop or two of 0.75 per cent sodium chloride on the gut and
break it open so that the protozoa in the hind gut are released into the
saline solution.
4. Lower a cover glass on the preparation, leaving some of the gut
fragments to support the cover glass. If necessary add additional saline
so that it reaches the edge of the cover glass.
5. Immediately examine the preparation under the microscope. Note
the appearance of the protozoa as viewed with the substage iris dia-
phragm closed down so that a refractive image is obtained. Open the
substage iris to the edge of the field of view, and note the color and ex-
tent of the stained areas in the various protozoa. Watch the protozoa for
22 GENERAL ZOOLOGICAL · MICROTECHNIQUES
a considerable period and notice if within 30 minutes or more the color

and/or extent of the staining varies. This exercise may be carried out
with blood by placing a drop of fresh blood on a cover glass and drop-
ping it on the surface of a slide which has been filmed with the stain.
The edge of the preparation should be sealed with paraffin to prevent
rapid evaporation. In order to obtain observations over any period of
time it is necessary to keep the slide warmed to about body temperature.
B. Methyl Green Fixing-Staining
Methyl green, combined with glacial acetic acid, serves to stain chro-
matin in fresh tissue. Unlike the supravital stains, the dye is actually
acting upon material which has been killed or fixed by the acetic acid in
the staining solution. The supravital stains, on the other hand, are
staining material which is still living, and as the cells die the staining
effect will change.
1. Prepare a termite protozoa preparation as directed in steps 2 to 4
in Section A . (Cultures of protozoa, etc., may also be used.)
2. Reduce the amount of saline solution so that only about one-half
of the area under the cover glass is flooded. If necessary touch a piece
of filter paper or a blotter to one edge of the cover glass to remove any
excess saline solution.
3. Add several drops of 1 per cent aqueous methyl green in 1 per cent
glacial acetic acid to one side of the cover glass so that the stain will
flow under the cover glass and mix with the material.
4. Examine the slide, being careful to set the microscope up for a
color image. How does the staining effect obtained with methyl green
compare with that obtained with neutral red and Janus green?
C. Demonstration Slides
Examine slides demonstrating various types of preparations. The fol-
lowing list suggests preparations which are usually available and serve
to illustrate different techniques.
1. Dry mount of Foraminifera shells on a black background (set up
with reflected light).
2. Ground bone or tooth section in air and/or in mounting medium
(compare with 13).
3. Unstained whole mount of a nematode or small arthropod in glyc-
erine jelly.
4. Stained whole mount of chick embryo, hydroid, etc.
5. Isolated smooth muscle cells from Rana intestine (compare with
12).
6. Mount of sponge spicules or sea cucumber skeletal plates.

7. Dried blood smear.
8. Dried and/or wet preparation of spermatozoa.
9. Wet smear of protozoa.
10. Salivary gland cell chromosome preparation of Drosophila.
11. Frozen section, stained for fat and mounted in glycerine jelly.
12. Paraffin section of Rana intestine (compare with 5).
13. Celloidin section of decalcified bone or tooth (compare with 2) .
3
Records
and
Labels
Before beginning a technique project, a system of records should be

set up which will provide an accurate history for each preparation. The
system should be designed for accuracy, permanence and simplicity.
The amount of detail required in the records will vary with the type of
project undertaken but certain necessary information might be con-
sidered mandatory. This information includes: (1) specific name of the
organism; (2) tissue; (3) fixative and date of fixation; (4) type of
preparation (e.g., smear, corrosion; or plane and thickness, if sectioned);
(5) stains; (6) date of completion. This information is essentially what
will be included, along ~ith the name of the preparator, on a paper
label on the completed slide.
There are many reasons to feel that a system of accession numbers is
ideal for maintaining slide records. A numerical designation provides a
simple and accurate system for marking even a large number of slides.
Permanence is assured if the number is etched or scratched on the slide.
This assumes that the original records are maintained with the prepara-
tions. Accession numbers are assigned in sequence, 1,2,3,4,5, etc., and
should never be repeated. The object may be further identified by
prefixing the numbers with the technician's initials, JJ-1, JJ-2, etc.
This is particularly advisable when a number of people are using the
same technique facilities, as in a class room. If a number of slides are
prepared from a given tissue block, these may be distinguished by af-
24
RECORDS AND LABEl,S 25
fixing a letter in alphabetical sequence after the accession number, JJ-

la, JJ-Ib.
When an accession system is set up the tissues should be catalogued in
numerical sequence. Such records should be kept in permanent, water-
proof ink (such as Higgins Eternal) in a note book, preferably bound
rather than loose-leaf. The catalogue should provide the information
listed in the first paragraph, as well as an indication of the number of
slides prepared from a given tissue block. For example:
No. 435. Rana pipiens; small intestine; Bouin's; 26-0ct.-1960 ; tram;.
sections in paraffiin at 8 microns; slides a-d, Harris' alum
hem. & eosin Y; slides e-f, Mallory's triple stain; slides g-h,
methylene blue & eosin Y. 3-Nov.-1960. Cleaned & labeled;
stored in box No. 51.
Since only the initial information (species, tissue, fixative and date) is
recorded at the first entry a few lines should be left between successive
numbers for the addition of information regarding sectioning, stain-
ing, and completion of the slides.
In addition to the basic information recorded in the slide catalogue,
it will be necessary to keep records regarding the actual processing of
the slides. Index cards are convenient for this type of record, which
should present a running account of the tissues' preparation. Notations
are made regarding the time when the tissue is introduced into each
solution. When time periods are so short that the material is processed
without other interruptions it may not be necessary to record time inter-
vals unless they represent a critical point of reference when subsequent
slides are processed, or the material is examined.
The Working.Label
The advisability of a numerical record system becomes apparent when
one considers the problem of maintaining a label with the material
throughout the processing in various reagents as well as on the slide. A
working-label should be prepared as soon as, or before, the material is
fixed. Such a label should be prepared on good quality paper and written
in black India ink. If an accession system has been established, the
working-label need only include the number, or the worker's initials
and the number. If an accession system has not been established, the
working-label should include the following information: (1) specific
name of the organism; (2) tissue; (3) fixative and date; (4) technician's
name or initials. The working-label should be processed with the ma-
terial (that is, in the containers with the tissue) and, therefore, should
be fairly small.
If the material is to be prepared as a whole mount, this label will
serve through the entire processing until the object is mounted on the
slide. When paraffin sections are to be prepared, precautions must be
taken to retain the label with the material when it is embedded in the
paraffin mass, attached to the lIlicrotome peg, cut into sections and
affixed to the slides. If the material is embedded in a paper boat (fig.
10) the accession number should be written on the outside of the boat
(before the material is embedded) . If the material is embedded in a dish,
the label is inserted into the paraffin mass. When the material is at-
tached to the microtome peg the label is attached to the shaft of the peg.
When the sections are cut, the paper on which the sections are placed
in the section tray should be appropriately labeled. Finally, when the
sections are affixed to glass slides, the accession number should be
marked on the slide with a carborundum or diamond pencil. The latter
is a good investment for anyone expecting to do any amount of technique
work. A carborundum pencil with "engravers points" is almost as ex-
pensive and much less satisfactory. A very inexpensive and effective
marker can be made- by embedding a carborundum crystal in sealing
wax, attached to some sort of holder (penholder, glass rod, etc.).
Black glass-marking ink may also be used. Be certain to apply it only
to thoroughly cleaned slides. This ink is removed by strong alkaline
solutions. Glass-marking ink is very useful when material is processed
on cover glasses. Slides with a frosted end may be marked with ordinary
India ink. Slides should not be marked with a grease pencil, since such
marks are usually lost in processing the slides.
Cleaning, Labeli,.ng and Storing the Finished Slide

The finished slide is usually provided with a small paper label pre-
senting the information outlined in the first paragraph. The label can-
not be permanently attached until the slide is perfectly clean. Every
effort should be made to avoid the use of excessive mountant and to
keep the slide clean during handling and drying. Protect the slide from
dust, handle it by the edges when possible and do not place it on dirty or
paraffin-coated surfaces.
Once the cover glass has been mounted, the slide should be allowed
to dry thoroughly before final cleaning. Any excess of mountant is then
removed from the slide with a razor blade. Care must be taken not to
slip the razor edge under the cover glass, or the preparation may be
damaged. The film of resin which remains may be wiped away with a
RECORDS AND LABELS 27
cloth moistened in xylol. It is best to then wipe the slide surface with a
cloth moistened in alcohol. If the mountant is perfectly hardened it may
be practical to dip the entire slide in a container of xylol for a moment
and then dip it in alcohol and wipe it clean. Do not attempt this with
slides which are not perfectly dry, or have been mounted in balsam.
Slides cleaned in this way should appear perfectly clean but enough
resin or xylol may still adhere to interfere with proper attachment of
the label. A final cleaning with Bon Ami paste, followed by polishing
with a clean cloth should immediately precede attachment of the label.
Water-soluble mountants are removed with a cloth moistened in water.
Only the best quality labels should be used and it is advisable to fill
out the data before attaching the label to the slide. Use a crow quill or
other fine pen and black India ink. In view of the limited area available,
the label data should be abbreviated. Thus a label for slide a from a tis-
sue block of Rana pipiens small intestine, assigned the accession number
435; fixed in Bouin's fluid; sectioned in paraffin in a transverse plane at
8 microns; stained in Harris' alum hematoxylin and eosin Y; prepared
by J. J. Jones and completed on the third of November 1960 would read,
on successive lines: No. 435a . . . Rana pipiens ... sm. intes .. .. par.
trans 8 1'- .•.• Bouin 's . .. Harris' alum hem .... eosin Y ... JJJones 3-
Nov.-'60. It may be advantageous to include other informat ion, such as
the ·mountant used . The label is usually placed on the left-hand side of
the slide. It is advisable to scratch the accession number on the right
hand end of the slide so it will not be covered by the paper label.
The completcd slides should be stored where they are protected from
dust , dirt and sun li ght. If stored in open boxes or trays the stains may
fade very rapidly. Ordinary slotted wooden or plastic boxes are adc-
quate. The slid es may he kept flat during periods of l:itorage hy standing
the boxes on end . Bal sam mounts must be kept fiat a · much of the timr
as possible or the material will gradua lly drift through the l110untant
even when it is relatively hard.
4
Fix atives
The initial step in the processing of an organism or tissue for perma-

nent microscopic preparations is fixation. The fixation process should
preserve the structure of the cells in as near a lifelike condition as
possible and prepare them for subsequent treatment. The importance
of fixation cannot be overemphasized, since poor fixation leadR to dis-
tortion, poor optical differentiation, either maceration or excessive
brittleness of the material, and may produce misleading artifacts.
An ideal fixative should have the ability to: (1) penetrate the ma-
terial rapidly; (2) kill the cell and prevent postmortem disintegra-
tion; (3) preserve the physical structure of the cell; (4) convert the
cell contents into stable compounds which will not be destroyed by
subsequent processing; (5) harden the material, without making it
brittle, so that it will withstand handling and subdivision; (6) increase
the refractive index of the material differentially so that the differ-
ences between both cellular and tissue constituents will be enhanced;
and (7) render the material receptive to stains.
Unfortunately, there is no reagent which is known to fulfill all these
requirements. Among those chemicals used as fixatives, many coagu-
late or precipitate cell protein but destroy or fail to stabilize carbo-
hydrates or fats. Some are excellent nuclear fixers but destroy cyto-
plasmic granules and other structures, etc. It is necessary, therefore,
to select a fixative that will not only fulfill as many of the "ideal" re-
28
FIXATIVES 29
quirements as possible but one which will meet the specific require-
ments of the study to be undertaken. A number of fixation techniques
may be necessary in order to obtain an adequate picture of the material.
The chemicals most frequently employed as fixers are acetic acid,
formalin (40 per cent formaldehyde), alcohol, picric acid, mercuric
chloride, potassium bichromate, chromic acid, and osmic acid. These
fixers are rarely used by themselves since by utilizing two or several,
more favorable fixation may be achieved through a combination of
their good qualities and a counteraction of their undesirable effects.
Thus, a good chromatic fixer (acetic acid) may be used in combination
with a good cytoplasmic fixer (chromic acid or potassium bichromate).
A reagent which tends to swell the material (acetic acid, potassium bi-
chromate) combined with one whir.h tends to shrink it (chromic acid,
alcohol, mercuric chloride), and so on.
The fixatives discussed below include only a few of the many which
have been devised by various workers. An effort has been made to present
representative fixatives of each type. In most instances these are what
might be called "classical" fixatives that have been used by many
workers on a wide variety of tissues. Some have a limited application
but are utilized on materials which are frequently the subject of
microscopic examination.
In the following discussion the combination fixatives are discussed
under the heading of that chemical which determines the postfixation
treatment required. Postjixation treutment refers to those procedures
necessary for the removal of the fixative.
It is generally advisable to mix fixatives just before use. Prepare
solutions in distilled water unless otherwise indicated. Specific sug-
gestions for stock solutions are given with the formulas. Thus, picric
acid and mercuric chloride are employed in saturated solutions; chromic
acid, in a 1 per cent solution; osmic acid, in a 2 per cent solution; etc.
Acetic Acid
Acetic acid precipitates proteins, especially those of the nucleus,
and penetrates very rapidly. It tends to distort the material by swelling
and destroys cytoplasmic structures such as mitochondria and the
Golgi apparatus, and fats and lipoids in general. It is widely used
in many of the combination fixatives listed below (e.g., Carnoy's,
Bouin's, acetic alcohol, Schaudinn's, etc.). It is usually used in ·com-
bination with other fixers which have a poor rate of penetration, or
do not fix nuclear constituents, or tend to shrink the tissue. No special
treatment is required to remove the acid from the tissue and in each
case, postfixation treatment is determined by the other chemicals em-
ployed with it in the fixa~ive.
Formalin
Formalin is one of the few fixers which is widely used as a simple
fixative. It is useful for the preservation of whole mount material,
leaving it pliable, transparent and receptive to stains. It is also recom-
mended for the fixation of nerve tissue, for material to be teased, be-
fore certain microchemical tests (iron), and in instances where fats and
lipoids are to be demonstrated. It is a good cytoplasmic fixative, pene-
trates well and hardens the material without making it brittle. Its
hardening properties are not adequate before paraffin embedding and
it is not recommended as a general histological fixative since much
better results may be obtained with the combination fixatives, many
of which include formalin.
Formalin is a 38 to 40 per cent aqueous solution of the gas formalde-
hyde. When used as a fixative it is prepared as follows :
Formalin .(40 per cent formaldehyde) . . . . . ............... 10 cc.
Distilled water . . .. . . . . . ...... 90 cc.
to give a 10 per cent formalin (4 per cent formaldehyde) solutioL
When used for the fixation of vertebrate tissues its effects are im-
proved by using physiological saline (0.9 per cent sodium chloride)
instead of water. When used for marine forms it should be made up with
sea water.
Formalin usually contains some formic acid and in certain tech-
niques it is necessary to neutralize the solution. "Neutral formalin"
may be prepared by adding a quantity of calcium carbonate to the
stock solution and shakIng occasionally after it is first added. The
calcium carbonate is then allowed to settle out and the solution is
filtered before use.
Material should be fixed for 24 to 48 hours and may be left for a
longer period. When fixation is complete, the tissue may be stored
in 5 per cent formalin until it is processed.
Postjixation Treatment
Before the material is processed, the formalin is removed by wash-
ing the tissues or organisms in water. Small objects (hydroids, em-
bryos, etc.) may be washed in several changes of water for 15 to 30
minutes in each change. Tissue blocks should be washed in running
FIXATIVES 31
water for 12 hours or overnight. If the material is to be stored in alcohol

it may be dehydrated through a graded series of alcohols (30 and 50
per cent for 30 minutes each) and stored in 70 or 80 per cent alcohol
without preliminary washing, since the alcohol will remove the forma-
lin.
•Alcohol
Alcohol is sometimes used as a simple fixative, primarily before
certain histochemical tests. Its use is indicated when the material to
be demonstrated is water-soluble (glycogen) or when other reagents
must be avoided. It is also used for the fixation of blood and certain other
smears. Its main disadvantages are that it tends to shrink and over-
harden the material. Ethyl alcohol of 95 or 100 per cent is used for tis-
sue fixation. Methyl alcohol is preferred for the fixation of smears and
may also be used for tissues. Hot 70 per cent alcohol is used for the
fixation of small nematodcs to be mounted entire and unstained in glyc-
erine jelly. Thirty per cent alcohol is used before maceration procedures.
Alcohol may be combined with glacial acetic acid and other reagents
to produce fixatives with tremendous penetration effects. Some of the
more generally used formulas are given here.
Acetic-Alcohol
Carnoy's Fixatives
Formula I Formula 2
Absolute alcohol. , . 60 cc. 60 cc.
Glacial acetic acid ... . 20 cc. 10 cc .
Chloroform ... .. ... . . ,. 30 cc.
Formula 1 is in more general use than Formula 2. Chloroform in-

creases the rate of penetration. These solutions are used for the
fixation of dense, impervious objects such as arthropods and nema-
todes, particularly Ascaris and Ascaris eggs. Fixation is very rapid.
Small blocks should bc removed in 30 minutes, larger blocks after
several hours.
Formalin-Acetic-Alcohol (F.A.A. or A.F.A.)
Lavdowsky's Mossman's Botanical
Formalin .. .. . . ....... .... . ... ...... . .. 10 cc. 10 cc. 5 cc.
Glacial acetic acid . . , ..... ......... 2 cc. 10 cc. 5 cc.
95 per cent alcohol . .... ... . .... ... 50 CC. 30 CC. 35 CC.
Distilled water ... ...... ........ 40 cc . 50 cc. 35 cc .
32 GENERAL ZOOLOGICAL MlCBO'I'ECRNIQUES
Lavdowllky'll mixture is excellent. for the fixat.ion of small embryos.

Mossman's hall a greater penet.ration effect. and is widely used for the
fixation of larger embtyo or blocks of tissue, particularly reproductive
tracts. F.A.A. is one of the 8tandard fixatives for t.he botanical ma-
terial. These fixative8 should be allowed to act for everal daY8.
Postjiration Treatment
Material hould be transferred to 70 per cent alcohol to remove
til xc ivc fixative and is then stored in 70 or 80 per cent alcohol.
o pecial tr atment is required to remove these fixatives.
Picric Acid
Picric acid, especially in combination with acetic acid and formalin ,
gives excellent general fixation for both whole mount material and
ti ue preparations. Nuclear fixation is excellent. It i not as favorable
for cytoplasmic tructure. Of the various formulas which have been
d'vised, Bouin's fluid is the most popular and gives excellent results
with a wide vari ty of ti ues and organisms.
Rouin's Fluid
Picric: "ci.l (~Ilt ur .. ted IIljU('OU3 solution) 75 cc.
Formalin 25 ce .
Uillrilll IlCl'tir IIcid ... 5 ce .
Matl'rilll an hr stored in thi olution for a considerable time with-

out over-hardenin~, uut the best staini ng results are achi(>\'ed if the
mat rial i proce d as soon a po ible after fixation. Fixation for 12
hours to !l veral days is recommended .
Alcoholic Bouin's may be prepared by ub lituting a saturated solu-
tion of picric acid in 70 per cent alcohol for the aqueous solution .
•'inee picric Reid is much more oluble in alcohol than in water, the
primary etTect of using alcohol i to increase the concentration of
tilt' picric ari{1. Thi fixative i recommended when particularly iru-
pen'ioll bj ct , ar to bt' fixed . The penetration rate of either solu-
tion may h greatly accel rated by heating.
On(' modification of Bouin's fluid which i useful for the fixation
of protozoa smears, and i al 0 r commended for the fixation and
d calcification of man bon ,i the following:
H ollande's Fluid
mbine the di tilled water, oopper acetate, and picric acid in a
mortar. Thi i nece ry in order to force this quantit.y of picric
Rcid into solution.
FIXATlVES 33
Distilled water . . ~c .
Copper acetate ... :I 6 nl .
Picric acid . . ".0 JIl\ .
Formalin ... 10 ec .
Glacial acetic acid 1.5 ('c .
POStfiIO/i07l Tr almf'7lt
~laterial which ha been fixed in picric ucid olutioll", hould b
washed with 70 per cent alcohol, ne\'(' r with watt'r. Till' tis ue 11-
COlnt' mushy and the cells y/\('uolliled if the IIlllt('rial i. (' ' J)() ro to
water or weak alcohol before the fixati\'e ha b n rrnlOV U. One
the picric acid ha been remoyed , as indi('uted by th los ' of til , yellow
color from tlt(· tis::-u ,the mat-erial can apparently be prOC('S$ d thruugh
aqueou olution. without detrimcntal l'fT('ct . MaLcrial fixed In nko-
holic Bouin' is tran ferred dirt·(·tly to 70 per c{'nL alcohol lind tr u d
with rep aLed chanJ!;es until the solution is no longer di colored by
the picric acid. ;'\1 ate rial fixed in aqU(,OllS ,.;ollition!> 1lI11~' hI' left for
a hort time in 30 and 50 per ('ent alcohol (30 minut(,1< (,lIeh fol' til-I'll('
blocks) and t1l('n washed in 70 per ('ent aleohoJ. If thC' matt'rilll il' not
fragile it may he transf('rr('d dirrctly mto 70 prr rent ule-ohol from till'
fixativr . Whcn thc material is to he proec. s d in dioxan', t.ht· picril'
acid ma~' h{' r(,lllo\'(·d in dioxall(' mIller I han in alcohol.
\Vhen material is to he Ill·()(· I·~ ~(·d by the pumflill nwthod , it i~ not
necessary to rcmove all of the picric acid bdorc' l'mb('dclin~ . !-imall
quantitie of picric a('id arc rrudily I'xtractc'd from th' ('rtton b -
fore staining. If the material i" rmhcdri<'d without r('rno\'ul of mo t of
the picric acid, how('\'er, it may be difficult to section .
;\Iaterial to be pro('('.~I< ('d in (,rlloidin doe. not SN'm to infiltrate
properly unl ess all of the pirrir arid i rrmovcd b fon' !'Illlwdding,
Objects to be preparl'd a~ whole mO\lnts IIhould al 0 b compl t ly
decolorized befor{' pro('('s~in~. Rc'mo\'nl of pirric acid i Qc('('l('rut .t!
by warming the alcohol 10 about 40° '. If th picric· arid ill not rl'-
moved by thi!' tr ntmrnt it. may be neC('~ ~a ry to add lithium car],onIl.U·
to the 70 p('r r('111 alcohol tls{'d for wl\I.hing.
Mercuric hloride
Mercuric chloride coagulates protein .. penetrates fairly W II, and
provides good general histological fixution . It may d troy d licnLe
structures, such as cilia, and ha a tendency to shrink th mal(>rial.
It i sometimes u ed alone in saturated solution wh n rulcar >ou struc-
t.ures are to be preserved, More frequentl y it is u d with th addition
of glacial acetic acid and oth r fixer.
It hould be noted that thc solubility of mercuric chlorid vor;
34 GENERAL ZOOLOGICAL IilCROTECHNlQUES
greatly with the solvent employed. Thus, in distilled water 100 cc.
will di880lve about 4.5 grams, wherea in sea water a saturated solu-
tion contains about 15 grams per 100 cc. The solubility is similarly in-
creased in saline solutions and in alcohol.
Considerable care should be exercised in handling the mercuric
chloride solutions, since they are extremely poisonous. Wash your
hands thoroughly after u ing the fixatives. Be particularly careful
to avoid inhalation of the powder when making up solutions. Metallic
in truments should not be brought into contact with the solutions, since
the instrument.s will be corroded and the solutions contaminated.
Wooden, horn, or glass forceps or spatulas should be used. Always pre-
pare solution wit.h distilled water.
Sublimate-Acetic
lituruted aqueoue mercuric chloride .. . .. 100 cc .
laciul acetic acid 5 cc.
This fixative is particularly good for flatworms to be prepared as whole

mounts. It is standard practice to use sca water for marine organi ms
and physiological saline with vertebrate tissues in preparing the satu-
rated mercuric chloride for sublimate-acetic.
Zenker's Fluid
Thi is one of the most useful mercuric chloride fixatives. When
cytopla mic granules are to bc preserved, the formula is modified by
'substituting formalin for glacial acetic acid. The original fommlas
al 0 included 1 gram of sodium sulfate per 100 ce. of solution. This
is now generally omitted.
z..nker'. Helly'.
Saturated aqueous mercuric chloride ..... 100 cc. 100 cc.
Potassium bichromate ....... . 2.5 gm . 2.5 gm .
Gll\cial acetic acid . .. . ...... ,.,. . ... , 5 cc.
Formalin .. ............. , . . 5-10 cc .
The mercuric chlorid and pota ium bichromate may be kept a a
tock solution to which either acetic acid or formalin i added ju t
before u e. H lIy' fluid is often referred to a "Zenker-formal." Thi
fixative i particularly u eful for material to be tained with the aniline
dye .
usa's Fluid
Anoth r popular mercuric chloride fixative i usa's, which com-
bin mercuric chloride with trichloroacetic acid as well as formalin
and ac tie acid.
FDC.ATl £S
turated aqueoUll mercuric chloride . ce.

Sodium chloride .. 0. 11 m.
Trichioroacetic acid . 2 .0gm .
Glacial acetic acid .. c.
Formalin 20 ce.
The effects of this fixative are similar to those of Zenker' . Trichloro-
acetic acid is a trong decalcifying agent. It also tends to sw II col-
lagen fibers if the material is washed in water. "'usa-fixed material
should be washed in 70 per cent alcohol nnd iodine-alcohol (see follow-
ing discll sion) a.fter processing through 30 and 50 per cent alcohols for
30 to 60 minutes in each.
chaudinll's Fluid
This alcoholic-mercuric chloride fixat.ive is one of the mo t popula.r
for the fixation of Pl'OtoZA . It is not n'('ornmended for tissues.
:::lilt urllted aqm'OllH m('rcuri c chloridr 66 cc .
115 per c('nt alcohol 33 ce.
Gbl'illl acct.ic IIcid . .5 ClC.
Fixation of protozoa smears is completed in 15 to 20 minutes.
Postfixation Tr crtment
In the CIl e of aqueou mcn'uric chloride fixatives (with the exception
of u a's as mentioned earlier), thp matt' rial Ah ould b washed over-
night in running water. The lissuc is then drhydrawd through 30
and 50 per crnt alcohols for 30 to 60 minutes in each and into 70 per cent
alcohol. All mercuric chloride-fixed tissues arc tht'n tr at<>d with iodine
in 70 per cent alcohol to assure complet( removal of th mercury . AI! the
iodine combines with the mercury, forming 11 soluble compound, the solu-
tion is decolorized. When all of the mercury ha b en removed the iodine
solution will retain the original color.
The iodine-alcohol ha ' been variously described as "port wine"
or "strong tea" in color. The following proportions make a usabl
solution.
Iodine, saturated in 70 per c nt alcohol ........... . 5 cc.

70 per cent alcohol ... .. 96 ee.
With large blocks of tiB8Ue, removal of the mercury may require a

week or morc. The iodine solution should be renewed as it becomes d -
colorized. When the solution retaim the iodine color, tran fer th
blocks to 80 per cent alcohol to remove traces of iodine and for storage
until the material is procesaed.
36 GENERAL ZOOLOGICAL MICRarECHNIQUES
In the case of alcoholic mercuric chloride fixath·e , the ti ue i

tran ferr d directly into 70 per cent alcohol and then into the iodine-
alcohol solution. Tissue block will require con iderable wa hing. Pro-
tozoa smears may be proces ed after a 20-minute treatment with
iodine-alcohol.
If the mercuric chloride is not removed from the ti ue it will inter-
fere with sectioning and, later, with staining. If t,he nuclei huve an
opaque appearance in water and will not stain, try treating the ec-
tions with iodine-alcohol.
Pota88ium Bichromate
Pots ium bi ·hromate is a good cytoplasmic fixer. I t penetrates well
and hardens the material without making it exre ~ i\"e1y hrittle, pro-
virlrd the ti ssue i. not I 'ft too long in th olution . It i ~ ol1e of th~
constituent:! of Zrnkrr's fluid, liHt('d earlie-r. Another· u,.r flll fixative
cont,aining pot,a sium hichromate is Smit,h's fluid . This is rx("ellent
for yolky urophilJian egg or larva since it, doe not tend to over-
hard n them, as do most other fixation fluid s.
8mith's Fluid
Pot68Hium hirhromale Ii.O 1(01 .
Formalin 10 cc.
Dietillt·d wat('r AA cc .
CllIl'i .. l IWl'tic I\cid .. 2.5 N'.
Prcpar(' the fixative iust before using. Fix for 12 to 24 hour,. Tn order
to avoid hard<>ning, yolky mat(' rial should 1)(' torrd in 5 prr cmt
formalin rather t.han in alcohol when t,he fixation pror s i completed.
Postfixation Treatment
Potu iurn bichromate fixative arc remov d by washing the ma-
terial in running water for] 2 hour or overnight.
Chromic Acid
hromic acid is excellent for the fixation of cytopla m and the
achromatic elements of cell division . It has a poor penetrut,ion rate
however, and tend to over-harden and hrink the material rather
rapidly . The mo tela ical chromic acid fixative ar Flemming' ·
flujd ,which ar consid red under" mic acid."
Chromo- itne
A combination of chromic acid with nitric acid and akohol has
b n recommended for the fixation of ey by some workers and
P'IXATIVES 37
as an embryological and general hi tologlcal fixative by oth rs. It i
also used as a fixative for marine organi m . Two version of thi
fixative are given here.
Perenyi's Fluid Ltc', flwd
Chromic acid . . . . 30 ('C. of o. 5';(. 30 cc. of I "~
Alcohol . 30 cc . of 95";, 40 ('c . or 1 ,0
~itric acid . . 40 cc . of 10% 30 ('c . of 20%
Karpechenko's FLuid
Combinations of chromic acid with formalin and glacial acrtie acid
are vcry u cful for the fixation of plant tissues for cytological studi ,
especially for the demonstration of division figure'. Nunwrous formula
are avai lable and are generally referred to a th NsvsHhin or Knrpe-
chenko fluids.
1 pe r ('('nt aqueous chromir I\('id 55 r(' .
Formlliin 40 rc .
Glacial acetic I1cid 5 ('C' .
Thi fixative shou ld be prepared just bt'forr it. is to he URNI . The 1ll1l-
terial i fixed for 24 hour~ to se"eral days .
Postjixntion Treatment
Wash aqueou s chromic acid-fixed mnterial in running water for
12 to 24 hour . Chromo-nitric-nleohol fixatin' liTe rl'movcd by several
change of 70 per rent alcohol.
o m;c Acid
o mic acid is generally considered to be on of the mo t. favorabl
fixer for preserving the cytoplasmic elt'm ntB of t.he ell in a n aT
lifelike condition. It i extremely poor in its penetrating capariti
and does not give good nuclear fixation. Jt i not to h' f('rOIDlnI'ndrd
for histological work.
The fume of this extr mely volatile solution ar very poi onoull and
are injuriou to the no .(. anti eyes. ] n "iew of this dang r, at< w II II
it co 1. a nd the fact that it ill not a good general fixati,·', it i not
recommended for u in general das work, not in g n ral te hniqu
procedures. It i included here for th b nefit of th t.udcnt of cytology.
Osmic acid solutions must be prepared with m ,ticllious care 0 that
no organic contaminate are introduced into th solution. The p per
label i carefully wa hed from the scaled cap ule of 0 mic acid crystals
and the capsule ( till sealed) is placed in a clean bottle in which th
solution i to be prepared. Bottle and capsule ar th n WI\ hed wit.h
38 FIXATIVES
chromic cleaning solution, after which they are thoroughly washed

with repeated changes of distilled water. The correct amount of glass-
distilled water is then added to the bottle and the osmic acid capsule
may be broken with a heavy glass rod which has also been carefully
cleaned. Osmic acid solutions are usually prepared in a 2 per cent
concentration. The solution srould be kept in a darkened cupboard or
box.
Flemming's fixatives are the best known of the osmic acid solutions.
There are two different formulas, the "strong" and "weak" solutions.
In either case it must be remembered that penetration is so poor
that only a few cell layers will be adequately fixed.
Flemming's Osmic Acid Fluids
W...k Formula Strong Formula
I per cent chromic acid .. 25 cc. 30 cc.
2 per cent o"mic acid 5 cc. 8 cc.
Acetic acid . .. 10 ce. of 1% 2 cc., glacial
Distilled water 60 ce.
Fix the material for 24 hours to several days.

Osmic Acid Vapors
Osmic acid vapors may be used for the fixation of mears of pro-
tozoa or tissues. The material is smeared on the slide, which is t11en in-
verted over a contain r of 0 mic acid. Fixation is very rapid (about 1
minute is adequate) but is only effective with thin preparations.
Postfixation Treatment
Wash osmic acid-fixed material in running water for 12 hours or
overnight. If the material ho. darkened in the fixative , a is usually
the ca e, it must be bleached hefore it is stained. mears are bleached
after wa hing and before staining by treating with full strength (about
3 per cent) hydrogen peroxide. erlions are bleached during the hy-
dration series by treating with a 1 : 1 solution of hydrogen peroxide
in 70 per cent alcohol or a full strength solution in water. Before
th material is stained the bleaching agent i removed by washing
the slides in running water for 30 to 60 minutes.
5
Fixation
In order to obtain as near a lifelike picture of thc material a po-

sible, it is essential that the fixative is applied to living or {r hly
killed organisms. Small animals may be killed and fixed at t.h sam
time by submerging them in the fixing fluid . Larger fonn rou t. first.
be killed, dissected and the tissues subdivided so that. th fixat.ive can
penetrate to t.he center of the tissue block b fore c lIular br ukdown
occurs. Fixation should be carried out in such a way that th gro
physical structure of the material is prcscrv d, as well c lIular d tail.
Contractile forms mu t be anesthetized or re trained.
stretching or compression mu t be avoided. Always u
of fixative, 30 to 50 times the volume of the ti u.
Tissues should be subdivided by placing them on a cork and cut.ting
them with a slicing motion with a sharp, ingle-edged ralor blade. Do
not u e excessive pressure. While the material is being ubdivided it
mu t never be allowed to dry. Keep on hand a g n rou supply of
phy iological saline with which to flood the material, and for rin ing
blood and debris from the object before placing it in t.he tixat.iv . Phy 10-
logical saline may be prepared from table salt (sodium chlorid ). For
invertebrates, use a 0.75 per cent solution; for warm blooded v rt.e-
brate8, a 0.9 per cent solution. The more complex Ring r's solutions
are not generally nee ary for t hnique purpo
When material is subdivided after a hort, initial fixation to h rden
it, it hould be flooded wit.h the fixative. In t.he cue of queous m r uric
39
chloride fixatives flood with saline. If the tissue is subdivided or trimmed

later, after storage ill alcohol, keep it wet with alcohol of the strength
from which the tissue was removed.
Subdivision of an animal should be made along structural lines.
Whenever possible, the plane of division of an excised tissue is desig-
nated on the basis of its position in the body. The standard planes of
subdivision and sectioning are transverse, frontal and sagittal (fig.
3). The term "sagittal section" refers to a section in the exact midline
of the animal; sections to either side of the midline, removed in
a sagittal plane, are referred to as "parasagittal." The terms "cross"
and "longitudinal " are more applicable to plant than to animal tissue,
although they are sometimes used for the latter. Sections of elongated,
tubular organs, such as the gut, oviduct, etc., are frequently referred
to as cross or longitudinal, on the basis of the long axis of the organ.
Sections of muscle masses may be described on the basis of the long
axis of the cells. Preparations of mesentery, skin, or bladder, which
TRANSVERSE
~ Q§:)
E
B
c F
FIG. 3. The subdivision of a -salamander larva (B) in the standard planes for
sectioning The saggital section (A) is cut in the ¢ane of the page; the frontal sec-
tion (C) 10 the plane indicated by C-C'; the transverse sections (D, E and F)
in the plane indicated by D--D', E--E', and F-F' .
FIXATION 41
are cut perpendicular to a flattened surface are generally referr d to

as "cross sections." Sections of such material, cut in the plane of the
flattened surface are referred to as tangential.
Anesthetizing and Killing
Invertebrates and larval vertebrates are generally anesthetized before
fixation, and the fixative itself is used as a killing agent. In most in-
stances these forms are aquatic and the anesthetic is added to the water
containing the animals. Anesthetization is indicated in the case of any
contractile form unless it can be mechanically restrained. Generally,
the anesthetic is added gradually and the time required for immobiliza-
tion of the animals depends upon the size of the organism ,the particular
anesthetic and its concentration. The animals must be closely watched
and fixed as soon as they no longer contract when stimulated by probing.
During anesthetization the animals should be placed in a minimum of
water. The fixative is then added directly to this medium, the diluted
solution removed after a few minutes, and fresh fixative added.
Although a great variety of anesthetics have been used for different
animals and may be required under special circumstances, most aquntic
animals may be anesthetized by one of the reagents discussed here.
Magnesium sulfate (epsom salts; MgSO.·7H 20) will successfully
paralyze most marine and fresh water forms. The crystals may be
placed directly into the medium containing the animals and allowed to
dissolve and diffuse through the water. It may also be introduced grad-
ually in a saturated solution in either fresh or sea water.
Magnesium chloride (MgCb'6H 2 0) may be used as an anesthetic
for marine or fresh water forms. For marine animals, use 7.5 per cent
magnesium chloride in an equal part of sea water. For fresh waler
forms use a 2.5 per cent solution.
Alcohol, slowly added via a siphon tube or drip bottle, may be used as
an anesthetic. Use a 10 to 50 per cent solution, depending upon the
speed of introduction.
Chloral hydrate is particularly good for some hydroids and for
nemerteans. It may be added in crystalline form to fresh ~r sea water
containing the animals.
Vertebrates are usually killed by a sharp blow on the head, or by
placing them in a closed container with chloroform, ether, or illuminat-
ing gas. The chloroform or ether should be introduced into the con-
tainer on cotton, and the atmosphere allowed to become saturated with
the fumes before the animal is introduced. Illuminating gas is introduced
via a tube after the animal bas been placed in the container. Use ether
or gas in a well ventilated area and keep away from open flames and
sparks. If the animal is small it may be decapitated with a pair of
heavy scissors. When relatively large or wild animals are to be killed,
their cages can be converted into killing boxes by wrapping them in
aluminum foil.
Fixation of Invertebrate8
Protozoa
Mass cultures of free living protozoans are fixed and handled as
whole mount material. The main problem is concentrating the protozoa.
If the culture is very thick, the fixative may be added to the culture,
the protozoa allowed to settle, the fluid decanted and fresh fixative
added. Gentle centrifugation may be employed to. aid concentration.
When the culture is not very thick and the protozoa tend to concentrate
in certain areas, such as toward a light, such areas of concentration
may be removed with a large pipette and transferred to a volume of
fixative. They are then handled as described above.
Amoebae should be allowed to expand in a thin film of water and,
then, fixed by forcefully squirting the fixative into the dish.
Intestinal protozoa and test-tube-slant cultures of the same are
prepared as smears. The material is smeared on the cover glass or
slide with a circular motion so that a fairly thin film is obtained. The
smear may be made with a wire loop, a stick, dissecting needle, or a
cotton swab moistened with saline. Termite protozoa may be smeared
with a needle, using the gut as a swab to spread the material over the
glass. The smear must be prepared rapidly and immediately dropped,
smear down, into a dish of fixing fluid. Cover glasses will float in the
solution. Slides must be supported off the bottom of the dish with glass
rods or strips, positioned so that they support the ends of the slides.
If the medium containing the protozoa is very fluid it may be necesary
to let the film "dry down" so that the excess fluid evaporates, but great
care must be taken that the organisms are not permitted to dry. If the
medium is very dry, it may be necessary to mix a drop of saline with
the material in order to spread it. Usually, dropping the preparation
face down in the fixative will result in most of the protozoa adhering
to the glass surface. If they float off it may be necessary to coat the
glass with a film of Mayer's albumen or Haupt's affixative (p. 164)
or to mix a small quantity of one of these with the material.
Fecal smears are prepared by spreading the material over the sur-
face of the slide with a stiff brush, such as a small paste brush. These
FIXATION 43
slides may be fixed in a vertical position since the material does not
tend to float off the slide as do intestinal smear preparations.
Poriferans
For the preparation of spicule mounts, preserve the material in
95 per cent alcohol. These may also be used for tissue preparations.
Material for sectioning may be fixed in Bouin's or Hollande's fluids,
either of which will remove calcareous spicules. Material may also be
fixed in 10 per cent formalin, but should be stored in alcohol. Silicious
sponges may often be sectioned without removal of the spicules. If it
is necessary to remove silicious spicules, place the material in 70 per
cent alcohol in a jar which has been coated on the inside with paraffin ;
add a few drops of hydrofluoric acid. Be extremely careful to avoid
inhalation of the hydrofluoric acid fumes . When desilicification is
complete, wash the material in alcohol.
Coelenterates
Hydra may be fixed in an extended condition by placing them in a
shallow dish of water and allowing them to become fully extended. A
quantity of hot Bouin's is then added suddenly from a large pipette.
Hydroids and anemones may be anesthetized with magnesium sul-
fate or magnesium chloride. Bouin's fluid is suitable for the fixation
of material to be sectioned. Large forms should be subdivided after
initial hardening in the fixative. Bouin's or formalin will give good
results with whole mount material.
Platyhelminthes
Turbellarian8 and Trematode8. Planarians should be starved before
fixation. Place on a glass plate in a film of water. When fully distended
add a drop or two of 2 per cent nitric acid; after a few minutes, place in
sublimate-acetic. If carmine-fed whole mount preparations are desired,
feed the animals raw liver into which has been mortared a quantity
of dry carmine. Proceed as described above.
Fasciola hepatica and similar large, tough forms should be fixed
between glass slides, held togetlier with rubber bands. Place the slides
in a dish of fixing fluid (sublimate-acetic or Zenker's) and force the
slides apart occasionally to permit access of the fixative to the material.
When the worms have hardened, remove from between the slides and
return to the fixative.
Bdelloura and similar delicate forms may be placed on a glass plate
and covered with a slide or cover glass, depending on the size of the
44 GENERAL ZOOLOGICAL NUCROTECHNIQUES
specimen. The weight should be sufficient to flatten without crushing

the material. Add the fixative at the edge of the cover glass with a
pipette, so that it will flow in to the specimen. When initial hardening
has occurred, remove the material to a dish of fixative .
Cestodes. The most effective method of fixation will depend upon
the size of the specimen. One of the following techniques should be ap-
plicable. A mercuric chloride fixative is to be preferred.
1. Flatten on a glass plate and cover as directed for Bdelloura.
2. Grasp one end of the worm and dip it into a container of fixative .
Remove it from the fixative, drawing it gently over the edge of the
container. Allow it to hang suspended in air for a short period (do not
let it dry out) and then repeat the process. Repeat until the material is
fairly hard and then place in the fixative .
3. Lay the specimen out on a glass plate and paint it with the fixative
with a small, soft brush. When initial hardening has occurred, transfer
it to a di sh of fixative .
Nemerteans
Anesthetize with chloral hydrate and fix in Bouin's fluid.
Bryozoa
Anesthetize with chloral hydrate or magnesium chloride solution.
Fix in Bouin's fluid or in 10 per cent formalin.
Nematodes
Small forms should be fixed by dropping them into hot 70 per cent
alcohol. This will usually fix them in a straightened condition. Large
forms may be fixed in hot Bouin's for general anatomy. Cut into 20-mm.
lengths when introducing into the fixative. For uterine contents of
Ascaris, dissect out the uteru ; and fix in hot Bouin's or in Carnoy's.
Annelids
One of the main problems encountered in sectioning annelids is the
presence of sand or soil in the gut. Marine forms should be kept in clean
sea water for several days. Lumbricus should be fed filter paper to
flush the soil from the gut. Place the worms in a covered dish with a
quantity. of moist filter paper for several days. It is best to starve them
for another day before fixation so that most of the paper will be elimi-
nated .
Anesthetize marine or fresh water forms with magnesium sulfate
FIXATION
or magnesium chloride. Anesthetize LumbriCtl8 by adding alcohol very

slowly to a shallow dish of water containing the worms.
Small specimens of either marine, fresh water or terrestrial annelids
may be fixed fairly straight by placing them on a gla s plate and paint-
ing them with the fixative until initial hardening; has occurred. Large
forms may be fixed as follows: run a stout thread through one end of
the anesthetized worm. A series of \vorms may be fixed at the same
time by looping the threads over a glass rod. Dip the worms into a tall
dish of fixing fluid; remove and hold them in air for as long as po sible
without letting them dry out. Repeat until initial hardening has oc-
curred and then place thcm in a fiat, shallow dish of fixative. Subdivide
large forms as required after initial hardening has occurred.
Hirudinea
Anesthetize with magnesium sulfate. Proceed as described for
Fasciola hepatica.
Arthropods
For whole mounts fix in alcohol. Place on a glass plate and arrange
the appendages in a natural position after killing (in the fixative) and
before hardening has occurred. For sections, fix in Bouin's fluid . Warm
the fixative for heavily chitinized or large forms , or use alroholi('
Bouin's or Carnoy's fluids. Soaking in Bouin's for a week at 30 to 40° C.
will sufficiently soften forms which are not too heavily chitinizcd RO
that they may be processed in paraffin .. edioning is greatly Rimplificd
by using individuals which have recently molted or metamorphosed.
Whenever possible, structures to be sectioned should be dissected out
of the arthropod before processing. If it is necessary to retain the
structures in sittL subdivide the body as much as possible to facilitate
penetration of the fixative.
Molluscans
Small forms may be fixed entire. Bouin's will usually decalcify small,
shelled forms if such material is left in the fixative for a long enough
period. With larger forms it is best to dissect out the organs of interest
and fix them separately. Material may be aneshetized with alcohol.
Radula preparations should be fixed , flattened on a glass plate or on a
piece of stiff paper, in 10 per cent formalin . Flat structures, such as
gills, should also be fixed in a flattened condition.
46 GENERAL ZOOLOGICAL MICBOTECHNIQUES
Echinoderms
These may be anesthetized with magnesium sulfate or magnesium
chloride solution. Large forms must be subdivided. Bouin's gives good
fixation for tissue preparations. Preserve in alcohol for skeletal plate
preparations.
Cleavage stages may be fixed in Bouin's or 10 per cent formalin.
Larvae with calcareous spicules should be fixed in 10 per cent neutral
formalin or saturated aqueous mercuric chloride.
Tunicates
Anesthetize with magnesium sulfate or magnesium chloride before
fixing. Colonial forms should be subdivided or the zooids dissected out
after anesthetization and before fixing. Tadpoles and adults may be
fixed in Bouin's or formalin.
Fixation of Larvae and Embryos
Ammocoetes larvae may be fixed by placing them on a glass plate
and painting them with the fixative with a soft brush. When hardened,
transfer to a container of the fixative. Bouin's fluid gives excellent
results.
Amphibian larvae will usually be fixed fairly straight if placed
directly into cold Bouin's. With other fixatives it is usually advisable
to anesthetize them before fixation or to handle them as described for
ammocoetes larvae. Yolky stages should be fixed in Smith's fluid .
Embryos should be dissected out of the jelly coat before fixation.
Reptile and bird embryos should be fixed according to the directions
in Exercise 4.
Mammalian embryos should be removed from the uterus and the
amnion opened before fixation. Embryos larger than 25 mm. crown-
rump length should be subdiviaed to facilitate penetration of the fixa-
tive. Use Bouin's or Lavdowsky's fixatives for smaller forms and Moss-
man's fluid for larger embryos. If the material must be left in gitu.
subdivide the uterus by cutting it across on either side of each embryo.
If the embryos are so small that areas of implantation are not yet
visible it may be necessary to fix the entire uterus for preparation of
serial sections. Early implantation stages may be located by opening
the uterus. The area of implantation, along with the embryo, may then
be removed, flattened by pinning it to a cork or board and floating this,
tissue side down, in the fixative. Ova or unimplanted embryos may be
located by cutting serial sections of the fallopian tubes and uterus or by
flushing the ova out with saline and handling them separately.
FIXATION 47
Fixation of Ti88ue Blocks
The problems and techniques of tissue fixation are essentially the
same for invertebrate and vertebrate materials. The ti sues mu t be
removed as soon as possible after the death of the animal and placed
in the fixative.
The initial subdivision of the tissue should be made in the same
plane as that in which it will later be sectioned; thus, an organ to be
sectioned in a transverse plane should be subdivided in a Lransver e
plane (fig. 4, C). In many cases the tissue may be so largc that it is
necessary to subdivide it in more than a single plane. It i important
that all cuts be made in structural planes and in such a way that the
tissue may be readily oriented for the sections required. If a block is
cut out as a nearly perfect cube (fig. 4, E-6) it becomes difficult to
distinguish the various faces of the block at the time of embedding and
sectioning. This difficulty can be avoided if the block has thrce (or at
least two) different dimensions. Thus a block may be cut so thaL each
face is rectangular in shape and the dimensions of each are different (fig.
4, E-l, 2 and 3) . A block of tissue 2 by 4 by 6 mm. will give sections in
one plane 2 by 4 mm. ; in a second plane, 2 by 6 mm.; and in a third, 4 by
6 mm. Such blocks can be readily oriented for the desired plane of sec-
tioning. A note should be made at the time of fixation regarding the
approximate dimensions of each plane.
A. General Procedure
1. Before killing the animals from which the tissues are to be re-
moved: (a) obtain the necessary fixatives and containers; (b) have on
hand a supply of physiological saline solution for rinsing blood and
debris from the tissue and to prevent drying of tissue surfaccs during
subdivision; (c) have labels prepared or the material on hand to pre-
pare labels; (d) have on hand the necessary instruments. Everything
should be arranged so no delay will occur once the animal has been
killed. If a large number of different tissues are to be fixed from a ingle
specimen it is advisable for several people to work togethcr so that the
tissues are processed in the shortest possible time.
2. Kill the animal.
3. As soon as the animal is dead it is desirable to drain as much blood
from the body as possible. Open the blood vessels in the neck by in-
cision or decapitation.
4. As quickly as possible remove the tissues to be fixed. Carefully
subdivide them as necessary, rinse them in saline solution and place in
the fixative with an appropriate label (Chapter 3).
SAGITTAL TRANSVERSE FRONTAL
A B c o
C;~
-- - ~ - -
3 6
ISAGITTAL
E
FIG. 4. Subdivision of excised organs in structural planes. Subdivision of a small
heart (A) in ssgittal (B), transverse (C), or frontal (D) planes. The initial sub-
division should be in the ssme plane as that intended for the sections. E, subdivision
of a tongue. A very small tongue might be fixed intact. A larger tongue might be
subdivided in ssgittal or transverse planes. A large tongue would require consider-
able subdivision as indicated by the numbered blocks. Tissue blocks I , t and Shave
been removed 80 that the sides of the blocks correspond to structural planes. They
are shaped so that they may be readily oriented for sectioning. Block 5 may be diffi..
cult to orient since it is an equal sided cube. Block 6 has not been properly removed
since the cut surfaces do not correspond to structural planes. This is also true of
block 4; in this instance, however, the block has been removed to permit sectioning
of a particular serie of 5trllctures (the vallate papillae).
48
FIXATION 49
5. Record the accession numbers and necessary information in the
catalogue (Chapter 3).
B. Suggestions for Specific Tissues
B lood Smears. See Chapter 9.
Bone. For stained, histological sections, remove the mu ele, etc.
(keeping flooded with, or submerged in, saline ) and cut into short
lengths with a bone saw; place in the fixative. If the fixative contains an
acid, partial or complete decalcification will occur, depending upon the
size of the bone, the volume and concentration of the acid, and the time
the material is left in the fixing fluid . Cover the container 100 ely or it
may be broken by the gas produced during the decalcification of the
material. Usually the fixative does not completely decalcify bone and
it is necessary to use a separate decalcifying fluid before sectioning.
After the tissue has received t he necessa ry postfixation treatment, it
should be taken to 70 per cent alcohol from which it i transferred into
an acid-alcohol solution. Two to four per cent hydrochloric acid in 70 or
80 per cent alcohol gives satisfactory results with most material. A 1
to 5 per cent solution of nitric acid in 70 per cent alcohol may al 0 ue
used. With large bones a considerable period of treatment may be
necessary and the acid-alcohol should be renewed every few days. When
completely decalcified the bone should be pliable and a pin should
penetrate readily into the material. After d calcification wash for a
week or more in several changes of 70 or 80 per cent alcohol to remove
all traces of the acid.
For ground bone sectiQns, remove muscle, etc., cut into the shortest
possible lengths and soak in water until all of the organic material has
been destroyed; or dry, subdivide and then soak (Exercise 14 ).
Cartilage. In the case of young animals to be prepared as whole
mounts fix in 10 per cent neutral formalin (Exercise 20). In adults, cut·
out blocks with or without surrounding areas and fix as usual. This
tissue is best processed in celloidin (Exercise 35).
Digestive System. If a number of organs are to be fixed , those of the
digestive tract should be processed first since breakdown occurs very
rapidly in the cells of the gut. Subdivide the tract into structural
units (esophogus, stomach, etc.). In the case of small animals, rinse out
the stomach, intestines and rectum with a stream of saline solution
introduced with a pipette. Subdivide into small sections or fill the lumen
with the fixative by means of a pipette and place in a volume of fixative
until some hardening bas occurred (about 30 minutes) , remove, sub-
divide and return the blocks to the fixative. When intestine blocks are
subdivided without initial hardening the ends tend to evert and should
be trimmed before processing.
Areas may be fixed in a distended condition by first tying off one end
of the gut. A fixative-filled pipette is then inserted into the opposite
end and a thread looped around the gut and enclosed pipette tip. The
lumen is filled with the fixative and, as the pipette is withdrawn, the
upper end of the intestine is closed by tightening the thread. Do not
overdistend. Place in the fixative and subdivide after initial hardening
has occurred.
Lnrge digestive tracts are usually opened, by cutting along their
length, washed in saline and pinned flat to a cork or wooden block.
These are then floated tissue-side-down in the fixative. Do not use metal
pins with mercuric chloride fixatives. After hardening, remove from
the block, subdivide and return to the fixative.
Eyes. When fixing eyes it is desirable to slice through the eyeball
either on the dorsal and ventral surfaces, or laterally, depending upon
the plane in which the material is to be sectioned.
Fat. Blocks of fat for frozen sections should be fixed in 10 per cent
formalin . Formalin-fixed mesenteric spreads are excellent for the dem-
onstration of fat cells.
Heart. Small hearts may be fixed intact. They should be rinsed
thoroughly with saline and flushed out with a pipette. The fixative
should be introduced into the cavities if possible, via the blood vessels,
using a hypodermic syringe in the case of very small forms, or a pipette
for larger forms . Large hearts should be subdivided as required by !Size.
Lung. Fix inflated by introducing the fixative via the bronchial tubes,
using a hypodermic needle or pipette. Subdivide as necessary after
initial hardening. Occasionally air pockets are trapped in the lung
and it may be necessary to use a vacuum during processing to remove
them. -
Mesentery. See Exercise 5.
Pancreas. The pancreas should be fixed immediately after the death
of the animal since breakdown is very rapid in the cells of this organ.
Fix in Helly's to retain cytoplasmic granules and to prepare the tissue
for differential staining of cell types (Exercise 46).
Pituitary. The pituitary should be fixed in Helly's fluid to retain
cytoplasmic granules and prepare the tissue for differential staining to
demonstrate cell types (Exercise 46).
Skin. Skin should be fixed flattened, by spreading like a mesentery,
if it is thin, or by pinning out on a cork or board if it is thick. In the case
FIXATION 51
of skin with hair it is advisable to trim off or even shave off the hair
unless it is a required part of the preparation.
Testis. Small testes may be fixed entire, although it is best to care-
fully slit the tunica before fixing. Larger testes, such as tho e of the rat,
are difficult objects to fix, since the tissue tends to fall apart if sub-
divided before fixation and is too large to obtain adequate fixation
with the entire organ. Carefully slice the testis transversely into two
portions if transverse sections are required. If sagittal sections are to be
prepared slice a portion from each lateral surface and place in the
fixative. Subdivide the blocks after initial hardening has occurred, and
return to the fixative. A second method is to inject some of the fixing
fluid directly into the testis with a hypodermic syringe, and place the
entire organ in the fixative for initial hardening. This method results
in considerable displacement of the tubules in the areas where the fixa-
tive is injected. A third method is to inject the fixative into the blood
vessels. This technique is recommended when a considerable amount of
work is to be done with such material. In general, however, it is rather
difficult.
Urinary Bladder. To obtain distended preparations, expel the con-
tents and distend the bladder with the fixative, introduced with a pipette
or syringe. In the case of large bladders, the material may be opened
out and handled as described for mesenteric spreads, or pinned to a
board and floated tissue-side-down in the fixative.
Uterus. In the case of small animals, the uterus or entire reproductive
tract can be removed, laid out on a piece of paper and flooded with the
fixative. If the material adheres to the paper, place it in a volume of
fixing fluid . If it does not adhere, place the tissue in a shallow dish and
just cover with the fixing fluid until hardening has occurred. The fixa-
tive may be introduced into the uterus as described for the digestive
system. With larger forms subdivide before fixation. Very large uteri
should be opened out, subdivided and flattened.
Exercise 3
Fixation of Invertebrates for Smears and Whole Mounts
The following exercises are designed to provide a variety of objects
for later processing. In most instances other forms may be substituted
from the local fauna of the area. When class time is too limited for the
group to carry out all procedures, some tissues may be provided already
fixed and washed for further processing by the students. It is strongly
52 GENERAL ZOOLOGICAL MlCROTECHNIQUES
recommended that the student fix as many tissues as pos ible, including
at least one wet smear, one embryo, one spread and several types of
tissues. The Buggested exercises can be readily modified or extended by
reference to the text.
A. Protozoa Smears
1. Prepare smears of termite protozoa on slides. For removal of the
termite gut, follow the directions given in Exercise 2. Termite and other
protozoa are spread as directed in the text (p. 42).
2. As each smear is prepared, drop the slide smear-side-down into
a shallow dish of Schaudinn's. The ends of the slides should be supported
off the bottom of the dish with glass rods or bars. Fix for 20 minutes;
keep the container covered.
3. Remove the slides from the fixative and wash in 70 per cent alcohol
for 5 minutes to remove most of the fixative .
4. Transfer the slides to iodine-alcohol (p.35) for 20 minutes.
5. Transfer the slides to 70 per cent alcohol to remove the iodine. Do
not use the alcohol used in step 3, since it will contain fixative. Leaye
the slides for 15 minutes.
6. Store slides in 70 per cent alcohol until processed. These slides may
be stained in iron hematoxylin as directed in Exercise 21 , or in Cason's
rapid Mallory stain as directed in Exercise 23.
B. ponge Spicules
1. Fix and store portions of some calcareous sponge in 95 per cent
alcohol.
2. Process as directed in Exercise 13.
C. Small Hydromedusae
1. Place ripe Obelia colonies in If measured amount of fresh sea water
at room temperature. This will usually result in the release of the
medusae. Remove the polyps (these may be used for section D).
2. Anesthetize by adding saturated magnesium sulfate in sea water.
3. As soon as the medusae no longer pulsate, add enough 100 per cent
formalin to convert the water containing the medusae to about 10 per
cent formalin .
4. Set aside and let the medusae settle to the bottom of the dish.
5. With a small pipette, collect the medusae from the bottom of the
large container and transfer them to a narrow vial. Allow them to
settle and decant off the fluid; add another pipette full of material, and
so on, until the medusae have been concentrated into the small vial.
FIXATION 53
6. Decant off the original fluid and add fresh 10 per cent formalin .
Leave for 2 hours or several days.
7. RepJace the fixative with 5 per cent formalin for storage. This
material may be processed according to the directions in Exercise 17.
D. Hydroids
Substitute fresh water bryozoans if hydroids are not available.
1. Place Obelia, Campanularia, or other hydroid in a measured
amount of sea water which is just sufficient to cover them.
2. Add to the water crystals of magnesium sulfate or an equal volume
of 7.5 per cent magnesium chloride.
3. Watch the animals closely; when they are fully extended and no
longer contract when the tentacles are touched with a needle, add
enough 100 per cent formalin to convert the sea water containing the
animals to about 10 per cent formalin .
4. Transfer to fresh formalin and leave 1 to 7 days.
5. Store in 5 per cent formalin until processed. This material may be
stained and mounted as directed in Exercise 16.
E. Platyhelminthes
1. Immobilize Plana ria, Fasciola, Bdelloura, or similar forms, ac-
cording to the directions given in the text (p. 43).
2. Fix in sublimate-acetic for 12 to 24 hours.
3. Wash in running water or through numerous changes of water
for 12 to 24 hours.
4. Dehydrate through a graded series of alcohols (30, 50, and 70 per
cent) for 30 to 60 minutes each.
5. Place in iodine-alcohol and leave until the solution is no longer
decolorized. If the solution becomes clear replace it with fresh iodine-
alcohol. Shake the bottle occasionally.
6. Wash with fresh 70 or 80 per cent alcohol to remove the iodine
(leave several hours to several days).
7. Store in fresh 80 per cent alcohol until processed. This material
may be stained in Grenacher's borax carmine according to the direc-
tions in Exercise 16.
F. Nematodes for Unstained, Glycerine Jelly Mounts

1. Place small nematodes directly into hot 70 per cent alcohol.
2. Store in a mixture of 70 per cent alcohol and glycerine 3: 1. Mount
according to the directions in Exercise 10.
G. Sea Cucumber for Body Wall Mounts or for Corrosion Preparations

1. For body wall mounts, anesthetize Leptosynapta in magnesium
chloride solution. Other sea cucumbers are more suitable for corrosion
preparations and may be fixed without anesthetization.
2. Fix in 95 per cent alcohol.
3. Store in 70 or 80 per cent alcohol until processed. Body wall
mounts may be prepared as directed in Exercise 12. Corrosion prepara-
tions are discussed in Exercise 13.
H. Arthropods
1. Fix a numb~r of small arthropods suitable for whole mounts in
95 per cent alcohol. Small marine, fresh water, or terrestrial forms arc
equally suitable.
2. Store in 80 per cent alcohol until processed. Such material may be
mounted unstained in glycerine jelly (Exercise 10) or in resin (Exercise
12) or stained in Grenacher's borax carmine and fast green (Exercise
16) . See also Berlese's gum-chloral mounts described in Exercise 11.
Exercise 4
Fixation of Bird and Reptile Embryos

The embryos of birds and reptiles require special handling because
of the large amount of yolky material present. The following method
will work equally well with either. For class work the chick embryo,
incubated for 48 hours is recommended.
1. Have on hand a large quantity of physiological saline warmed
to incubator temperature (for chick embryos about 103° F) .
2. Open e. fertilized, incubated egg by carefully cracking the broad
end of the shell with a blunt instrument.
3. Carefully pick away the cracked shell and underlying membrane.
Keep your forceps close against the shell to avoid puncturing the
yolk. Break the shell down, working around and around the edge.
As the shell is removed, work out some of the albumen. Continue to
break away the shell and remove the albumen until the yolk rests in
the bottom of the shell.
4. Submerge the shell with the yolk into a dish of warm saline solu-
tion and tip the yolk out into the solution.
5. If necessary, rotate the yolk gently so that the embryo lies on
top.
6. Plunge a pair of forceps into the yolk just beyond the sinus ter-
FIXATION 55
minalis. Hold these firmly in your left hand and do not release them
until the embryo has been removed.
7. With a pair of fine, sharp scissors, cut around the edge of the sinus
terminal is, meanwhile holding the yolk steady with your forceps.
Begin and end the cut at the point held by the forceps.
8. Remove the embryo from the yolk by directing a stream of saline
solution between the embryo and' the yolk with a small pipette. Do
not release your hold on the forceps restraining the yolk until the
embryo is removed . Do not eject the solution forcefully from the
pipette and be careful not to suck the embryo up into ti)e pipette. Notice
if the delicate vitelline membrane, lying over the embryo, comes free at
this time. If not it must be removed later. Pull the yolk mass away
from the area where the embryo lies. The yolk may now be released.
9. Submerge a syracuse watch glass in the saline solution and care-
fully pull the embryo into the dish with your forceps. Hold the embryo
in the dish as you remove it from the solution.
10. Check to see that the embryo is lying in the dish with the
yolky side down. Turn it over if necessary before removing the saline
solution. .
11. If a quantity of yolk was introduced with the embryo, wash it
away by changing the saline solution as many times as is necessary.
12. Remove the vitelline membrane, which lies over the upper surface
of the embryo. Usually it can be pulled free with a pair of forceps. If
dull scissors were used it may be fused to the underlying layers and
difficult to remove.
13. Carefully remove as much of the saline as possible, drawing it
off with a pipette, held just beyond the edge of the material. Work
the pipette around and around the dish so that the embryo is perfectly
flattened and not pulled to one side.
14. Add Lavdowsky's fixative, a drop at a time, with the tip of the
pipette as close above and as near the center of the embryo as possible.
Let each drop spread out before adding the next. Add just enough to
completely cover the embryo but not enough to float it.
15. Cover the dish and let the embryo harden for about 1 hour. Be
sure it is not permitted to dry out.
16. Add additional fixative without disturbing the position of the
embryo. Cover the dish, sealing the edges shut with a film of vaseline.
Leave in the fixative for several days.
17. Remove the fixative and store in 70 per cent alcohol until pro-
cessed. This material may be stained in Grenacher's alum carmine as
directed in Exercise 15.
An alternate method of removing the embryo is dependent upon its

situation on the upper surface of the yolk when the shell is removed.
Place the egg shell in a holder and without submerging the y olk in saline
cut the embryo free. Then slowly lower the yolk into a dish of saline
solution and the embryo will float off on the surface of the solution .
. Complete processing as directed above.
Exercse 5
Mesenteric Spread
1. Kill a mouse, rat or other small mammal and expose the viscera
by making a mid-ventral incision from the anus to the level of the
diaphragm.
2. Tie off the gut above the stomach and below the large intestine
and remove the gut with attached mesenteries to a dish of saline solu-
tion. Change the saline to remove excessive blood.
3. Bring up under a section of the mesentery a short length of
glass tubing with fired ends. Slip over this, and the intervening mes-
entery, a slightly larger glass tube around which has been doubled a
small rubber band (fig. 5). Push the rubber band off onto the small
tube so that the mesentery is held against it. Remove the larger tube.
4. Trim off the mesentery just beyond the rubber band.
5. Empty the saline from the tube and place it with the attached
mesentery into the fixative. Tip the tube so that it will be filled with
the fixative. I f the material is to be processed for the demonstration of
fat, fix in lO per cent formalin. If it is to be processed for routine stain-
ing, fix in Bouin's.
TUBE
RUBBER BANO-
A B c
FIC . 5. Preparation of a mesenteric spread on a glass tube (lee Exercise lj) .
FIXATIOX 57
6. Formalin-fixed material should be stored in 5 p r cent formalin

until it is processed and washed in water just before staining. Bouin"
fixed spreads should be dehydrated through 30 and 50 per cent alcohol
for 10 minutes in each (do not leaye longer), washed in everal changes
of 70 per cent alcohol and stored in alcohol until proce ed. Thi ma-
terial may be stained in :Mallory's triple stain as directed in Exerci e
18. Formalin-fixed material may be stained in sudan II as directed in
Exercise 40, Section B.
Exercise 6 .
Fixation of Animal Tissue8
Prepare a number of tissues, preferably including oille from in-
vertebrates, amphibians, and mammals. Before proceeding. select ap-
propriate fixatives and prepare an outline of the steps neees ary for
postfixation treatment and processing each ti sue to a point where
it can be stored (usually 80 per cent alcohol) .
In the case of tissues fixed with reagents which sr remO\'ed by water,
it is best to wash in running water if possible. The material may be
placed in a loose-fitting cheesecloth bag (along with the working-label)
and washed in a pan of water under a faucet . Tip the pan slightly so
that t,he water runs in one end and out the other. The bags should be
weighted, if necessary, to make sure they are not washed out of the
pan. This provides better circulation of water than trying to wash in
a small container. It also allows for simultaneous washing of a number
of tissues. Delicate objects should be washed by placing them in a
relatively large volume of water and then changing this occasionally
over the period recommended to remove the fixath·e. After washing.
the tissues are partially dehydrated by processing through 30 and 50 per
cent alcohols for 30 to 90 minutes each . depending upon the . ize of
the tissue, and stored in 70 or 80 per cent alcohol.
Tissues which must be washed in alcohol (following picric acid I or
in water and/ or iodine-alcohol (following mercuric chloride fixatives I
are simply placed in fresh 70 or 80 per cent alcohol for torage upon
completion of the postfixation treatment,
Exercise 7
Fixation of Plant Ti88ues
The fixation of plant material involves the same basic procedure
which hayc been described for animal tissues. Many fixatives may be
used equally well for plants and animals. One of the most widely u ed
58 GENERAL ZOOLOGICAL )(ICBOTECHNIQUES
fixatives for plant materials is formalin-acetic-alcohol, "F.A.A.," (p.

31). The problem of penetration is somewhat greater with many plant
tissues than with animal tissues of the same size and it is advisable to
provide cut surfaces, even with small blocks, to facilitate penetration
of the fixing fluid. Subdivision of leaves, stems, roots, etc. should be
made with, or at right angles to, the growth axis of the material.
This text is primarily concerned with zoological materials and only
a few techniques directly applicable to plant materials are included.
These are intended for use in courses which are predominantly zoo-
logical in interest but where time permits a consideration of both types
of material. Students who are primarily concerned with processing
plant tissues should consult texts considering botanical techniques.
The exercise on onion root tips is recommended for courses which are
otherwise zoological in approach.
A . Materials for Whole Mounts
Small leaves, flowers, etc. may be fixed in F.A.A., washed in 50
per cent alcohol and stored in 70 per cent alcohol until they are
processed. Filamentous algae may be fixed in 10 per cent formalin and
stored in 5 per cent formalin. Bouin's may also be used. These materials
may be processed as directed for zoological materials, either for un-
stained or stained whole mounts. In the case of material fixed in forma-
lin or other aqueous media, the material is washed in water and then
dehydrated through an extended series of alcohols in 10 or 15 per cent
grades (10, 20 or 15, 30 per cent, etc.) to 70 per cent alcohol, where
it may be stored. Botanical material is subject to considerable dis-
tortion if mounted directly from the dealcoholizing-clearing agent into
thick balsam. It is advisable to infiltrate such material with dilute
balsam as described for whole mounts of minute objects (Exercise 17) .
B. Materials for Sections
Portions of herbaceous and woody stems and leaves may be fixed
in F.A.A., washed in 50 per cent alcohol and stored in 70 per cent
alcohol. Herbaceous material may be processed in paraffin, using the
tertiary butyl alcohol method (Exercise 25, Section B) . Woody stems
should be processed in celloidin (Exercise 35) . If materials are fixed in
aqueous fixatives, such as Karpechenko's, they should be washed in
running water and dehydrated through an extended series of alcohols.
A series using 5 per cent grades from water to 30 per cent alcohol and
10 per cent grades from 30 to 70 per cent alcohol will usually produce
good results. The material is left for 30 to 90 minutes in each change,
FIXATION 59
depending upon the size of the tissue block, and may be left for a
longer period (overnight) to facilitate scheduling of the work.
C. Onion Root Tips
This material is excellent for class work with Heidenhain's iron
hematoxylin. There are numerous division figures , the cell are rela-
tively large, and the material can be processed by routine embedding
methods.
1. Submerge the root end of an onion bulb in water. Individual
onions may be placed in jars, suspended by a tripod of tooth pick in -
serted around the circumference of the bulb. The mouth of the jar
should be large enough to permit some air circulation around the onion .
If the water becomes foul or cloudy it should be changed.
2. Cut the root tips when they are 10 to 15 mm. long. Let them fall
into a dish of freshly prepared Karpechenko's fluid (p. 37) . If the
tips are cut between noon and 1 p.m., a large number of division figure
will usually be obtained. Fix the tissue for 24 hours to several days.
3. Wash in running water for 12 hours or overnight.
4. Dehydrate through a graded series of alcohols (15, 30 and 50
per cent and into 70 per cent alcohol where they may be stored until
further processing. Twenty minutes in each solution is usually adequate.
This material may be processed by the usual methods (Exercise 24 ) and
embedded in paraffin. Root tips should be stained li ghtly with eosin
just before they are processed into paraffin so that they will be visible
in the paraffin mass.
6
Unstained
Preparations
Certain materials are commonly mounted unstaincd, and studied on

the basis of a refractive image. These include objects which are difficult
to stain and/ or where the detail provided by a color image and higher
magnifications is not required. Thus small organisms, such as nematodes
and many arthropods and arthropod parts, which are to be utilized for
general morphologic or taxonomic work may be prepared in this way .
Other classes of un tained material' include spicules, hair, feathers, and
ground sections of bones or teeth.
Objects which are unstained and unpigmented must be mounted in a
medium having a refractive index (p. 7) differing from that of the
material. On the other hand, t.he refractive indices of the object and
the mountant should be sufficiently simi lar to produce transparency
adequate for observation by transmitted light. The refractive index of
fixed tissue is about 1.54. The mountant, therefore, should have an R.I.
which is lower or higher than this.
There are three general types of unstained preparation" : those in
which the material i mounted in air; those utilizing a water-solubl e
mountant; and tho e using an oil-soluble (resinous) mounting medium.
Air (R.I., 1.00) is used in preparation of dry secreted structures.
Water-soluble mountan.ts are most frequently used for unstained prepa-
rations and have refractive indices ranging from about 1.40 (glycerine)
to 1.48 (glycerine jelly). Certain water-soluble mountants such as
Berle e's gum-chloral are killer-fixer mountants and provide simple one-
60
UNSTAINED PREPARATIONS 61
step methods for mounting small arthropods. Resinous media are Ie

frequently used for unstained preparations than are aqueous media.
Euparal (R.I., 1.48) is miscible with 95 per cent alcohol, but Hydrax
(R.I., 1.82) and almost all other rcsins require complete dehydration
through absolute alcohol, and dealcoholization with some agent which
is miscible with both the alcohol and the reSIn. Pigment.ed objects may
be mounted in resins which have refractive indices approximating that
of the tissue (c.g. Canada balsam , R.I., 1.54) .
T he Slide Ringer
The preparation of dry mounts and mounts in aqueous media is
greatly simplified by the use of a slide ringer. This instrument is used
for preparing a cell within which the material is mounted, and for ob-
taining an effective fleal, especially for aqueous preparations. The
slide ringer (fig. 6, A) consists of a turntable and hand rest. The turn-
table is provided with clamps for holding the slide in position and is
marked with a series of guide rings, one within the other, approximating
the sizes of round cover glasses.
A. Preparation of a Cell
1. Wash a slide with 70 per cent alcohol or Bon Ami and wipe clean.
2. Center the slide on the turntable and hold it in position with
T UR N
TABLE-
c
SLIDE RINGER
A
FlO. 6. A, the slide ringer; B, method of applying the llelll or ring; C, cr06l! sec-
tion of a sealed preparation.
the clamps. Determine wh.ich of the guide lines corresponds to the size
of the cover glass to be used. The cell wall should be prepared so that
its outer edge will coincide with the edge of the cover glass. A width of
1 to 2 mm. is usually sufficient.
3. Have on hand a clean "camel's hair" brush (No. 2 to 5), a con-
tainer of ringing solution (Gold Size varnish, Canada balsam or other
resinous mounting medium) and a container of solvent for the ringing
compound.
4. Place the ringer so that the hand rest is toward you. With your
right hand spin the table vigorously toward you in a clockwise direction.
Quickly dip the brush into the ringing solution, brush off the excess on
the lip of the container and place the brush tip at "9 o'clock" (fig.
6, B) . The brush tip should be held slightly in from the point which you
have predetermined to correspond to the edge of the cover glass. Your
hand should be supported on the hand rest. As the slide spins, a perfect
ring of media should be left on the slide surface. (If you are left-handed,
spin the table with your left hand in a counterclockwise direction and
hold the brush at "3 o'clock.")
After a little practice you 'Yill find that the width of the wall can be
varied by using different brushes and varying the pressure applied to
the brush. The height of the ring may be increased by applying
numerous layers of ringing material. In general it is best to prepare a
number of cells and let them dry completely before using them. Protect
them from dust.
Irregular rings with thick and thin areas are caused by: (1) ex-
cessive or over-thin media which results in a large blob where the brush
is first applied; (2) irregular pressure on the brush; (3) dry ringing
material on the brush. The brush should be carefully cleaned in a
suitable solvent between each application of ringing material.
B. Sealing Procedure
Mounts made with aqueous mounting media must be carefully sealed
so that the mountant will not evaporate. Air and resin mounts may also
be sealed. Such mounts are best prepared with round cover glasses and
sealed with the aid of the slide ringer. Any quick drying paint with
good elasticity, such as nail polish, model aeroplane dope, or a good
varnish such as Gold Size, may be used as a sealer.
1. Clean the preparation very carefully, scraping away any excessive
mountant with a razor and then carefully cleaning the slide with a
cloth moistened with water (in the case of aqueous mountants) or
xylol (in the case of resin mounts). The surface of the cover glass must
also be clean. It is very difficult to remove aqueous mountants from

the cover glass surface without damaging the preparation and every
effort should be made to keep it clean at the time the material is
mounted.
2. Place the slide on the turntable so that the cover glass is perfectly
centered as indicated by the guide rings.
3. Spin the table as described in Section A and apply the sealing
compound so that it extends over the edge of the cover glass, down
over the wall of mountant and on to the slide (fig. 6, C) . Check the seal
carefully and repeat the process if necessary. It is advisable to make
two complete seals to insure permanence. The use of colored sealing
compounds greatly simplifies checking the completeness of the seal and,
when two seals are made, use two colors.
The errors indicated for spinning a cell apply here. If the seal does
not follow the edge of the cover glass, the preparation was not perfectly
centered on the slide ringer. Failure of the sealer to adhere to the glass
occurs when the slide was not properly cleaned. Such areas will have a
mirrorlike appearance when the inverted slide is examined under a
light. Scrape off the ring, clean the slide surface and re-seal. Do not
attempt to trim the ring since this will usually break the seal. It may
be simpler to remount the material rather than attempting to repair the
initial attempts.
Exercise 8
Dry Mounts
Dry mounts are limited to objects which are already dry or may
be dried without distortion. The material is mounted on a slide under
a cover glass which is supp'.>rted off of the material and held in place
by a wall, thus enclosing the object in a cell of air. If the object is thin
enough to transmit light, prepare it as a transparent mount as directed
in' Section A. If the material is extremely opaque or too thick to transmit
light, it is prepared as an opaque mount according to the directions in
SectionB.
A. Transparent Mounts
Lepidoptera wing cales, mall membranous wing , hair, feathers,
ground sections of bone or tooth, etc., may be prepared as follows.
1. Prepare cells for round cover glasses using a slide ringer as de-
scribed earlier. Cells for quare cover glas es may be prepared freehand.
by drawing an outline of the cover glass on a piece of paper and using
this as a pattern when preparing the cell. The wall should be as low 88
possible, although deep enough to accommodate the material.
2. Place the material to be mounted in the center of the cell. Lepidop-
tera wing scales may be transferred by pressing on the wing with an
eraser and then pressing it on the slide surface; do not twist. Other
objects may be handled with a brush or forceps or on the tip of a
needle. Material suspended in alcohol or xylol (such as corrosion
products, Exercise 13) may be transferred in a drop of the fluid by
means of a pipette. Be sure the material is completely dried before
mounting the cover glass. Moisture left in the material will condense on
the under side of the cover glass and obscure the preparation.
Objects may be held in place in the bottom of the cell by a very thin
layer of mounting medium or varnish applied just before the object is
introduced. This is useful with larger objects, but must be kept at a
minimum so that it does not infiltrate the material.
3. Warm a cover glass in a soot-free flam e and quickly place it over
the material so that it is centered on the wall. Press it firmly into place
by applying pressure around the edge of the cover glass where it is
supported by the wall. Use a blunt instrument such as the handle of a
dissecting needle. Excessivc pressure or pressure on the unsupported
portion of the glass will brcak the cover. If the seal is not complete,
invert the slide and warm thc CO\'cr glass area slightly over a flame.
Ovcrheating will break the glass or cause the medium to flow in to the
material.
4. Optional. Seal the preparation, using the turntable for round cover
glasses as described earlier.
If the ringing compound flows into the center of the air cell, the
obj ect will be infiltrated and will be difficult to observe. The medium
will flow in: (1) if the cover glass is applied before the wall is sufficiently
dried or, (2) if the sealing material is overheated while attaching the
cover glass. For this latter reason it is better to heat the cover glass
instead of the slide when making the seal.
B. Opaque Mounts
Foraminifera shells, ground tooth or bone sections, and other opaque
objects which are mounted dry must be studied by reflected light, and
are ea ier to observe if they are placed on a colored background rather
than on a clear slide. This background may be provided by paint,
01' by mounting on opaque paper.
a
1. Prepare a cell, using a turntable for round cover glasses or making
a freehand ('Pll for square royer gla!<ses.
UNSTAtl'iED PREPARATIONS liS
2. Cover the bottom of the cell with ~ome quick-drying paint. . e

black paint for white objrct , white paint for colored objects.
3. Place the material to be mounted on the paint while it is still
"tacky." If the paint is too wet it will engulf the material.
4. When the paint is completely dry, mount a cover gin S over the
material using the procedure described in cction A. Alternately, the
paint may be applied to the bottom of the slide and the objects may be
held in place with a very thin lay r of mQunting medium applied just
before the obj ect is in troduced.
b
Mounts on opaque paper backgrounds are very useful for making
multiple preparations of shells of various species of Foraminifera on
a single slide.
1. Prepare a cell as follows (fig. 7, A). Rule off a 22- by 60-mm.
strip of paper so that a 2- to 3-mm. border is left on all four sides and
the center is grilled into the required number of squares. For purposes of
·'0
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FlO , 7. Types of whole mount preparations, A, dry mount in a paper cell . B, thick
mount in a cell of glass strips. C, mount of thin, fragile material with the cover glass
supported by a tripod of cover glass chips. D, mount of minute obj ects with the
cover glass supported by short lengths of capillary tubing. E , thi ck mount in a glass
or aluminum cell. F, double cover glass mount of material in glycerine jelly, sealed
with Canada balsam; G, cross section of double cover glass mount.
66 GENERAL ZOOLOGICAL MlCBOTECHNIQUES
labeling, the squares may be numbered. Such mounting papers may be

prepared by ruling heavy black paper with white ink (or white paper
with black ink). Fasten the paper to the slide, leaving about 2 mm.
at the right-hand side of the slide and a label space to the left. The paper
may be attached with Canada balsam painted around the bottom
margin of the paper just before it is placed on the slide. 'Cut thin card-
board into strips 2 to 3 mm. wide and attach these around the margin
of the paper background to form a cell. The size can, of course, be
varied, but should correspond to an available size of cover glass.
2. Sort through samples of material (such as sea sand) containing
Foraminifera shells, and group them into different types.
3. Paint one of the squares on the ruled paper with a thin coat of
Canada balsam and attach a number of sbells of a given type, orient-
ing tbem so that various views are presented for observation. Proceed to
mount a number of specimens in each square. The material may be
handled on the tip of a needle which has been very slightly moistened by
touching it to the tongue.
4. Paint a layer of Canada balsam on the upper edge of the card-
board frame surrounding the mounting area and cover with a cover glass.
Alternately, the frame may be extended to the ends of the slide and
the preparation covered with a second slide, which may be held in
place by binding the edges with tape such as that used for projection
slides.
Exercise 9
Glycerine Mounts
Glycerine is frequently used as a mounting medium for small nema-
to'des and helminth eggs in fecal samples. For temporary preparations,
a 1: 1 mixture of glycerine in water is usually used. For permanent
mounts it is best to use pure glycerine, and even then permanency is
not insured because of the consistency of the medium. Glycerine jelly
(Exercise 10) is much more satisfactory for permanent mounts of this
type of material.
The material may be mounted directly from water or alcohol (after
fixation). This is usually adequate when a mixture of glycerine and
water is employed for temporary mounts. For permanent mounts
in pure glycerine it is best to gradually infiltrate tbe material with
glycerine to prevent distortion. Infiltration is accomplished by placing
the material in a mixture of water and glycerine, or alcohol and
glycerine, and evaporating off the water or alcohol. Evaporation may
UNSTAINED PREPARATlON 67
be hastened by warming, but if it is too rapid the material may collap e.
The rate of evaporation will have to be adjusted to accommodate the
objects being processed.
1. Fix the material in alcohol (see Chapter 5) or u e material which
was fixed by other methods, washed as required and proce sed into
alcohol. .
2. From 70 or 80 per cent alcohol, transfer the material into a mixture
of glycerine and 80 per cent alcohol, 1: 2 by volume. Leave the container
open so that the alcohol will evaporate. Reduce the volume by one-half
to two-thirds before proceeding. The container should be protected from
dust.
3. Prepare a cclI on the center of a clean slide.
For temporary mounts, the cell wall may be prepared with warm
paraffin or glycerine jelly. Round cells are prepared by dipping the open
end of a vial of ~ uitable size into the melted ringing solution and
transferring a ring of the solution to the slide. Such rings solidify when
cool. For permanent preparations the cell wall should be prepared with
varnish or balsam, using the slide ringer as described earlier. The cell
wall should be high enough to support the cover glass up off the matt!rial.
4. Place a small drop of glycerine in the center of the cell. Transfer
into this the material to be mounted. Small objects such as nematodes
may be handled on the tip of a needle. Helminth eggs in fecal samples
may be transferred with a small pipette or wire loop. If necessary add
more glycerine so the cell will be completely fill ed when the cover glass
is put in place.
5. Film a clean cover glass with moisture by holding it over a beaker
of steaming water or by breathing on it. Lower it horizontally (fig. 8, A)
onto the preparation. centering it on the wall. (The moisture film helps
to prevent bubbles and if the cover glass is not lowered horizontally the
material wiII be displaced in the cell.) If bubbles are trapped in the
glycerine, or if there was not enough glycerine to fill the cell, remove
the cover glass, wash it with water, add more glycerine and recover.
After some practice you wiII be able to estimate the correct amount of
glycerine required to fill a cell.
6. If any glycerine has exuded onto the slide, remove it very care-
fully and wash the slide surface with a cloth moistened with water.
If there is glycerine on the cover glass it will probably be necessary
to mount a clean one.
7. Place the clean slide on a slide ringer and seal the preparation with
orne quick-drying paint. (Follow the directions given earlier in this
chapter.) Be sure the seal extends on to the edge of the cover glass, over
68 GENERAL ZOOLOGlCAL MlCROTECHNIQUES
the wall of the cell and on to the side (fig. 6, C) . If the preparation was
made with a square cover glass, paint around the edge of the cover glass
freehand.
Exerci8e 10
Glycerine Jelly Mounts
Glycerine jelly is solid when cool and is to be preferred over glycerine
for permanent preparations. As in the case of glycerine, the material
may be mounted directly from water or alcohol, but the results are
generally better if the object is first infiltrated with glycerine.
Of the many formulas for glycerine jelly, the following is both satis-
factory to use and easily prepared.
Kaiser's Glycerine Jelly
Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 gm.
Distilled water ....................... . .. . .... .... . ... ..... 60 cc.
Glycerine ........ . . . .... . . ............ . . . ............. . .. . 70 cc.
Concentrated phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 gm.
Dissolve the gelatin in water, heating just enough to dissolve the
gelatin i add the glycerine and phenol. Phenol crystals (0.25 gram per
100 cc. of glycerine jelly) may be used in place of the concentrated
solution of phenol. For thick mounts it is best to evaporate this solution
CG- ~ l~ F
MM - . ~O MM-
,, I
A SLiOE B
I
I
<'
CG- '6
C
FIa. 8. Methods of mounting the cover glass. A, for loose material such as a whole
mount. B, for sections or smears, when the material is attached to the slide . C, the
inverted slide technique for minute objects (see Exercise 17).
down before using. Such preliminary thickening greatly reduce later
shrinkage of the mountant. In preparing, evaporating, or melting glyc-
erine jelly, excessive heat should be avoided or the material will not
solidify when cooled. Evaporation may be accomplished in an oven
maintained between 55 and 60° C. Always melt the jelly in a hot water
bath, never over a direct flame .
A. Mounting in Glycerine Jelly

1. Fix the material in alcohel (Chapter 5) or use material fixed by
other methods, washed as required and processed into 70 or 80 per cent
alcohol.
2. From alcohol, place the material into a mixture of glycerine and
80 per cent alcohol, 1:2 by volume. Leave the container open but pro-
tected from dust. Permit the alcohol to evaporate so that the solution
is reduced by one-half to two-thirds before proceeding. Evaporation
may be hastened by warming the vial or placing it in a convection oven .
3. Since the glycerine jelly solidifies on cooling it is not necessary to
mount the objects in a cell. If the material is very fragil e or very thick it
is best to support the cover glass off of the object by introducing chips
of glass in a tripod arrangement around the material (fig. 7, C). This
will prevent crushing llnd will keep the cover glass from tipping. The
glass chips need not be large and flhould have a thickness approximating
that of the material.
4. Place a clean slide on a warming plate such as that us d for
spreading paraffin sections. The temperature of the plate should be
between 40 and 55° C.
5. Melt down some glycerine jelly in a hot water bath. If there are
bubbles on the surface of the jelly, scoop off the surface layer before
melting. Allow plenty of time for the solution to liqu efy and for any
bubbles which are present to escape from the solution.
6. Expel the air from a small rubber-bulbed pipette which you have
first warmed in hot water. With the air expelled, insert the pipette into
the glycerine jeJly and suck up some of the solution from the bottom of
the container. This procedure will avoid introducing bubbles into the
solution. Place one to several drops of glycerine jelly on the c nter of the
warmed slide. The correct amount will vary with the size of the cover
glass and the thickness of the preparation. (The pipette may be cleaned
by flushing with warm water.)
7. Transfer the object from the glycerine into the glycerine jelly.
Try to carryover as little glycerine as possible. Arrange the object in the
center of the jelly, using a dissecting needle or fine forceps. If the object
is thick introduce several glass chips around it to support the cover
glass.
8. Lower a cover glass horizuntally until it is just above the surface
of the warm glycerine jelly. Hold it just above the jelly surface until
you can see that a film of water vapor has condensed on the lower sur-
face, then lower it on to the jelly. The moisture film will prevent
bubbles. Alternately, the covE!r glass may be water-filmed by holding
it over a steaming beaker of water.
If insufficient glycerine jelly was used, large air pockets will form
when the cover glass is mounted. If the material was mounted in a cell,
the cover glass must be removed, washed in water, additional glycerine
jelly added and the cover glass replaced. If the material is not in a cell,
additional glycerine jelly may be added at the point where one edge of
t,he air pocket is in contact with the edge of the cover glass. If the jelly
is added at the mid-point of such an air space, a large bubble will be
trapped in the preparation. If air bubbles are trapped in the glycerine
jelly, it is usually necessary to remove the cover glass, burst the bubbles
with a warm needle, and recover. Occasional bubbles can sometimes be
removed by sucking them out with a fine tipped pipette.
9. Keep the slide perfectly fiat, remove it from the slide warmer
and set it aside to cool.
10. Carefully scrape away any excessive glycerine jelly from ' the slide
and clean with a cloth moistened with water. The cover glass must als(l
be clean and great care must be taken not to cru~h the preparation
or displace the cover glass during cleaning. Make every effort to keep
the cover glass clean while mounting it.
11. Place the slide on a turntable and carefully seal the preparation
with Gold Size varnish or any quick-drying paint as directed earlier.
If unthickened glycerine jelly IS used in preparing the mount the seal
should be completed the same day that the mount is made.
B. Alternate Method for Small Objects
1. Fix the material and infiltrate with glycerine as described in
Section A.
2. Place a large (22 mm.) round cover glass on a warming plate and
on the center of this a small drop of warm glycerine jelly.
3. Transfer the material from the glycerine to the glycerine jelly on
the cover glass.
4. Lower a smaller cover glass (14 to 16 mm.) on to the preparation '
so that it is centered on the large cover glass with the material sarid-
wiched between. The glycerine jelly should flow to the edge of the smaller
cover glass.
5. Place a drop of Canada balsam on the small cover glass and bring
the center of a clean slide into contact with the drop of mountant. Do
not press down on the cover glass, but rather let the balsam flow out
between the slide and the cover glass. Invert the slide. The mountant
should flow to the edge of the large cover glass, which is now on top.
The small cover glass is attached to the slide and the preparation is
automatically sealed by the ring of balsam between the outer edge of
the small and large cover glasses (fig. 7, F and G).
Exercise 11
Berlese's Gum-Choral Mounts
This procedure is extremely useful when mounting large numbers of
small arthropods from soil or debTlS samples. It i a rapid and effective
method of preserving them for later reference and identification. The
material need not be previously fixed; although fixed material may be
used, the best results are obtained if the animals are placed in the
mountant while they are still alive.
Berlese's Gum-Chloral }If ountant
Distilled water . 20 ee .
Gum a.ra.bic ..... . .. ...... ,.,.,.".,",", " "'.,',. ,. 15 gm .
Glucose syrup . , , . , , , .. , , , , , , . , " , , , , , . , , , , , , , JO cc.
Combine these and then add:

Chloral hydrate to satum't ion
Glacial acetic acid . , , . , , , , , , . , , , , , , , , ... ' "', . .. ,.,' 5 c(! .
The mounts are prepared as follows:

1. Place a drop of gum-chloral on the center of a clean slide. If
the animals are not too fragile or too large the cover glass may be
mounted without any supoprt so that it will distend the material
slightly. Fragile obj ects should be mounted in cells such as those de-
scribed earlier. The use of shallo\\' cells, prepared with the slide ringer,
will facilitate sealing the preparation.
2. Place the animal in the drop of mountant. It will usually be killed
in an extended condition. Arrange the appendages as necessary and cen-
ter the object on the slide.
3. Breathe on the lower surface of a clean cover glass and cover the
object; keep the cover glass horizontal to the slide so that the material
will not be displaced.
4. Allow the slide to dry for about a week. Add more mountant if
necessary. Carefully clean away any excess mountant from the slide,
using a cloth moistened with water. Seal the preparation with some
quick-drying paint.
Exercise 12
Resin Mounts of Unstained. Materials
Resin mounts are definitely superior to any aqueous preparations
from the standpoint of permanence. In the case of pigmented materials,
the resin also provides better optical qualities. Unpigmented objects
may be mounted in resins such as Euparal or Hydrax, having relatively
low or high refractive indices, as compared with the tissue. Pigmented
objects are mounted in resins having refractive indices which approxi-
mate those of the tissues. In these instances the image is provided by the
pigment. It may be necessary to bleach heavily pigmented forms to in-
crease their transparency. Arthropod exoskeleton mounts may be im-
proved by first destroying the soft tissues. The material may then be
mounted in a flattened condition, thus increasing the transparency of
large ~bjects as well as permitting observation with higher magnification
lens systems.
A. Soft Tissues
The following exercise is based upon the use of some small arthropods
or arthropod larvae, but may be adapted to other types of material.
1. Preserve in alcohol, Carnoy's fluid, or another suitable fixative
(Chapter 5) . Wash as required by the fixative employed.
2. Optional. For flat exoskeleton mounts of arthropods, destroy the
soft tissues by soaking in 20 per cent potassium hydroxide in water
(handle with care). Wash w~l in water to remove the corrosive before
processing.
3. Optional. Heavily pigmented forms may be partially bleached by
one of the following methods. (a) From water, transfer the material
into 3 per cent hydrogen peroxide; when sufficiently bleached, wash well
in water and dehydrate to 70 per cent alcohol. (b) From alcohol, trans-
fer the material into sodium hyperchlorite in 70 per cent alcohol. Add 1
drop of 5 per cent sodium hyperchlorite to each 2 cc. of alcohol. When
the material is sufficiently bleached, wash in at least three changes of
70 per cent alcohol, leaving at least 30 minutes in each.
4. From 70 or 80 per cent alcohol transfer the material to 95 per cent
alcohol for 1 hour or more.
5. Complete dehydration through two changes of 100 per cent alcohol
for 1 hour or more in each change. (For Euparal mounts of uDpigmen~d

materials, proceed to step 7.)
6. Dealcoholize the material with a solvent which is miscible with
both the alcohol and the mountant. Toluol , xylol, terpineol, beechwood
creosote-xylol, or carbol-xylol may be used with most resinous mounting
media. The first two tend to make the material more bTittle than do the
others and have almost no affinity for water, so that.the material mu t
be perfectly dehydrated before it will "clear." Beechwood creosote-
xylol and carbol-xylol will remove considerable amounts of water.
Leave the material for 30 minutes to several hours in each of two changes
of the solution, depending upon the size of the material. The object
should become perfectly transparent or "clear." White, opaque areas
or a general opaqueness of the material indicates that the object was
not sufficiently dehydrated. Return such material to fresh 100 per cent
alcohol and again dealcoholize; or treat them with carbol-xylol before
proceeding.
7. Unless the objects are too fragile or too thick, it is best to mount
the cover glass directly upon the material. Small fragile objects should
be mounted in cells of glass or metal (fig. 7, E) which have been attached
to the slide with balsam. The depth of the cell should approximate the
thickness of the material.
Place a drop of mountant on the slide. Transfer the object into this
drop and immediately cover with additional mountant. Do not let the
material dry. Place the cover glass on the preparation and keep the slide
fiat until the mountant has hardened. If bubbles are trapped in the
preparation, they will usually escape if the slide is kept warm and fiat
for several hours or overnight. Warming will also hasten hardening
of the mountant.
In the case of thick preparations in Canada balsam, the mountant
should be thickened beforehand (see the Appendix) and the balsam and
slide are warmed at the time the material is mounted . Upon cooling,
the preparation will be e sentially solid.
B. Dry Materials
Many dry objects may be mounted in resins even though they are
unstained and unpigmented. Thus spicules, skeletal plates and small
bones usually have very high refractive indices and will be visible in
balsam or may be mounted in one of the other resins which experiment
shows to be satisfactory. Feathers and hair may also be mounted in this
way.
Since the materials are not distorted by drying, they may be mounted
without any preliminary treatment, provided they are perfectly dry.

With minute objects it may be easier to cover them with the mountant if
a drop of xylol or toluol is placed on the material beforehand. This will
prevent the collection of numbers of bubbles around the object when the
mountant is added. If bubbles do form, warm the slide for a few hours.
Exercise 13
Corrosion Techniques
Sponge spicules, skeletal plates of sea cucumbers, placoid scales, etc.
Corrosion involves the destruction of soft tissues to permit isolation of
hard secreted structures such as spicules or skeletal plates. Calcareous
or chitinous structures are usually isolated from soft tissues by destroy-
ing the latter w,ith potassium hydroxide i silicious structures, by treating
the object with nitric acid. Both solutions should be handled with great
care to avoid spilling or splashing. The treatment of arthropod exo-
skeletons has already been considered in Exercise 12, Section A.
1. Fix the material so that the secreted structures will be preserved.
Alcohol is a good preservative for such material or, in many cases, the
object may be dried. Acid must be avoided.
2. Place the material in a corrosive fluid in a test tube or centrifuge
tube (Pyrex) .
Use 10 to 20 per cent potassium hydroxide for calcareous structures
and nitric acid for silicious structures. Since some gases are generated
during the corrosion process it is best to cover the container loosply or
leave it open but protected from dust. (If ordinary vials are used,
handle the container carefully in case it is damaged by the heat of the
reaction.) Usually it is well to let the corrosion proceed slowly at room
. temperature.' It may be hastened by using a more concentrated solution
(up to 35 per cent potassium .hydroxide) or by gentle heating.
3. After several days (at room temperature) shake the container to
determine if corrosion has been completed. The material should fall
apart and minute spicules or plates may be observed suspended in the
solution. If corrosion is not complete, replace the corrosive with a fresh
solution. If the material separates, set the container aside and let the
spicules or plates settle to the bottom of the container.
4. Pour off as much of the solution as you can without losing the
material. It is best to pour the solution off into a second container in
case you accidently pour away the material. Fill the container with
water and set it aside to let the material settle. The procedure may be
shortened by washing minute objects in centrifuge tubes and centrifug-
ing the material in!tcad of waiting for it to settle.
5. Replace the water with 95 per cent alcohol. ( ince the material is
not fragile, gradual dehydration is not necessary.)
6. Small objects are transferred to the slide with a pipette. Place a
drop of fluid containing the material on the center of a clean slide and
let the alcohol evaporate completely. This may be hastened by warming.
Minute objects may be mounted dry (Exercise 8) or these and larger
objects may be mounted in a suitable resin (Exercise 12, Section B).
Exercise 14-
Ground Sections of Bone or Tooth
1. Strip the bone of all flesh and dry thoroughly. Some workers pre-
fer to complete steps 2, 3 and 4 before drying.
2. Clamp the bone (or tooth) in a small vise, and with a fine blade
(a hacksaw will suffice) subdivide it into the thinnest possible sections,
keeping the cuts as parallel as possible.
3. Place the strips in water and let them macerate for several weeks
or longer to destroy all of the soft tissue.
4. Wash thoroughly in running water to remove the macerated soft
tissue.
5. If the bone is spongy, dry it thoroughly, place it in xylol and then
transfer it to moderately thick balsam. Dry the balsam down until
it is hard when cool. This may be accomplished very rapidly in a con-
vection oven. The bone may be placed on a slide in a drop of balsam,
or in a small dish of the resin, depending upon its size.
6. Carefully rub the bone over a flat, wet, sharpening stone. Apply
light, even pressure with the finger tip and turn the material frequently.
The process should be continued until the section is very transparent.
It is usually advisable to start the grinding on a fairly coarse stone or
even a flat file, especially if the initial block is rather thick. The grind-
ing should be finished on a very smooth stone or glass plate so that the
surface of the section will not be scoured. Wash the section to remove
grinding dust and then dry thoroughly.
7. Remove the . balsam from the spongy bone by soaking it in xylol.
8. (a) Air mount the sections (Exercise 8) using either a transparent
or opaque mount, depending upon the thickness of the section. A com-
bination slide may be readily prepared by painting one-half of the cell
black and leaving the opposite side clear. The tissue is mounted over
the junction of the two areas. Attach the bone with a very thin layer of
vaIVish or balsam, placing the bone in the film while the latter is just
tacky enough to hold it in place. (b) The ideal mount for dense bone is
one in which the matrix is cleared while the haversian systems remain
"black" with trapped air. Although it may require many attempts,

the results obtained justify the effort. Use thickened balsam (p. 216)
which is further hardened by placing a drop of it on a slide and another
drop on a cover glass and leaving these in a paraffin oven or on a slide
warmer for several days (protect from paraffin and dust) . Remove the
slide and cover glass and let them cool. Place the bone section on the
hardened balsam on the slide and place the cover glass on top. Put the
slide on a slide warmer and apply firm pressure to the cover glass with a
clean finger tip. As soon as the balsam warms enough for the balsam on
the cover glass and the slide to flow together, remove from the warmer
and place on an ice cube. If examination shows that the bone is com-
pletely cleared and the haversian canals invisible, soak the preparation
off in xylol, dry and begin again.
7
Stains
When one considers the multiplicity of dyes available, there is a re-

markable limitation to those which are generally used in either zoo-
logical or botanical techniques. Each of these dyes is subject to many
modifications of staining solutions and procedure for application. One
of the main sources of variation in staining has been in the dyes them-
selves. This problem has been minimized by the establi hment of the
Biological Stain Commission , which tests lots of certain dyes produced
by various manufacturers. Commission-certified dyes hould be em-
ployed whenever possible. 'Ffte PI8UI1' nhapl.4lor is primarily concerned
with the general characteristic of certain stains and their formulas .
It must be emphasized that many other stains and formulas are availa-
ble. The student is referred to Conn, Biological. tains, for the chemical
structure and general properties of dyes utilized as biological stains.
?"Staining techniques are probably the most difficult for the beginner
to master and arc dependent more upon experience than upon direction.
The situation is complicated by the fact that staining effects vary
with different tissues, after different fixatives, different periods of
storage and, a mentioned earlier, different lots of dyes. The student
should follow the directions as closely as possible, compare the re-
ults with demonstration slides and then attempt to modify his tech-
nique to improve on the initial attempts. You will find suggestions at the
end of the exercises regarding common difficulties encountered with
different stains and suggestions for how these may be corrected.
77
Stains are generally classified on the basis of function, as nuclear

(chromatin) or cytoplasmic. These are also referred to as primary
stains and counterstains, respectively. It must be borne in mind, how-
ever, that most stains will color all of the tissue unless they are utilized
in a certain way. It might be said that nuclear dyes have a greater
affinity for chromatin than for cytoplasm. In many instances the nuclear
stain is combined with a mordant which has an affinity for both the
chromatin and the dye and serves to bind the latter to the chromatin
while permitting its removal from the cytoplasm. The mordant is
usually a metallic salt and may be used before the dye is applied to
the tissue, or may be combined with the dye in the staining solution.
Some nuclear dyes may also have affinities for certain types of cyto-
plasmic structures and may have polychromatic effects, staining various
structures various shades or tones. This polychrome effect also occurs
with many counterstains. The majority of the nuclea r dyes are basic,
whereas most of the cytoplasmic dyes are acid.
-f.4There are two general types of staining procedures, progressive and
regressive. In progressive staining the material is exposed to the stain
until the desired degree of coloring is obtained. In regressive staining
an excess of dye is allowed to accumulate in the tissue and the material
is then destained until the proper degree en differentiation is produced.
Although this latter method may seem unnecessarily laborious, it gen-
erally produces a much better nuclear differentiation than do pro-
gre sive methods. It is the usual procedure for chromatin stains. Coun-
terstains are almost always used progressively. In general the nuclear
stain is applied to the tissue before the counterstain . .,_,..
The Natural Dyell 8od-StaHlS .,.. ~
The natural dyes are obtained from natural sources rather than
being synthesized in the laboratory. These include two of the most
important nuclear dyes, carmine and hematoxylin.
Carmine .,. oJ..
Carmine is produced by combining alum with cochineal, obtained
from the dried bodies of the cochineal bug. Carmine is most common It
used for whole mount preparations but may be utilized as a primary
stain for sections. The clarity of a good whole mount results from the
limitation of the carmine to the nuclei with the cytoplasmic areas com-
pletely or relatively free of the dye. The structures of the organisms
are visible on the basis of the different concentrations of nuclei in the
various organs and tissues. When a whole mount fails to show sharp
STAINS 79
detail it usually indicates that the stain is too diffu e, coloring th

cytoplasm as well as the nuclei. It may al 0 indicate that the tain
did not penetrate sufficiently or that the material was poorly fixed.
Of the many formulas for carmine stains, two example are given
here which will erve for most purposes and will illu trate th types
of carmine stains generally employed.
Grenacher's Alum Carmine
Distilled water . . ........... 100 CC .
Aluminum ammonium sulfate .,. ................ 3.0 gm .
Carmine ... , . . ""'" 1.0 gm .
Combine and boil for 15 minutes or until the carmine i dissolved. It may
be possible to increase the amount of carmine in solution by increasing
the amount of aluminum ammonium sulfate to 5 grams and the carmine
to 2 grams. When the solution has cooled, it is filtered .
Alum carmine is used progrcssivcly for whole mounts (Exercise 15
and 17) or for bulk staining (Exercise 41). It does not usually over-
stain but may be destained in 0.1 pcr cent hydrochloric acid in 70 per
cent alcohol, following the method described for Grenacher's borax
carmine. Large dense objects are usually not counterstained. mall
objects and sections may be counter tained with fast green or similar
dyes.
Grenacher's Borax Carmine
Distilled water , . 100 ce .
Borax ... 4 .0 gm .
Ca.rmine .. " . 2.0 gm .
Mix and allow to stand for several days, stirring daily. Thc solution may
be boiled to hasten preparation. Mix with 100 cc. of 70 per cent alcohol
and allow to stand for several days, stirring daily. Filter before use.
Grenacher's borax carmine may be used as a whole mount stain
(Exercise 16), for staining tissues in bulk (Exercise-n) or a a pri-
mary stain for sections. It is used regressively and differentiated in
a weak solution of hydrochloric acid in 70 per cent alcohol. For whole
mount preparations with dense parenchymatous tissue it is generally
used without a couRterstain. On sections and on object without paren-
chyma, and especially for materials containing chitin, it i eff ctively
combined with fast green as described in Exercise 16. When working
with large whole mount materials it i important that the material i '
stained for a long period (several days to a week), ince the penetration
rate of the dye is very slow.
80 GENERAL ZOOLOGlCAL MlCROTECHNIQUES
Hematoxylin
Hematoxylin is obtained from the South American logwood tree,
and produces a very sharp blue to black chromatin stain when oxidized
to hematein and combined with a mordant. Heidenhain's iron hema-
toxylin technique provides a very beautiful and critical chromatin stain
for cytological work. The alum hematoxylins are more generally useful
in hi stological preparations in combination with counter tains such as
eosin or erythrosin. In the case of the alum hematoxylins the mordant
(aluminum ammonium sulfate). is combi ned with the dye in the tain-
ing olution.
Ehrlich's Acid Hematoxylin
Distillp.d water . 100 cc.
100 per cent alcohol 100 ce .
Glycerine . 100 cc.
Glacial acetic acid .. 10 cc.
Hematoxylin . 2 gm.
Aluminum ammonium su lfate , to exceSR (ahout 20 gm. )
Dissolve the hcmatoxylin in alcohol, add the acid, then the water and
glycerine. Allow the solution to ripen in the sunlight for about 2 months,
opening the bottle occasionally. Th e solution should assume a deep
red color. The aluminum ammonium sulfate may be added before or
aftpr ripening. When the solution is ripened , store in a closed bottle
on a dark shelf. This stain is quite stable and will keep for long
periods.
This stain may be used regressively as described for Harris' alum
hematoxylin (Exercise 33) . It may also be used as a progressive stain
for sections (Exercise 32) or for staining tissues in bulk (Exercise 41).
The times and cffects of staining may be varied by diluting the stain
with distilled water or with a saturated solution of aluminum ammonium
ulfatc. The latter technique tends to restrict the stain to the chromatin .
It is important that the material is washed very thoroughly in running
water or severe fading of the stain will occur after mounting.
Harris' Alum H ematoxylin
Hematoxylin . . .. .............. . . . . . . . . . .. . ...... . 0 .5 gm.
Aluminum ammonium sulfate ... . . . . . . . . . . . . .. . . . . . . . . . 20.0 gm .
Distilled wat r . . . ..................... ... ....... .. . 100 cr .
Mercuric oxide ... 0 .5 gm .
Boil the hematoxylin and a.mmonium alum together in the distilled

water nnd add the mercuric oxide. Prepare the stain at least 1 week be-
STAINS 1
fore use. The solution will keep for a considerable period and may b
reused. If a scum develops on the solution it should be filtered.
This stain is most effective when used regressively (Exercise 33). It
may be used progressively in a dilute solution, diluting the stock 1: 100,
more or less, with distilled water or with 20 per cent aluminum am-
monium sulfate. When used regressively the material is destained in
0.5 to 1 per cent hydrochloric acid in distilled water The material must
then be washed and "blued" in a basic solution (ordinary tap water
is usually effective) before further processing. When the dilute solution
is prepared with a quantity of aluminum ammonium sulfate the slides
should be washed thoroughly in running water to remove all trace
of the mordant.
Heidenhain's Iron Hematoxylin
10 per cent hematoxylin in 100 per cent alcohol (aged) ... . . 5 cc .
Distilled water ...................................... ..... , 100 oc .
Many of the problems which arise in this technique may be attributed
to the preparation of the staining and mordanting solutions. If the
staining solution is made up directly in water (0.5 gram of hematoxylin
in 100 cc. of distilled water) it will require several weeks to ripen when
exposed to air, and may pass rather rapidly from an under-ripened to
an over-ripened condition. The. staining solution may also be prepared
from hematein, in which case it is theoretically ready for use im-
mediately since the hematoxylin has already been oxidized. Such a solu-
tion, however, is frequently over-ripe, the hematein oxidizing further
and losing its staining capacity. The most satisfactory results are
obtained by ageing the hema.toxylin in 100 per cent alcohol. Enough
hematoxylin is placed in a glass stoppered bottle of 100 per cent alcohol
to make up a 10 per cent solution of stain. The bottle is then closed
but not sealed and is left on a window sill or sunlit shelf for 6 to
8 months or more. It may then be tested in aqueous dilution, and if it
produces a good stain the bottle of stock solution may be sealed and
stored in a dark cupboard. This stock will keep for 6 months or more.
Before the stain is used, the stock solution is diluted with distilled
water and allowed to ripen for about 1 week (with the bottle closed).
This staining solution should not be muddy in appearance, but a clear '
brownish fluid.
In Heidenhain's technique the tissue is treated with a separate
mordanting solution of 2 to 4 per cent ferric ammonium sulfate ("iron
alum") before staining. The material is then overstained in the hema-
toxylin and is differentiated in a 2 to 4 per cent solution of the mordant.
GENERAL ZOOLOGICAL MICROTECHNIQUES
Separate solutions of iron alum should be used for mordanting and

for de taining. The ferric ammonium sulfate should be freshly pre-
pared (within 1 week of using) and only violet crystals of iron alum
should be used. The working solutions of iron alum should be changed
each day or each period of work. The tissue may also be destained in
1 per cent hydrochloric acid in water or alcohol, or in a saturated solu-
tion of picric acid in 95 per cent alcohol. This latter technique may be
employed if the picric acid is to be used as a counterstain. In general
the results obtained by differentiation with iron alum provide the
greatest contrast between chromatin and cytoplasm, since the stain
is removed from the cytoplasm while leaving a heavy "lake" of dye in
the chromatin (unless, of course, it is over-destained) .
Iron hematoxylin is frequently used without any counterstain. It may
be combined with fast green or with orange G. In Masson's technique
it is employed with acid fuchsin and fast green and may be similarly
combined with safranin 0, with or without fast green. Eosin and
erythrosin do not usually produce good counterstain effects with iron
hematoxylin. The chromatin tends to stain blue with short periods
of staining in iron hematoxylin and black after long exposure to the
dye. In addition to its use as a chromatin stain, hematoxylin may be
used to great advantage in the demonstration of muscle striations,
mitochondria, cell walls in plants, etc.
The Coal Tar Dyes and Stains

Of the large number of coal tar dyes available only a few will be con-
sidered here. Some of these dyes are acid and others are basic. Some
are employed as chromatin stains, others as counterstains and some
are utilized as either chromatin or cytoplasmic stains depending upon
the technique.
Acid Fuchsin
Acid fuchsin is generally employed in a 0.2 to 1.0 per cent aqueous
solution as the primary stain in Mallory's triple stain. It may be used
in a 1 per cent solution in 95 per cent alcohol as a counterstain follow-
ing hematoxylin. Acid fuchsin is also used in Masson's technique in
combination with hematoxylin and fast green. The stain is selectively
removed from connective tissue by treatment with phosphomolybdic or
phosphotungstic acids. It may be destained in the higher alcohols or
in basic solutions. When used without hematoxylin, the fuchsin should
stain the chromatin bright red and the cytoplasm varying shades of pink.
Basic fuchsin may be substituted for acid fuchsin.
STAINS 83
Alizarin Red S
Alizarin red S is an acid dye most commonly employed as a stain
for calcified structures such as bone (Exercise 19). The stain is u ed
in concentrations of 0.0025 to 0.01 per cent in 1 per cent potassium
hydroxide.
I
Aniline Blue WS
Aniline blue . . .... . ........ . ................ . 0 .5 gm .
Oxalic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . 2.0 gm .
Distilled water . . .. .. . ............................ . .. 100 cc .
Aniline blue may be used as a simple counterstain following hema-

toxylin.1t may also be used following acid fuchsin and phosphomolybdic
acid as a counterstain for connective ti sue. It is usually employed
along with orange G in the second staining solutions in Mallory's triple
and Heidenhain's Mallory-azan techniques.
Aniline blue is extremely water-soluble and is often removed by
a water rinse. If the water rinse is replaced by 2 per cent phosphomolyb-
dic acid , however, it is possible to remove the superficial stain without
removing it from the tissue. Failure to rinse the slide after staining
and before dehydration may result in unsightly precipitates on the ma-
terial. In view of its solubility in water and the lower alcohols, the de-
hydration rate must be accelerated or the series of alcohols shortened in
order to retain the stain in the tissue.
Azocarmine G
Azocarmine G . . .................. .. .. . 1.0 gm .
1 per cent glacial acetic acid ........... " .. 100 cc.
Azocarmine is an acid dye utilized as a nuclear stain. It is most com-

monly employed in Heidenhain's Mallory-azan technique and imparts
a brilliant red color to the chromatin. The stain is differentiated in
aniline-alcohol (Exercise 46).
Basic Fuchsin
Basic fuchsin is best known for its use in Feulgen's nuclear staining
technique (Exercise 43). The staining solution is prepared as follows .
Basic fuchsin . . .. ...... .. .... . ......... " .. . ... . . . . .. . . . 1.0 gm .
Distilled water . ...................... , " ..... . ....... . . 100 cc.
Boil, filter, and add 30 cc. of normal hydrochloric acid and 1 gram of
anhydrous sodium bisulfite or potassium metabisulfite. Let the solution
stand in the dark for 24 hours. The solution produced should be a clear
yellowish fluid.
An aqueous solution (0.2 to 1 per cent) of basic fuchsin may be utilized
in place of acid fuchsin in Mallory's techniques or as a general choma tin
stain with a fast green counterstain.
J
Crystal Violet
Crystal violet is a basic dye employed as a nuclear stain. The
stain is applied as a 1 per cent solution in distilled water. It washes out
quickly in water and the lower alcohols and an accelerated dehydra-
tion schedule is necessary. Its most popular use is as a spindle dye in
Flemming's triple stain (Exercise 44).
Eosin Y
Eosin Y (yellowish) is widely used as a counterstain following alum
hematoxylin. It may be used in aqueous solution but generally pro-
vides the best results with the least difficulty in a 0.2 per cent solution
in 95 per cent alcohol (Exercises 32 and 33). Eosin should impart a
bright, clear pink color to the cytoplasm without interfering with the
clear blue of the chromatin stain. The stain is acid l and if the sections
are left in the solution for an excessive period the blue color of the
hematoxylin is changed to purple and then to a weak pink shade. A
10- to I5-second staining period should suffice. The main source of
error in using the stain is in leaving the slides in 95 per cent alcohol
after staining. The slides should be rinsed quickly through two changes
of 95 per cent alcohol and placed in 100 per cent alcohol before they
are examined. Eosin is removed by basic solutions. Eosin B (bluish) may
be substituted for eosin Y following alum hematoxylin stains.
Eosin Y is also utilized with methylene blue chloride as a general
blood and tissue stain (Exercise 42). The eosin is applied in a 0.2 per
cent aqueous solution before the methylene blue. These two dyes are
the basic ingredients of both Wright's and Giemsa's stains.
Erythrosin
Erythrosin is similar in effect and use to eosin, and is generally em-
ployed in place of eosin by botanists. It is utilized in a 0.5 to 1.0
per cent aqueous solution in a 0.2 per cent solution in 95 per cent alcohol.
Erythrosin B is used more commonly than erythrosin Y.
Fast Green
Fast green is an acid dye which makes a very effective counter-
stain for who.le~!:IJ material prepared with carmine (Exerci e 16),
STAINS 85
for sections stained with iron hematoxylin (Exercise 31) or safranin 0

(Exercise 37), and in combination with iron hematoxylin and acid
fuchsin in Masson's technique (Exercise 45). It is easy to apply, al-
though some caution must be exercised to avoid excessive staining. Al-
though sometimes used in aqueous solution it is most effectively applied
in a weak (not more than 0.2 per cent) solution in 95 per cent alcohol.
The material should be rinsed in 95 per cent alcohol before being placed
in 100 per cent alcohol after staining or precipitates will be left on the
slide. The green cOlor of the dye may be altered to blue by rinsing the
slides in alkaline alcohol (add a pinch of lithium carbonate to the
alcohol wash) . The blue phase is very effective with carmine stains.
Feulgen's Nuclear Stain /
Feulgen's stain for chromatin involves hydrolysis of the material in
hydrochloric acid and staining in reduced basic fuchsin (Exercise 43)
The material may be mounted without a counterstain or may be used
with fast green or orange G.
Flemming's Triple Stain
Staining solution I . . . . . . .. 1 per cent aqueous safranin 0
Staining solution II ..... . . 1 per cent aqueous crystal violet
Staining solution III. ..... Orange G saturated in 95 per cent al-
cohol
The safranin 0 is applied first and is differentiated with 1 per cent hy-
drochloric acid in water until only the chromatin retains the dye. After
washing, the slides are stained with crystal violet until the spindles
are violet. The slides must be removed before the chromatin takes up
the dye. The material is dehydrated quickly to 95 per cent alcohol, and
then counterstained in orange G. The main problem encountered with
this technique is in retaining the crystal violet stain and considerable
experimentation may be necessary to obtain the desired results. D -
tails of the procedure are presented in Exercise 44.
Giemsa's Stain
Giemsa's stain is a special combination of eosin Y, azures and meth-
ylene blue chloride, and has a differential staining effect on blood and
other tissues. The stain is usually obtained in a stock solution which
is diluted with distilled water (1 part of stock to 10 parts of water'
or with a phosphate buffered solution of pH 6.4 to 7.2. The procedure
for staining is described in Exercise 22.
In dried blood smears the nuclei stain varying shades of dark blue
to deep purple. The cytoplasm of the erythrocytes should be a clear,
bright pink. The cytoplasm of the neutrophiles is a paler pink to bluish

pink i the cytoplasm of other leukocytes and the lymphocytes is light
blue. In the granular leukocytes (polymorphonuclear leukocytes) the
eosinophiles have large, round cytoplasmic granules which stain a bright
red to orange red; the basophiles have granules of variable size which
stain a purple to violet shade; the heterophiles in man (neutrophiles)
have numerous, small, lilac-red granules. The size, numbers, and stain-
ing reactions of the heterophilic granules vary with different species of
animals. In blood parasites the nuclei stain red and the cytoplasm
blue.
H eidenhain' s M allory-azan
Although Mallory-azan is one of the more difficult techniques it is
indispensable for the demonstration of cell types in pituitary and pan-
creatic tissues. The technique is similar to Mallory's triple stain, with
azocarmine G being utilized in place of acid fuchsin.
Staining solution I. ... .. 1 per cent azocarmine G in acetic acid (p.
83)
Staining solution II
Aniline blue ...................................... . 0 .5 gm.
Orange G . ............ . ...... . .. . . . ............... . 2.0 gm.
Oxalic acid ....... '" ...... . . . ....................... . 2.0 gm.
Distilled water ....................... . .............. . 100 cc.
Heidenhain's formula omits the oxalic acid and includes 8 cc. of glacial
acetic acid per 100 cc. of solution. Some workers add a few cubic centi-
meters of 5 per cent phosphotungstic acid to the second staining solu-
tion. The procedure for this staining technique is presented in Exercise
46.
Janus Green
Janus green is a basic dye that has specific affinities for mitochondria
in fresh, living tissue. The material may be submerged in a dilute
(1 : 10,000 to 1 : 30,000) solution of stain in physiological saline; or the
dye may be filmed on the slide as directed in Exercise 2, Section A. The
mitochondria should appear blue after 10 to 20 minutes of exposure to
the stain.
Mallory's Triple Stain
Staining solution I. ..... 0.2 per cent acid fuchsin in distilled water
Staining solution II
Aniline blue WS .............•.••••............... . . .. 0.5 gm.
Orange G .. . ....... . ............ .' ... .. .. ... ......... . 2.0 gm.
Oxalic acid . ..... . ................................ ... . 2.0 gm.
Distilled water ..... . ... . ............ . .......... : .... . 100 cc.
STAINS 7
Many procedures call for the inclusion of 1 gram of phosphomolybdto

acid in place of the oxalic acid in the second staining solution. In this
case the slides are transferred directly from the first stain into the
second stain. If 2 per cent phosphomolybdic (or phosphotungstio) acid
is used between the two stains, however, a greater control of the stain-
ing effect is usually obtained (Exercise 34). The phosphomolybdic acid
removes the acid fuchsin from the connective tissue and lightens it in
the cytoplasm without removing it from the chromatin. It al~o aids in
improving the aniline blue staining of the connective tissue and the
staining effect may be varied by the length of time the tissue is left
in the acid before staining.
Among·the various multiple stains, Mallory's triple usually produces
the best results with most tissues, even for the inexperienced technician.
The slides have a clarity and transparency which is often lacking with
some of the triple stains. It is useful for demonstrating various tissue
elements in complex structures and is not intended as a critical stain
for cytological work. The chromatin should be ~ deep pink or red,
the cytoplasm varying shades of pink, the connective tissue should be
blue and the blood cells orange. If blood elements are not an im-
portant part of the preparation it may be desirable to omit the orange
G from the second staining solution. Mallory'S triple stain is mo. t
readily employed with celloidin sections.
Cason' 8 Mallory
Phosphotungstic acid ........... '. .. .. . ......... . ....... . 1.0 gm .

Orange G . . ...... ....... .. : ...... . .... . ........ . .. . ... . 2.0 gm.
Aniline blue WS .......... . ...... ................... ... . 1.0 gm.
Acid fuchsin .... .. .......... . ................ .... ..... . 3.0 gm.
Distilled water ................ . ............... .. . . ..... . 200 cc.
Cason (Stain Technology, 1950, Vol. 25, p. 225-226) adds the acid and
the dyes to the distilled water, one by one, stirring to dissolve each as
it is added.
Cadon described this rapid technique for protozoa and paraffin sec-
tions. It has proved very useful for dried sperm and vaginal smears that
would not stain with Wright's or Giemsa's stains. The main disadvan-
tage of this technique is that when it is unsuccessful on certain sections
there is little possibility of varying the procedure to improve the re-
sults. As with the usual Mallory's triple stain, the results are improved
if the tissue is treated with phosphomolybdic acid before staining and
rinsed with phosphomolybdic acid after staining. Directions for staining
sperm and vaginal smears with this technique are given in Exercise 23.
88 GENERAL ZOOLOGICAL IUCROTECHNIQUES
Masson's Triple Stain

Staining solution I ........ 0.5 per cent hematoxylin in 5 per cent al-
cohol
Staining solution II . . . . . .. 0.2 per cent acid fuchsin in water
Staining solution III .. .. .. 0.2 per cent fast green in 95 per cent al-
cohol
This technique utilizes Heidenhain's iron hematoxylin as a primary
stain, employing ferric ammonium sulfate as a mordanting and differen-
tiating solution. The material is then stained in acid fuchsin and the
connective tissue is differentiated in phosphomolybdic or phosphotung-
stic acid. Finally the connective tissue is stained with fast green in 95
per cent alcohol (or with aniline blue in aqueous solution). The direc-
tions for this procedure are given in Exercise 45.
Methylene Blue Chloride
Methylene blue chloride is a basic dye used as a nuclear stain. It is
commonly employed in a 0.2 to 1 per cent aqueous solution with an eosin
Y counterstain (Exercise 42). The combination of eosin Y and methyl-
ene blue is the basis for both Wright's and Giesma's stains. In the aged
methylene blue solution which is generally recommended, some of the
dye is apparently converted to the azures which are constituents of
Giemsa's. The presence of several oxidation products gives the stain
a polychrome effect, so that it stains in various shades of blue, _purple
and lilac. The primary difficulty encountered with methylene blue stain-
ing is in retaining the dye during dehydration, and an abbreviated de-
hydration schedule may be necessary.
Methylene blue is also utilized as a bacterial stain, a vital dye for
nervous tissue, and as a pH indicator. It may be substituted for toluidine
blue as a cartilage stain (Exercise,)O) .
M ethyl Green
Methyl green is a basic dye derived from crystal violet. It is employed
as a fixing-staining solution for chromatin in living tissue (1 per cent
methyl green in 1 per cent aqueous acetic acid). This procedure is
described in Exercise 2, Section B .
Neutral Red
Neutral red is a slightly basic dye and a pH indicator. It is yellow in
basic solutions, red in slightly acid solutions. Its primary use in tech-
nique is as a vital or supravital dye. The dye may be applied to living
STAINS 89
tissue in dilutions of 1: 10,000 to 1: 100,000 in physiological saline solu-

tion, or it may be applied as a film (Exercise 1, Section A), with or with-
out Janus green. In general the stain has an affinity for cytoplasmic
granules and inclusions in the living cell, staining the nuclei only a the
cells die.
Orange G
Orange G is an acid dye which is employed with aniline blue in the
second staining solutions of Mallory's triple and Heidenhain's Mallory-
azan techniques. It is widely used as a general counterstain, especially
following iron hematoxylin. The dye may be introduced as a 2 or 3 per
cent aqueous solution or as a saturated solution in 95 per cent alcohol.
Picric Acid
Picric acid may be employed as a counterstain and is commonly used
following iron hematoxylin. When so employed, the counterstaining-
destaining procedures may be consolidated since the picric acid will dif-
ferentiate the hematoxylin. If the hematoxylin is differentiated before-
hand with iron alum or an acid, it should be left somewhat darker than
finally required. Other uses for picric acid include combinations with
acid fuchsin or crystal violet. The acid is introduced as a saturated solu-
tion in 95 per cent alcohol. It may be lightened or removed by washing
in alcohol.
Safranin 0
Safranin 0 is a basic dye which is usually employed as a primary stain
with a fast green counterstain (Exercise 37, Section B). As in the case
of acid fuchsin, safran in 0 stains certain cytoplasmic structures and
may be employed as a secondary stain with iron or alum hematoxylin,
with or without fast green. Its use in Flemming's stain has already been
mentioned. This stain is widely used on botanical material.
There are a number of different staining solutions, but a simple 1 to
1.5 per cent solution in distilled water is effective. The stain may be dif-
ferentiated in aqueous or alcoholic hydrochloric acid (1 per cent). It is
also removed by 0.5 per cent picric acid in 95 per cent alcohol. In either
case the acid should be removed after differentiation. If fast green is
used as a counterstain, the safranin should be left somewhat darker
than required since it is destained by the fast green. Certain procedures
call for differentiation of the stain in a 0.2 per cent solution of fast
gree~ in 95 per cent alcohol.
Sudan II
Sudan II is an acid dye which is soluble in fats and is used as a fat
"stain" (Exercise 40, Section B). It has a low solubility in alcohol
(about 0.4 per cent) and is introduced as a saturated solution in alcohol.
The dye is taken up as a solution in the fat. Sudan III and sudan IV have
similar effects and are applied in the same manner as sudan II.
Toluidine Blue 0
Toluidine blue 0 is a basic dye similar to methylene blue and it may
be substituted for the laUer in staining techniques. It is employed as It
nuclear stain in It 1 per cent solution in ·water and is differentiated in
95 per cent alcohol. This dye is also utilized as a cartilage stain in the
Van Wij he technique.
Van Wijhe's Stain
Van Wijhe's stain is a 0.1 per cent solution of toluidine blue 0 in 0.1
per cent hydrochloric acid in 70 per cent alcohol. It is employed as a
cartilage stain as directed in Exercise 20. The material is differentiated
in a weak solution of hydrochloric acid in 70 per cent alcohol until only
the cartilage is stained.
Wright's Stain
Wright's stain is a combination of oxidized methylene blue and eo:sin
Y and is similar in effect and use to Giemsa's stain. The material is first
treated with the concentrated staining solution, which serves to fix
the material; the concentrated stain is then diluted with distilled water
as directed in Exercise 22.
8
Stained
Whole
Mounts
Whole mounts may be prepared of a great variety of organisms which

are small enough to be mounted on a slide and thin enough to be studied
by transmitted light. It should be noted that in aodition to mount.s of
entire organisms, this "class" of preparations includes portions, such as
appendages or dissections of organ systems, of larger animals.
The carmine stains are usually preferred for thick whole mount prep-
arations, smce they have a greater transparency than other nuclear dyes.
The criterion of a good whole mount is the limitation of carmine to the
nuclei so that the organs are visible as areas of varying shades of red
depending upon the concentration of the nuclei. The more diffuse the
stain, the less sharply the organs will be defined. The primary problem
in preparing whole mounts is in distinguishing the proper degree of
staining. The material is often so thick that it is impossible to examine
it under high power during processing; both because of the opaqueness
of the material in water or alcohol, and because the thickness of the ob-
ject does not permit adequate clearance for the high-dry objective. It
becomes necessary, therefore, to judge the degree of staining or destain-
tng by the gross appearance of the material. This is a. matter of experi-
ence. As a rough guide, the carmine-stained object should present a. very
pale pink appearance in water or alcohol. This will become a brilliant
red upon clearing.
Very small, thin objects may be stained with hematoxylin, but if too
many layers of cells are present the density of the hematoxylin "lake"
91
92 GENERAL ZOOLOGICAL III1CROTECHNIQUFS
deposited in the chromatin will interfere with the transparency of the

preparation. Whole mounts of protozoa are usually stained with hema-
toxlyin and these may be processed in bulk as considered in this chapter,
or as smears (Chapter 10). In the case of unicellular organisms, minute
larvae, etc., the material may be stained as critically as thin sections of
larger organisms.
Counterstains do not usually improve large whole mounts except
when noncellular structures are present. Thus hydroids possessing a
noncellular sheath or perisarc, bryozoa, arthropods and similar forms
may be advantageously counterstained. In the case of unicellular forms
prepared as whole mounts, the counterstain is employed to demonstrate
the cytoplasm.
Exercise 15
Grenacher's Alum Carmine
1. Use the chick embryo fixed in Lavdowsky's fluid (Exercise 4) or
other embryos suitably fixed and washed. In processing the material the
embryo may be left in the original container until step 9, and the solu-
tions changed; or the embryo may be transferred through the solutions
in a series of flat dishes. The embryo should be lifted on a spatula or
stiff piece of paper and must be handled very carefully.
2. Hydrate the material. If stored in 70 per cent alcohol, process
through 50 and 30 per cent alcohol for 10 minutes in each change and into
water.
3. After 10 minutes, transfer to alum carmine and leave for about 30
minutes. Be sure the material is completely submerged in the stain.
4. Check the extent of the staining by transferring the embryo to
water and observing its gross appearance. When properly stained, it will
be a pale pink color. If necessary,. return the embryo to the stain.
5. When sufficiently stained, rinse well in distilled water to remove
all superficial stain.
6. Dehydrate through a graded series of alcohols (30, 50 and 70 per
cent) leaving for 10 to 20 minutes in each change. Store in 70 per cent
alcohol or complete.
7. If the border of the preparation is irregular, or the over-all size is
excessive for mounting, trim it before proceeding. It is usually easiest
to pick the embryo up on a sheet of paper and trim it, and the paper, with
a pair of sharp scissors. Be careful not to let the embryo dry out. When
returned to the solution, the paper will usually float away from t.he em-
bryo. (If dull scissors are used it may be difficult to free the embryo
from the paper.) With early stages (through 52 hours) the embryonic
STAINED WHOLE MOUNTS 93
area is usually trimmed just beyond the edge of the SinU8 tenninalis.
With later stages it may be necessary to trim closer to the embryo in
order to mount it within a standard sized cell.
8. Complete dehydration through 95 per cent alcohol and two change
of 100 per cent alcohol, leaving for about 20 minutes in each solution.
9. Pick the embryo up on a piece of stiff paper and tran fer it into a
dish of xylol, toluol or carbo I-xylol (see the Appendix). The embryo
should be perfectly .flattened when it is introduced into the dealcoholiz-
ing-clearing solution, since it will become hardened; if it is wrinkled or
folded at the time of transfer it will not be possible to flatten it. Within
about 10 minutes the material should be perfectly transparent or
"cleared." If there are opaque areas the embryo has not been propcrly
dehydrated and must be returned to 100 per cent alcohol or transferred
to carbol-xylol or beechwood creosote-xylol. Failure to obtain complete
dehydration and clearing is frequently due to placing the dealcoholizing
agent in a wet dish. Only a perfectly dry container should be used. As
soon as the embryo has cleared the stain will assume a brilliant red ap-
pearance.
10. Process the material through a second change of clearing solution
to insure complete removal of the alcohol. If carbol-xylol was lIsed for
initial dealcoholization, use xylol for the second change.
11. Attach a glass or aluminum mounting cell of appropriate size to
the center of a clean slide (fig. 7, E) with a layer of balsam. The cell
shou ld be slightly deeper than the embryo.
12. Place a drop of moderately thick balsam in the bottom of the
cciI. Carefully transfer the embryo from the clearing solution into the
cell, centering it within the cell wall. The long axis of the embryo should
be at right angles to the long axis of the slide. The embryo should be
placed in the cell dorsal-side-up with the ventral or yolky side next to
the slide surface. The initial placement of the embryo should be correct,
since it is now extremely fragile and almost impossible to arrange in the
balsam without shattering it completely. The embryo will be quite stiff
and the most successful procedure for transferring and placing it is as
follows . pick the embryo up on a piece of stiff paper moistened with
the clearing solution; let one edge of the embryo protrude slightly be-
yond the edge of the paper; bring this edge into contact with the balsam
at one side of the mounting cell and slowly withdraw the paper, leaving
the embryo upon the layer of balsam. Do not let the embryo dry. Imme-
diately cover the embryo with balsam.
13. Fill ~he cell with balsam. If numerous bubble. Are trapped in the
mountant, place a drop of xylol on the surface or warm the slide and
leave uncovered until the bubbles escape.
94 GENERAL ZOOLOGICAL KICROTECHNlQUES
14. Grasp a clean cover glass with a pair of forceps and dip one end
of the glass in xylol. Bring this moistened edge into contact with the
balsam at one side of the cell. Slowly lower the cover glass to the oppo-
site side. (If thin balsam is used for mounting it may be necessary to
lower the cover glass horizontally to avoid displacement of the ma-
terial. In this case bubbles may be trapped beneath it and these may
be removed by leaving the slide flat on a warmer or in an oven overnight
or longer.)
The preparation must be kept fiat until the balsam is completely
solidified. With thick balsam mounts this may require a very long
period. Drying may be hastened by heating in a convection oven. In
any case it is best to store thick balsam whole mounts fiat in trays or in
slide boxes kept on end, since the objects will gradually drift to one
side if the material is left on edge for long periods.
Exercise 16
Grenacher's Borax Carmine

The use of Grenacher's borax carmine is essentially the same for
large or small objects. The .best results are obtained if the material is
heavily overstained and then destained until the cytoplasm is free of the
dye. The larger the object, the longer the staining period required for
satisfactory results. With small objects the staining and destaining may
be checked under the microscope, but with larger forms it is necessary
to rely on the superficial appearance of the material until it has been
cleared. A fast green or other counterstain is recommended for forms
with a perisarc, exo keleton, tunic or similar noncellular structures,
but it should be avoided with densely parenchymatous forms . For class
work, hydroids or small arthropods, and flukes or tapeworms are satis-
factory for processing by this method.
1. Fix the material according to the recommendations given in Chap-
ter 5. Wash the fixed tissue as required by the type of fixative emFloyed.
2. Optional. Bleach pigmented material as described in Exercise 12.
3. Transfer the material into 50 per cent alcohol. If the objects are
in water, run them through 30 per cent alcohol for 10 to 30 minutes and
then into 50 per cent alcohol.
4. Stain in Grenacher's borax carmine. Small hydroids, flatworms,
tunicates, arthropods, etc., may be adequately stained in 30 to 90 min-
utes. Large larvae, flukes, tapeworms, annelids, etc., may require one to
severnl days for good staining results. If in doubt. stain for a long period.
The material may be left in the stain for as long as a week.
5. Remove from the stain and wash in 50 per cent alcohol to remove
the superficial stain.
6. Destain in 1 per cent hydrochloric acid in 70 per cent alcohol. With
small fonns the process of destaining may be ob erved under the micro-
scope. With animals possessing a noncellular sheath or exo keleton the
material should be destained until such areas are completely free of the
dye; destaining must be stopped, however, before the dye has been ex-
tracted from the nuclei. With large forms, stained for several days, de-
staining may require a day or more. Renew the destaining solution every
day.
7. Wash the material in fresh 70 per cent alcohol to remove the acid
and arrest the destaining process; 30 minutes will usually suffice.
8. Transfer to 95 per cent alcohol; leave small forms for 10 to 15 min-
utes, large forms for 30 to 60 minutes.
9. Optional. Counterstain in fast green in 95 per cent alcohol. Use 1 or
2 drops of a saturated solution of fast green in 10 cc. of 95 per cent al-
cohol. Leave the object in the stain for a few seconds, transfcr to 95 per
cent alcohol and examine. Noncellular areas should be pale grecn. If
necessary return tu the stain, remove to 95 per cent alcohol, examine,
and repeat until the required degree of staining is obtained. If staining
is very slow increase the concentration of the staining solution; if too
rapid dilute the stain further before proccssing additional material. A
light stain is usually preferable and in any case it sh ould be limited to
the noncellular layers.
10. Complete dehydration through two changes of 100 per cent alco-
hol. Leave small obj ects for lO to 15 minutes, larger organi ms for 30
to 60 minutes, in each change.
11. Dealcoholize and clear in xylol, toluol or carbol-xylol, using two
changes and the time intervals indicated for alcohol. The material
should be perfectly cleared. If opaque areas per ist, water i present in
the material and it must be returned to 100 per cent alcohol. Be sure to
put the dealcoholizing agents in perfectly dry containers.
When the material has cleared it may be critically cxamined for tain-
ing effect. If it was not destained enough, return it down through the
series of solutions to 70 per cent alc0hol and repeat the dcstaining proc-
ess. If the fast green is too light it may be increased by returning the
material down through the series of solutions to the stain. uch material
must then be reprocessed through the dehydration and dealcoholizing
solution.
12. Mount the material in Canada balsam. Place ome of the balsam
on the slide, introduce the material and immediately cover it with bal
sam before it has an opportunity to dry.
Small objects, such as hydroids, may be mounted without a cell, but

it is advisable to support the cover glass off of them with cover glass
chips or lengths of capillary tubing so that the material will not be
crushed (fig. 7, D) . Tapeworms, flukes and similarly flattened and rela-
tively tough material may be mounted without any cover glass support
provided the cover can be prevented from tipping. With this type of ma-
terial it is frequently helpful to place a small lead weight on the center
of the cover glass until the preparation has hardened. Very thick, ir-
reg;ular or delicate forms should be mounted in a cell, or the cover glass
should be supported by glass strips placed around the object (fig. 7, C).
In case of large forms it is desirable to mount the material in thick-
ened balsam (see the Appendix). In this case the objects should be in-
filtrated with balsam before they are mounted. This may be accom-
plished by placing the material in the usual 50 per cent solution and
permitting the solvent to evaporate off. Evaporation may be hastened
by heating on a warming table or in an oven. Protect the material from
dust. The slide, mounting balsam, and the balsam containing the ma-
terial are then warmed before mounting and upon cooling the prepara-
tion will be essentially solid. See Exercise 15 for further remarks regard-
ing balsam mounts.
Exercise 17
Whole Mounts of Minute Objects
Protozoa, small medusae, echinoderm and molluscan developmental
stages and similar materials may be handl ed as minute whole mounts.
These objects are transparent enough so that they may be stained as
critically as sections of larger organisms. Such material may be stained
by any of the usual methods. Progressive techniques are usually satis-
factory and are relatively simple iii procedure. It should be noted that
such material may be sectioned in paraffin (Exercise 26) , and upon
affixation to slides is easier to process through regressive staining tech-
niques than are free objects in vials.
When considerable quantities of material are available it may be
handled in small, narrow vials and processed by sucking off each solu-
tion and replacing it with the next one. The material must be permitted
to settle between each change The proce s may be hastened by. using
centrifuge tubes and settling the objects with gentle centrifugation; this
is particularly u eful with protozoa. Since it is not possible to com-
pletely remove each olution without the ri k of losing some of the
material, it is necessary to increase the number of washes and changes
of dehydrating and dealcoholizing solutions. If the material tends to
compact in the bottom of the container it is necessary to agitate it

gently upon the addition of each solution. This is particularly necessary
if centrifugation is employed.
When only a limited quantity of material is available it may be proc-
essed in a depression slide i the material being examined under a dis-
secting microscope while the solutions are carefully removed with a fine
tipped pipette. The material should be covered with a cover glass to
prevent evaporation of the solutions. If the material is to be left for
any period of time the cover glass should be sealed in place with a thin
film of vaseline.
1. Fix the material in 10 per cent formalin or other fixatives. Carry
out the necessary postfixation treatment. In the case of picric acid fix-
atives, be sure to remove all traces of picric acid from the material be-
fore proceeding.
2. If the material is in water proceed directly to step 3. If the material
was stored in 5 per cent formalin wash in several changes of distilled
water before staining. If the material was stored in 70 per cent alcohol,
hydrate through a graded series of alcohols (60, 50, 40, 30, 20, 10 per
cent) and into distilled water. Leave the material for 5 minutes in each
change, or as much longer as is required for the material to settle to the
bottom of the container.
3. Stain the material in one of the following :
Alum carmine. Stain in the usual solution of alum carmine, ex-
amining the material at lO-minute intervals to determine the correct
staining time. The stain must be replaced with distilled water in order
to observe the extent of the staining. If staining is too rapid the alum
carmine may be diluted with one or more parts of distilled water. The
chromatin should be red.
Harris' alum hemotoxylin. Add lor 2 drops of Harris' alum hema-
toxylin to 10 ce. of distilled water. Submerge the material in the stain
and leave for 24 hours. Rinse in distilled water and examine. If the ma-
terial is not sufficiently stained, return to a fresh staining solution. The
concentration of the hematoxylin and the length of the staining period
may be varied. The chromatin should be blue to purple.
4. Wash the material with several changes of distilled water to re-
move all superficial stain. Hematoxylin-stained material should be left
in tap water (or a weak soda solution) to bring out the bright blue color
of the dye; 5 to 15 minutes will suffice.
5. Dehydrate the material through a graded series of alcohols (10, 20,
30, 40, 50, 60, 70, 80 per cent) into 95 per cent alcohol. Leave for 5
minutes in each change, or as much longer as is required for the material
to settle to the bottom of the container.
98 GENERAL ZOOLOGICAL lollCROTECHNlQUES
6. Optional. Counterstain in a dilute solution of fast green in 95 per

cent alcohol. Use about 1 drop of a saturated solution of fast green to 10
cc. of 95 per cent alcohol. Cover the material with the stain and remove
it as soon as the material settles. Replace the stain with 95 per cent al-
cohol and examine the materilll under the microscope (place a sample
of the material in a shallow depression sl~de). The cytoplasm should be
a light green. If necessary return to the stain and repeat the process.
The concentration of the fast green may be varied to suit the require-
ments of the material. Although fast green is not usually employed in
combination with alum hematoxylin, it provides a very satisfactory
counterstain in whole mount preparations of this type. If preferred a
counterstain of eosin Y or orange G may be substituted, using a very
dilute solution in 95 per cent alcohol.
7. Wash the material through three changes of 95 per cent alcohol,
removmg each as soon as the material settles. All of the fast green should
be out of the solution or it may precipitate on the material when it is
placed in 100 per cent alcohol. Do not leave the material in 95 per cent
alcohol for long periods or the fast green may be extracted.
8. Complete dehydration through 3 changes of 100 per cent alcohol,
leaving 5 minutes in each change.
9. Dealcoholize and clear by replacing the 100 per cent alcohol with
three changes of xylol or toluol, leaving the material for about 5 minutes
in each change.
10. Transfer the material into a small syracuse watch glass filled with
thin (20 per cent) Canada balsam.
11. Permit the thin balsam to thicken by evaporation, leaving the
container open but protected from dust. The process may be hastened on
a warm plate (between 40 and 50° C.) or further speeded in a convection
oven. Permit the balsam to thicken to a point where it is essentially
solid when cool. -
12. Warm the thickened balsam containing the material, and with a
wire loop transfer a sample of the specimens to a clean cover glass on
a warming plate. If a single sample is to be mounted on the slide, place
the material in the nenter of the cover glass. If a mUltiple preparation
is to be made place the sample of the material off center and successive
samples in a counterclockwise direction around the cover glass. Thus,
in the case of echinoderm developmental stages, a single stage or a
mixture of variou stages may be mounted in the center; or uncleaved,
2, 4, 8, 16 and 32 celled stag~s might be mounted in normal sequence on
a single cover glass.
13. Place very small chips of cover glass or short lengths of capillary
tubing around the material to prevent the weight of the cover glass
from crushing the objects.
14. Place a drop of balsam on the center of a slide. Invert tht' slide
and bring the drop of balsam into contact with the cover glass (fig. 8,
C). D o not press down upon the cover glass but lower the slide just
enough to make sufficient contact so that the cover glass will be lifted
with the slide when the latter is withdrawn. Leave the slide inverted
until the balsam flows to the cover glass edge. It is advisable to use
round cover glasses for this mounting procedure.
15. Turn the slide right-side-up and store flat until dry. If bubbles are
trapped in the preparation leave the slide on the warmer at about 35° C.
until they escape. When the material is mounted in this way it' will re-
tain almost exactly the position in which it was originally placed on the
cover glass. If mounted on the slide in a quantity of balsam, minute ob-
jects tend to flow outward with the balsam and may escape from be-
neath the cover glass.
Exercise 18
Mesenteric Spread in Mallory's Triple Stain
The mesenteric spread prepared as directp.d in Exercise 5 may be
stained by anyone of a number of dyes, but is perhaps most effectively
processed in Mallory's triple stain. (Formalin-fixed material to be used
for the demonstration of fat should be processed as directed for frozen
sections in Exercise 40.) The results will be much more satisfactory if
the spread is left on the fixing tube. during staining and dehydration.
This produces a flat preparation which is otherwise difficult to obtain.
The disadvantage of leaving the material attached to the tube is that
the progress of staining and destaining cannot be checked under the
microscope. Staining must be based on a time schedule developed by
trial and error, with a rough check provided by the gross appearance
of the material. Fortunately the results obtained with this type of ma-
terial and this combination of stains are fairly uniform and the timing
given below will usuallly produce excellent results.
1. Fix the material in Bouin's fluid as directed in Exercise 5. Wash in
70 per cent alcohol until all of the picric acid has been removed ; removal
may be hastened by warming.
2. Hydrate the material through 50 and 30 per cent alcohols and into
distilled water, leaving for 5 minutes in each change. Transfer the ma-
terial on the fixing tube. At each change, tip the tube and pour out the
solution, then tip the tube in the subsequent solution so that the tube is
refilled. This procedure should be followed' throughout processing.
100 GENERAL ZOOLOGICAL MICROTECHNIQUFS
3. Place the spread in 0.2 per cent acid fuchsin (Mallory's I) and
leave for 5 minutes. When removed from the stain the tissue should ap-
pear generally red.
4. Transfer to 1 per cent phosphomolybdic acid and leave for 10
minutes. When removed from the acid the tissue will be a very pale pink,
except for fatty areas which will still be red.
5. Stain in aniline blue and orange G (Mallory's II) for 1 minute.
6. Rinse well in a fresh solution of 1 per cent phosphomolybdic acid.
The general appearance of the tissue should now be blue, unless large
areas of fat are present; these will be red.
7. Dehydrate through 50 and 70 per cent alcohol and into 95 per cent
alcohol, leaving about 2 minutes in each change. Some of the aniline
blue will wash out in the first two solutions.
8. Complete dehydration through two changes of 100 per cent alcohol,
leaving for 5 minutes in each.
9. Transfer the tube with attached mesentery to. a shallow dish of
xylol or toluol. Remove the rubber from the preparation and carefully
work the spread off of the tube. Do not let the preparation dry. Trim the
spread for mounting. Cut away the areas which were folded up over the
sides of the tube. The spread may be quickly trimmed by lifting it on
a strip of paper and cutting spread and paper with a pair of sharp scis-
sors.
10. Complete dealcoholization and clearing in a second change of
xylol or toluol.
11. Mount the spread as follows: place a small drop of balsam on the
center of a clean slide; lift the spread on a strip of paper; invert the
paper and place the spread on the balsam. Press firmly on the spread
area with a finger tip (do not move the paper) and then carefully lift
the paper from one end to the other_If the spread tends to stick to the
paper, catch one edge of it with a dissecting needle and hold it down
as the paper is removed. Immediately cover the spread with another
small drop of balsam and put the cover glass in place.
The connective tissue fibers should be blue, nuclei and fat cells red,
and blood cells orange.
Exercise 19
Alizarin R ed S for Bone
A number of stains are utilized for staining bone in :situ in small ani-
mals, young animals or portions (e.g. paws, tails, bacula) of larger
forms. Of these, alizarin red S seems the most widely used. The tech- .
nique consists of treating the tissue with a relatively weak solution oj
potassium hydroxide, which clears the muscle (renders it transparent)

and helps to limit the stain to the bone. After staining the material is
usually stored in glycerine, but small objects may be mounted on slides
in glycerine, glycerine jelly or in a resin.
1. Fix the material in 95 per cent alcohol. Dried material may also
be used. Large or hairy forms should be skinned.
2. Soak in 2 per cent potassium hydroxide until the bone is visible
through the muscle; this usually requires 24 hours or more.
3. Transfer to alizarin red S, 1 : 10,000 in 1 or 2 per cent pota sium
hydroxide. Leave in the solution until the bone is red. This usually re-
quires 24 hours or more. If the staining solution becomes c!)lorless before
the bone is adequately stained, replace with a fresh solution.
4. Transfer the material to 80 per cent alcohol. If the muscles have
picked up the stain, leave the material in 80 per cent alcohol until this
stain is removed.
5. Mount the material in glycerine (Exercise 9) or glycerine jelly
(Exercise 10) . The material may also be mounted in balsam by proc-
essing through 95 per cent alcohol and two changes of 100 per cent al-
cohol to complete dehydration; then dealcoholize and clear in two
changes of xylol or toluol. Leave for 30 to 90 minutes in each solution.
See Exercises 12 and 15 for remarks regarding balsam moun ting.
Note that unlike the corrosion techniques discussed in Exercise 13,
the flesh is not destroyed by the weaker potassium hydroxide solution
used here. When a number of bones are involved it is advantageous to
process the material intact to hold the separate bones in position.
Exercise 20
Toluidine Blue for Cartilage
Cartilagenous areas in embryos and larvae as well as in small mature
forms may be demonstrated in whole mounts by differential staining in
toluidine blue. Other cartilage stains include methylene blue, nile blue
and gentian violet. This procedure is known as Van Wijhe's technique.
1. Fix in 10 per cent neutral formalin . Wash in water after fixation .
2. Bleach pigmented material in 3 per cent hydrogen peroxide. Wash
in water after bleaching.
3. Dehydrate to 70 per cent alcohol, processing through 30 and 50
per cent alcohols for 10 to 30 minutes in each depending upon size.
4. Stain in 0.25 per cent toluidine blue in 1 per cent hydrochloric acid
in 70 per cent alcohol. Staining will usually require 24 hOUTS or more.
5. Wash in 70 per cent alcohol to remove the superficial stain.
102 GENERAL ZOOLOGICAL KICBOO'ECHNIQUES
6. Destain in 0.25 per cent hydrochloric acid in 70 per cent alcohol

until the blue stain has been removed from the muscle.
7. Wash in 70 or 80 per cent alcohol to stop destaining and remove the
acid.
8. Dehydrate through 95 per cent alcohol and two changes of 100
per cent alcohol, leaving for 30 minutes or more in each change.
9. Dealcoholize and clear in xylol or toluol; two changes for 30 min-
utes or more in each change.
10. Mount in Canada balsam. See Exercises 12 and 15 for remarks re-
garding balsam mounting.
9
Processing
Materials on
Slides
Whole mount materials, discussed in earlier chapters, are proces ed

through various -solutions in small containers. Smears and tissue sec-
tions, to be discussed in subsequent chapters, are processed attached
to slides, and special containers and procedures are necessary. This
chapter is concerned with the general techniques which apply equally
well to smears and to sections.
Cleaning Slides and Cover Glasses
Slides should always be washed before they are used, even when they
are new. This is particularly true of slides to be used for the prepara-
tion of smears and the attachment of sections. If the slides are not
clean the material will tend to peel off when they are processed. It is im-
possible to obtain an even smear, especially of blood, if the slides are
not scrupulously clean.
Slides may be washed in 70 per cent alcohol and used for the prepara-
tion of most smears (other than blood) and for other types of prepara-
tions. A good procedure is to place a number of slides, one by one, in a
jar of alcohol and leave them until they 'are needed. Just before use they
are removed and dried with a clean, lint-free cloth.
If the slides are so dirty that they do not clean well with alcohol,
or if they are to be used ' for blood smears, clean them first with an
abrasive glass cleaner. (Do not use glass cleaners which contain wax,
etc.) Rub the cleaner on the slides, let it dry, polish off the abrasive
103
and then place the slides in alcohol, to be removed and dried just before
use. This is satisfactory for most procedures. Keep in mind that the
slides will not be properly cleaned if the alcohol or towels are dirty.
Once the slide has been cleaned, handle it only by the edges, never by
the flat surfaces.
Occasionally new slides are so dirty that they must be cleaned with
a dichromate cleaning solution or with strong nitric or hydrochloric
acid. Such solutions must be handled with extreme care to avoid splash-
ing, contact with the skin, etc. The slides are placed in the solution, one
by one, and left over night. They should be thoroughly rinsed in running
tap water, placed in clean alcohol and dried before using. Such treat-
ment is generally required before reusing old slides. In general old slides
are not recommended for affixation of sections or for the preparation of
blood smears, since small scratches will interfere with proper attach-
ment and spreading. Such slides may be used for whole mounts or for
protozoa smears. Old slides of resin mounts should be soaked in xylol
before washing.
Cover glasses may also be cleaned by dipping in alcohol and wiping
dry. It is advisable to use a thin cloth (such as an old, clean, hand-
kerchief) for drying cover glasses since one is less apt to break them
than with a heavy cloth. If the cover glasses are soaked in alcohol be
sure to drop them in one by one or they may be difficult to separate.
Cover glasses may also be cleaned in dichromate or acid cleaning solil-
tions if required. Be sure you have an adequate supply of cover glasses
on hand before you begin removing slides from-the final clearing solution
for mounting. A convenient method of storing cover glasses is to stretch
a small spring over a cardboard in the bottom of a covered dish and
insert the cleaned cover glasses in an upright position in the slightly
extended spring. They may then be readily removed with your forceps
when you are ready to mount them on the slide.
Containers and General Procedures
When processing slides with materials attached, several points should
be kept in mind:
1. The material must not be permitted to dry out; be sure the solu-
tions are deep enough to cover the material on the slides.
2. The surface of the slide bearing the material must not oe allowed
to contact the surface of the jar, other slides, etc.
3. Every effort should be made to avoid excessive contamination of
the solutions. Racks of slides should be drained when changed from
one dish to another (although the slides must not be permitted to ~ry) .
PBOCESSING MATERIALS ON SLmES 105
Slides run in pairs, back-to-back, should be separated upon removal
from one solution and introduced one at a time into the next solution.
4. Containers should be correctly and clearly labeled on the jar-
not just on the lid.
5. Containers should be kept covered as much of the time as pos ible.
6. Containers should be set out in an orderly fashion in the sequence
in which they will be used.
7. Solutions should be changed whenever necessary, but excessive
replacement is costly and poor technique. Change water washes fre-
quently.
8. Solutions should always be placed in dry, clean containers.
Small numbers of slides may be processed through 32 by 90 mm. or
larger shell vials ; small jelly glasses with screw caps are also suitable.
The vials should be supported in some type of rack or wooden block,
since they are easily tipped. Rubber stoppers should be used with such
vials, or corks covered with aluminum foil. Rubber stoppers must not
be used for containers of dealcoholizing-deparaffinating solutions since
these will attack the rubber. Two ' slides, placed back to back with the
tissue out, may be processed through the vials at one time. As noted
earlier the slides should be separated between solutions. Slides may be
kept separated in such containers by placing a paper clip at the top of
each or alternate slides. This is practical only if the clips are kept above
the level of the solutions. The clips permit processing of larger numbers
of slides at one time.
Coplin jars which are grooved to hold five slides separated from one
another, are more convenient for processing slides than are vials. The
complement of slides may be increased to nine, with the extra four
angled between successive slides in the jar. This is to be preferred over
running ten slides back-to-back.
Since shell vials are relatively inexpensive, the student is often pro-
vided with a number of vials and a few Coplin j are. The latter should be
utilized at those points in the technique where the slides are to be stored
from one period to the next (as in 70 per cent alcohol) or where the
longest period of treatment is called for -(as in the stain, 100 per cent
alcohol, xylol, etc.).
Large groups of slides are most conveniently processed in glass or
metal racks which hold 10 to 19 slides. Such racks permit transfer of all
of the slides as a unit from solution to solution. Such'racks of slides may
be processed in rectangular staining jars, or in less expensive "refriger-
ator" dishes of appropriate size and provided with lids. Plastic dishes
are very useful for aqueous solutions and alcohols of 95 per cent or lcs .
106 GENEItAL ZOOLOGICAL )(JCBCY.l'ECHNIQUFS
Dried blood smears may be processed on cover glasses without special

equipment. It is not practical, however, to use cover glasses for wet
smears or sections unless you are equipped to handle them during
processing. Small "Columbia" jars or special racks of glass or porcelain
are almost prerequisite.
Mounting the Cover Glas8
With most whole mount preparations it is advisable to lower the cover
glass horizontally (fig. 8, A) to avoid displacement of the material.
When the object is attached to the slide, however, the cover glass is
lowered from one side to the other so that the mounting medium flows
ahead of it (fig. 8, B). The procedure must be completed rather rapidly
so that the material will not dry. It is important that only a minimum
of mountant remains between the slide and the cover glass. If the
amount is excessive it will have the same effect as a cover glass which
is too thick- it may not allow for adequate clearance and it may inter-
fere with the optical qualities of the preparation.
Balsam is rather slow drying and is not generally used for thin prepa-
rations. There are many synthetic resins. available on the market and
the one selected should fulfill the following requirements. It should be
neutral ; as colorless as possible; have a suitable refractive index ; dry
quickly; have enough resiliency when dry so that it does not shatter; and
flow easily ahead of the cover glass. This latter quality will vary some-
what with the thickness of the solution. With most of these resins it is
difficult to remove trapped bubbles (whereas they are readily removed
from balsam mounts by careful warming), and every effort must be
made to avoid trapping bubbles between cover glass and slide.
The following procedure should provide a well mounted, clean prepa-
ration:
1. Have on hand clean cover glasses, a bottle of mounting medium,
clean, dry forceps and a clean cloth.
2. Remove one slide fr.om the last solution (the second change of
dealcoholizing-clearing fluid) . Be sure you are holding the slide tissue-
or smear-side-up. If the slide has been scratched with an accession
number, the number should be on the same side of the slide as the
material so that you can detect the scratches with your finger nail (the
scratches are difficult to see when the slide is flooded with clearing
solution). If the slide is not scratched, hold the slide at about chin
level in front of a light source and tip the far side of the slide down
slightly. If the tissue is on the upper surface of the slide you can detect
an image or shadow of it on the lower surface of the slide. This may
PROCESSING MATERIALS ON SLIDES 107
take some practice but, once mastered, provides a quick and infallible
check of the position of the tissue. It is advisable to get in the habit of
checking every slide even if you are sure of the position of the tissue.
3. Without draining or wiping the slide, place a drop of mountant to
one side of the material at about the position that one end of the cover
glass will occupy when it is in place. .
4. Grasp a cover glass by one edge with a pair of forceps. Bring the
opposite end into contact with the drop of mountant j hold the cover
glass at an angle of about 45° with the mountant between the cover
and the slide. Hold the cover glass at an angle for a moment until
the drop of mountant has flowed out along the edge of the cover glass
that is resting on the slide. Lower the cover glass slowly on to the
preparation so that the mountant will flow ahead of the descending glass
without trapipng any air bubbles between the cover and slide. The
entire process must be carried out before the tissue can dry. If the
mountant is too thick, and does not flow smoothly over the slide, it
should be thinned. Alternately the edge of the cover glass which is
brought into contact with the drop of mountant may be dipped into
clean xylol before it is placed on the slide. Over dilution of the mountant
must be avoided or large air pockets will develop when the preparation
is dried.
If there is a possibility that excessive mountant was used, blow
straight down on the cover glass as soon as it is in position. This will
tend to drive excessive mountant out from under the cover glass and
on to the ends of the slide where it can be easily removed. If you attempt
to remove excessive mountant by pressing on the cover glass you may
damage the tissue or break the cover glass and the excessive mountant
will tend to climb on to the top of the cover glass where it is difficult to
remove.
5. With a clean cloth wipe the bottom and upper ends of the slide
clean, being careful not to displace the cover glass. If excessive mountant
is not readily removed, moisten the cloth slightly with xylol and wipe
the slide clean. Set the slide aside to harden, keeping it flat. Drying may
be hastened by warming.
If bubbles have been trapped in the preparation or if air pockets
develop when the preparation has dried, it is best to remount the ma-
terial. Place the slide in a container of xylol and leave until the cover
glass falls off of its own accord.
10
Smears
Smear preparations vary from whole mounts of minute organisms to

isolation preparations of cells from "fluid" and soft tissues. In all cases
the material is spread in a thin film on a slide or cover glass before
fixing and processing. Unlike other preparations, smears, especially
those of blood, are frequently prepared by drying the film. Wet smears,
however, must not be permitted to dry at any point during their prepa-
ration.
For purposes of discussion, smears have been grouped into three
general classes: protozoa or fecal smears; blood smears; and sperm,
vaginal or tissue smears. In each instance a different staining procedure
has been outlined. It should be kept in mind that the staining techniques
are more or less interchangeable and, in addition, most smears may be
processed by the staining procedures discussed for paraffin or celloidin
sections. .
Exer ci8e 2 1
Protozoa, Fecal, and Other Wet Sm ears in Iron Hematoxylin
1. Fix protozoa or fecal smears as directed in Exercise 3. Sperm or
tissue smears (Exercise 23) may also be processed as directed below.
2. Complete the necessary postfixation treatment, washing mercury-
fixed material in iodine-alcohol and picric acid-fixed material in 70 per
cent alcohol.
lOS
SMEARS 109
3. Hydrate the material by passing it through 50 and 30 per cent
alcohols and into distilled water. Leave about 3 to 5 minutes in each
solution.
4. Mordant in fresh 2 per cent ferric ammonium sulfate for 20 to 30
minutes.
5. Working with one slide at a time, wash the slides through three
changes of distilled water, agitating the slide while in each change and
leaving it 20 to 30 seconds in each. Replace the distilled water washes
as often as they become cloudy.
6. Collect the slides in 0.5 per cent hematoxylin, prepared from a
ripened stock solution of 10 per cent hematoxylin in 100 per cent alcohol.
Stain for 20 to 30 minutes or longer.
If the slides have not been properly washed a black, flocculent pre-
cipitate will form and settle to the bottom of the container, leaving a
yellowish green solution. If the material is overwashed, it will not pick
up the stain. A noticable darkening of the smear should occur in about
5 minutes. With repeated use, a fine black precipitate will gradually
develop in the solution. If the solution becomes yellowish green, it must
be replaced with fresh stain.
7. Transfer· the slides to distilled water and change the solution at
least once to remove the superficial stain from the slide.
8. Destain the material in 2 to 4 per cent iron alum. The stronger the
solution, the greater the rate of differentiation and the less control you
will have over the process.
Work with one slide at a time. Place the first slide in the destaining
solution for about 30 seconds, rinse well in a beaker of distilled water
and examine under the microscope. Protect the microscope stage with a
glass plate and make every effort to keep the objective dry. Should the
lens contact the fluid, wipe it at once with lens paper. Be sure the
microscope is set up for a color image, or it will be impossible to judge
the destaining. Do not let the slide dry.
If the nuclei appear as solid black masses the material is not suffi-
ciently destained. Return to the iron alum ' for another short period,
rinse and re-examine. When the material is properly differentiated it
will be possible to distinguish some detail of nuclear structure. The
nucleolus will usually be clearly visible and the chromatin will appear
as granular or threadlike structures, although in water the image will
not be sharply defined. The color imag~ of the chromatin should be blue
or black. When the material is over-destained the nuclear structure will
be visible as a brown or yellow color image be,cause of the presence of
the iron alum. Remember that the color of the stain is blue or black and
110. GENERAL ZOOLOGICAL MICROTECHNIQUES
that finishing the slide will sharpen the image but will not reveal
structure of nuclei which appear as solid black masses in water. If these
two points are kept in mind one can quickly learn to differentiate the
material properly with a little experience. In the case of preparations
of various types of protozoa, as in termite intestinal preparations, it is
necessary to concentrate on one type while destaining, since different
species will destain at different rates.
When you feel that the first slide is properly differentiated, wash it in
distilled water and then place it in running tap water. Proceed to differ-
entiate the second slide. The initial destaining period of the second
slide may be adjusted on the basis of the time required to destain the
first. The second, however, should not be left in the iron alum for the
total time required to destain the first. For example, if the first slide was
destained for ten, 30-second intervals, or a total of 5 minutes, the second
slide might be left for 3 minutes before initial observation. If left for
5 minutes it will probably be over-destained.
9. Wash the destained slides for 20 to 45 minutes (or longer) in run-
ning water. This insures complete removal of the iron alum, which will
otherwise impart a muddy brownish color to the finished preparation.
10. Dehydrate the slides through a graded series of aicohols (usually
30, 50, 70 and 95 per cent) and two changes of 100 per cent alcohol,
leaving for 3 minutes each in the lower alcohols and for 5 minutes each
in the absolute alcohols. ' If chunks of intestine still adhere to the slide
(in the case of termite protozoa smears) remove these during processing.
11. Dealcoholize and clear in xylol, using two changes for 5 minutes
each.
12. Working with one slide at a time, mount the cover glass on each
preparation, following the directions given in Chapter 9. Keep the slide
flat until it is dry.
Exercise 22
Blood Smears
Do not attempt to prepare blood smears until you have a supply of
slides carefully cleaned as directed in Chapter 9.
Special Problems and Procedures
Invertebrate blood. Since the corpuscles are usually relatively few
it is best to prepare thick rather than thin smears.
Amphibians. Becau&e of the large quantity of mucus encountered
when attempting to obtain blood from superficial sources, it is best to
8MEABS 111
obtain blood from the heart. Kill the animal with a sharp blow on the
head. Make a medial-ventral incision and expo e the heart. Mak a
small incision in the tip of the heart (in small forms snip off the end)
and collect the blood on the slide.
Rodents and Other Small Mammals. Blood may be obtained
by snipping a small portion from the end of the tail after first re training
or anesthetizing the animal.
Holding Blood Samples. When blood samples are required from ani-
mals killed in the field, it is sometimes difficult to make the smears
immediately, although this procedure is to be preferred. A sample of
blood may be collected from the heart of a recently killed animal and
introduced into a small vial which has been previously provided with
a drop of anticoagulant such as heparin. The sample is then kept cool
until it can be processed upon return to the laboratory.
A. Thin Smears
1. Wipe the area from which the blood is to be obtained with cotton
soaked in alcohol. Sterilize a needle or thin, sharp scapel blade and
puncture the skin. Wipe away the first blood which exudes. As soon as
another drop forms, touch a slide to the blood so that a small drop is
deposited toward one end of the slide surface. Grasp a second, clean
slide at one end, holding it by the long edges. Place the opposite end on
the surface of the first slide, between the blood drop and the slide c('nter,
holding it at an angle of 30 to 45° to the blood drop (fig. 9, A). Draw the
angled slide back toward the blood until the slide edge and blood contact
one another (fig. 9, B) . The blood will flow out laterally along the edge
of the angled slide. Immediately push the angled slide away from the
blood drop, movil!g it quickly and smoothly to the opposite end of the
flat slide (fig. 9, C). The blood will be drawn out behind it in a thin film.
The entire process must be carri~d out before the blood clots.
The angled slide should rest lightly. upon the surf ace of the flat slide;
if pressure is applied, the blood cells will be broken. If the film is too
thick (so that adjacent cells overlap one another) decrease the angle
and/or move the slide more rapidly. If the film is too thin, increase
the angle and/or move more slowly. The second smear may be made on
the surface of the slide used to spread the first, and so on, provided you
have not touched the surface of the slide with your fingers.
2. Wave the slide vigorously through the air so that the film dries
quickly. The slides may be stored before staining but should be pro-
tected from dust. The best staining results are obtained with freshly
prepared films.
I
A
~
,
C
Fla. 9. Preparing blood or spermatozoa ernears. A-C, method of spreading the
material on the surface of one slide with the edge of a second slide. D-E, method of
preparing smears on cover glasses. Corners 1-1 and 2-f are held in the finger tips of
opposite hands and the cover glasses are slipped apart in the plane of their surfaces.
3. Stain with Wright's or Giemsa's as described below. Results (p.

85) should be essentially the same, although the colors are usually more
sharply defined with Giemsa's.
Wright'a atain. Place the slides smear-side-up on a level rack over
a pan or sink. The rack may consist of two glass rods or wooden strips
spaced to support the ends of the slides. Flood each ' slide with several
drops of Wright's stain and let this act for 2 minutes. This fixes the
material. Alternately the material may be fixed in absolute methyl
alcohol. Add twice as much distiJIed water as there is stain on each slide,
being careful not to let it flood over the slide edge. The staining effect
may be varied by ' changing the amount of distilled water. Let the
diluted stain act for 5 minutes. Flood the slides with distilled water,
drain and dry.
Glem ••'. alain. Fix the slides in absolute methyl alcohol for 5 min-
utes. Remove, drain and dry. Place in Giemsa's stain which has been
diluted with distilled water or distilled water and a buffered solution,
using 1 cc. of stain to 50 cc. of diluent. If only a few slides are being
processed, the sta.in (which is very costly) should be conserved by 60od-
MEARS 113
ing the slides instead of submerging them. Use 1 drop of stain to 1 cc. of
diluent. Place the slides flat and flood with the solution. The slides may
be covered to reduce evaporation of the stain.
Stain for 20 to 30 minutes. Rinse a slide in di tilled water and ex-
amine. The nuclei should be varying shades of deep blue or purple. The
cytoplasm of the erythrocytes 'should be pink. If it is grey the stain is
not sufficiently acid. If the stain is light, return to the staining solution
and recheck about every 10 minutes. Rinse the slides in distilled water,
drain and dry.
4. Slides to be observed with an' immersion system may be left un-
covered and examined with the immersion oil on the slide surface. uch
slides are then dipped in xylol to remove the oil. (Do not wipe them or
the film will be removed!) If the smears are to be examined with a dry
lens system they should be covered with a thin film of mounting medium
and a cover glass. Unless the mountant is neutral, rapid and pro-
nounced discoloration of these stains will occur.
B. Thick Smears
When the purpose of the blood smear is not to observe the blood
cells but to examine the sample for parasites, a thick smear is indicated.
Thick preparations are also preferable when small numbers of cor-
puscles are present, as in many invertebrates.
1. Obtain the blood as directed in Section A. Place several drops of
blood in the center of a clean slide and spread the blood with a circular
motion with a glass rod. A combination slide may be prepared by
placing the blood toward onc end of the slide, preparing a thin film from
the edge of it as described in Section A , and then spreading the bulk of
the blood as described above. Air dry the smear.
2. Thick smears for the demonstration of parasites are hemolyzed
in distilled water for 15 minutes before fixing. In the case of combina-
tion slides, only the end bearing the thick smear is submerged in water.
Remove and dry.
3. Fix, stain and mount the material as described in Section A, using
either Wright's or Giemsa's stains. For staining parasites the staining
time in Giemsa's should be increased, especially if undulating mem-
branes are to be demonstrated.
C. Cover Gla88 Preparation,

Blood smears may be prepared on cover glaBse as well as on slides.
Thin smears, prepared as described below, tend to show a more uniform
distribution of leukocytes than do thin smears on slides.
114 GENERAL ZOOLOGICAL MICllOTECHNIQUES
Thin smeartl. Place a small drop of blood on the center of a clean,

square cover glass. Immediately drop a second cover glass on the first,
positioning it so that the corners protrude beyond the sides of the first
(fig. 9, D). The blood will spread out between the two covers which
are then slipped apart, pulling in opposite directions in the plane of their
surfaces (fig. 9, E). Air dry and process the smears as described in
Section A.
Thick Smears. Prepare as if on slides.
Exercl8e 23
Sperm, Vaginal and Tis8ue Smears
Cason's Mallory's triple stain (p. 87) has proved particularly
effective for processing dried sperm and vaginal smears. In many in-
stances preparations which proved unreceptive to the usually employed
Giemsa's or Wright's stains are easily processed by this method. Al-
though wet smears of sperm and vaginal epithelium are preferable when
any detail of structure is required, the dry smear is effective for the
demonstration of the presence or absence of spermatocytes and may be
readily prepared and handled in the field . Under laboratory conditions
it is better to prepare wet smears.
A . Preparation of Smears
Spermatozoa may be obtained directly from the testis, but in general
it is better to obtain them from the ducts or storage areas between
the testis and the external genital orifice. Some amphibians have an
enlarged mesonephric duct in which the sperm are stored, and this
provides a good source of material for smears. In many birds sperm
storage occurs in an enlargement of the vas deferens, near the cloacal
opening. In other birds and in reptiles the sperm are obtained from
the epididymis. In most cases the sperm may be readily obtained by ex-
posing and opening the indicated structure and transferring a drop of
the fluid contents to a clean slide. Spread the material with the edge
of a second slide as described for thin smears of blood (Exercise 22,
ection A). If the fluid is very thick it may be diluted with physio-
logical aline olution before spreading.
Soft tissues such as testis and epididymis may be smeared on a slide
by removing the organ, cutting across it with a sharp instrument and
drawing the cut surface over a clean slide. Other tissues frequently
used for smear preparations include spleen and bone marrow.
VaginaZ smears are obtained in one of several ways. The material
may be collected with a flattened glass rod of appropriate size and the
SMEABS 115
material spread over the slide with the rod. With l~ger animals a
pipette with a curved tip may be used to collect the material, and a
drop of the fluid is placed at one end of the slide and pread in the
manner described for blood smears. Thicker preparations may be ob-
tained by spreading the material with a needle or glass rod. Some
workers prefer to use a cotton swab which is slightly moistened with
saline solution, inserted into the vagina, gently rotated, withdrawn
and then rolled over the surface of a slide.
B. Fixation Methods
Air dry the material by waving the slide vigorously through the air.
Such preparations may then be fixed before staining by submerging
in absolute methyl alcohol for 5 minutes.
H eat fix by drying the film smear-side-up above a small flame, such
as that provided by an alcohol lamp.
Wet fix the material by submerging the preparation in a fixing fluid
as soon as it is spread and before it has dried. If the material is very
fluid it may be desirable to let it "dry down;" that is, to let the excess
fluid evaporate off before fixing, but it must not be permitted to dry.
Saturated aqueous mercuric chloride is frequently u ed for the fixation
of such smears. Other standard fixatives may be employed. Fixation
will occur very rapidly with this type of preparation and 5 to 10 minutes
will usually suffice. The material should then be given the necessary
postfixation treatment recommended for the fixative employed. Vaginal
smears are usually fixed in a 1: 1 mixture of 95 per cent alcohol and
ether. After several minutes in the fixative transfer the smears to 70
per cent alcohol where they are stored until processed.
C. Staining Dry Smears

Dry smears may be processed in Wright's or Giemsa's stains as de-
scribed for dried blood smears, or in Cason's Mallory'S triple stain, as
described here.
1. Place the smears in 2 per cent phosphomolybdic acid for 5 to 30
minutes.
2. Stain in Cason's Mallory's triple stain (p. 87) for 5 minutes.
3. Remove and rinse thoroughly in 2 per cent phosphomolybdic acid
and examine. If not yet stained, return to the staining solution.
4. Rinse very briefly in distilled water and allow to dry.
5. When thoroughly dry, add a drop of mounting medium and cover
the preparation with a cover glass.
D: Staining Wet Smears

Wet smears may be processed by any of the usual staining methods
described for sections, or in Cason's Mallory's triple stain as described
here.
1. Assuming the material has 'been fixed, washed, and is stored in
70 per cent alcohol, hydrate through 50 and 30 per cent alcohols and
into water, leaving for 3 to 5 minutes in each solution, If already in
water proceed directly to step 2.
2. Mordant in 2 per cent phosphomolybdic acid for 5 to 30 minutes.
3. Transfer directly into the staining solution (Cason's Mallory's
triple stain, p. 87) and stain for 5 minutes or more.
4. Rinse in phosphomolybdic acid and examine. If not stained enough,
return to the staining solution.
5. If properly stained, dehydrate rapidly though 50 and 70 per cent
alcohols, leaving for 30 seconds in each solution and gently agitating the
slide.
6. Complete dehydration in 95 per cent alcohol and two changes in
100 per cent alcohol, leaving for 5 minutes in each solution.
7. Dealcoholize and clear through two changes of xylol, leaving for
5 minutes in each change.
S. Remove the slides one at a time and mount the cover glasses as
directed in Chapter 9.
This 'procedure also gives excellent results with protozoa smears.
11
The
Paraffin
Method
1. Infiltra tion and Embedding
The subdivision of tissue blocks into thin sections is most frequently

accomplished by infiltrating the tissue with paraffin and sectioning
the resulting block on a rotary microtome. This method is relatively
rapid, provides adequate support for most tissues, and has thc added
advantage of yielding ribbons of sections which greatly facilitate the
preparation of serial sections. Certain tissues, such as liver and spleen,
tend to over-harden in the dealcoholizing reagents usually employed,
and these reagent · and the temperatures used may distort delicate
objects. Very large blocks are difficult to infiltrate adequately or to
section once they are infiltrated. Den e structures such as large masses
of cartilage and decalcified bone or teeth may not be supported suf-
ficiently in paraffin. In these cases celloidin embedding (Chapter 14)
is indicated , especially when serial sections are not required. When fats
are to be demonstrated the tissue must be processed as frozen sections
(Chapter 15) since these substances are removed by the reagents used in
both paraffin and celloidin techniques.
General Steps
Dehydration. Since paraffin is not miscible with water, the tissue
must be perfectly dehydrated before it can be infiltrated with paraffin
which must permeate the material, not just surround it. Dehydration is
usually accomplished by replacing the water in the tis ue with ethyl
alcohol. Dehydration should be carried out gradually to avoid distor-
117
tion of the tissue. Usually a series consisting of 30, 50, 70, 95 and two
changes of 100 per cent alcohols produces excellent results. If distortion
occurs, the series may be extended by introducing intermediate solu-
tions. Thus, 15, 30, 40, 50, 60, 70 and 80 per cent alcohols may be sub-
stituted before 95 per cent alcohol. The series can, of course, be further
extended if necessary. The average block of tissue which does not ex-
ceed 6 mm. in any dimension is adequately permeated after 20 to 30
minutes in each solution. Larger blocks of tissue must be left for pro-
portionally longer periods. In general it is best not to leave tissues over-
night in alcohols below 70 per cent, since they will tend to macerate,
or in 100 per cent alcohol, where they may over-harden and become
too brittle to section. If the block is to be stored overnight or longer
during dehydration, the schedule should be planned so that the tissue
is left in 70 per cent alcohol. If long storage is anticipated, 80 per cent
alcohol is to be preferred. Although ethyl alcohol is the standard solu-
tion for dehydration, methyl alcohol, iso-propyl alcohol or acetone may
be substituted.
Dealcoholization. Since alcohol is not miscible with paraffin it
must in turn be removed from the tissue and replaced with a solvent
which is miscible with both the alcohol and paraffin. Toluol, xylol and
chloroform are the most commonly used dealcoholizing agents. Toluol
is' to be preferred since it does not make the tissues as brittle as does
xylol, and i not as unpleasant to work with and has better penetrating
powers than chloroform. Dealcoholization is usually accomplished by
transferring the tissue from the second change of 100 per cent alcohol
into a 1: 1 mixture of 100 per cent alcohol and toluol. The tissue is
th n processed through two changes of pure toluol. The times. required
will approximate those for the dehydration solutions. For delicate
structures, such as embryos, it may be desirable to extend the series by
introducing 100 per cent alcohol and toluol mixtures of 2: 1, 1: 1 and
1: 2 before pure toluol. This series can be further extended if necessary.
The procedure is the same when using xylol or chloroform, although it
is advisable to double the times in each solution when using the latter.
Benzene and n-butyl alcohol are al 0 used as dealcohoJizing agents.
Dioxane may be substituted for both alcohol and toluol as described
in Exercise 25, Section A.
Material such as liver, spleen and yolky embryos, which become very
brittle in the standard dealcoholizing agents, may be processed through
terpineol or cedar oil. These are not recommended for general work since
they are difficult to remove from the material, but with extra changes
of infiltrating paraffin they impart excellent cutting qualities to many
PARAFFiN METHOD. I 119
difficult tissues. Such material may al 0 be proce ed in tertiary butyl

alcohol as outlined in Exercise 25, Section B.
Dealcoholization is frequently referred to a "clearing." Thi re ults
from the fact that many of the dealcoholizing agents have a high r -
fractive index and render the tissue transparent. Although this eff ct is
incidental, it does provide a method of determining the completeness of
dealcoholization. You will soon learn to recognize the tran lucent ap-
pearance which indicates proper processing. It i al 0 indicative of com-
plete dehydration, since areas which retain any trace of water will
have a white or opaque appearance even after long exposure to the de-
alcoholizing solution. Such tissues must be returned to 100 per cent
alcohol to complete dehydration.
Infiltration. Infiltration consists of replacing the dealcoholizing
agent with paraffin. The tissue is transferred into a solution of the de-
alcoholizing agent which has been saturated with paraffin. This mixture,
loosely covered, is placed in a paraffin oven at a temperature ju t above
the melting point of the paraffin. It is then left until the temperature
of the solution has been raised to that of the oven. The tissue is then
processed through two changes of pure paraffin, kept in the oven at
temperatures above the melting point of the paraffin. For the average
tissue block, 20 to 40 minutes in each change will suffice. Large block
must be processed for longer periods. If the material was dealcohoJized
in terpineol or another heavy oil, it shou ld be run through 5 or 6 change
(or more) of paraffin. The process may be hastened considerably by
transferring the tissue from the heavy oil into toluol-paraffin for initial
infiltration, although this may result in some hardening of the ti ue
block.
Paraffin
The embedding paraffins which are available from biological supply
houses give excellent results for most tissues and should be obtain d
if at all possible in preference to plain paraffins such as thosc designed
for canning. The paraffin can be obtained in various melting point
grades from about 4{)-42° C. to 64-66° C. Paraffin with a melting point
range of 56-58° C. is the most useful under average conditions for
sections cut between 5 and 15 microns. High melting point paraffins are
used for thinner sections or when cutting at warm room temperatur .
Low melting point paraffins are neces ary for very thick sections. Very
low melting point paraffins may be useful when it is desirable to avoid
high oven temperatures, but the blocks are very difficult to section un-
less a cold room is available. Avoid, whenever pos ible, the use of 60-
120 GENERAL ZOOLOGICAL MlCROTECHNlQUES
62 C. or higher melting point paraffins, since tissues are literally cooked

0
at these temperatures. If the oven is maintained at 58 0 C. or lower,

tissues can be left in the infiltrating paraffins for considerable periods
without over-hardening or distortion. This means that even very large
blocks can be infiltrated. If the oven is not reliable or the temperature
is maintained above 58 0 C., the tissue should be processed in the short-
est possible time. Thus, use 52-54 C. melting point paraffin for 16- to
0
25-micron sections when working at average room temperatures. Set

the oven at 55 to 58 C. Use 56-58 C. melting point paraffin for 5- to
0 0
15-micron sections when working at average room temperatures; set the

oven at 58 0 C. Use 60-62 0 C. melting point paraffin for 1- to 5-micron
sections when working at average room temperatures, set the oven at 62 0
C. and remove the material from the oven as quickly as possible. Use
lower melting point paraffins for low room temperatures or thicker sec-
tions; higher melting point paraffin for high room temperatures.
Melting and Handling Paraffin

There is a remarkable disregard for. the dangers of handling hot paraf-
fin. The following suggestions should help to avoid fire or explosion.
1. The safest way to melt paraffin is in a double boiler over a water
bath.
2. Never 'heat paraffin in a glass container such as a beaker. This is
especially dangerous if previously melted paraffin has solidified in a
beaker and is then remelted; such beakers will frequently break.
3. Metal cups with a pouring lip and a handle (such as ordinary
kitchen measuring cups) are convenient receptacles for paraffin. The
handle may be wrapped with cloth.
4. If you are melting paraffin and arc called away "for a few mo-
ments" always turn off the source. of heat just in case you are delayed.
Failure to observe this rule is probably the most common cause of
paraffin fires.
5. Should a paraffin fire start do not attempt to extinguish it with
water. Smother it with a CO 2 extinguisher, sand, or, if necessary, a
heavy wet cloth.
6. Whenever possible handle paraffin over a fireproof surface. Do
not cover table areas with newspaper when embedding. Instead, use
some fireproof material such as aluminum foil or a glass plate to protect
the working surface.
The paraffin may be melted in the paraffin oven at the temperatures
to be used for infiltration. This is a slow process and considerable time
(usually overnight) must be allowed. If the paraffin is melted before it
PARAFFIN METHOD. I 121
is placed in the oven it should only be heated until it is just melted; or,
if overheated, let it cool until a scum begins to form on the surface and
the sides of the container before placing it in the oven. If you place the
tissue in hot paraffin it will cook the tissue even if the oven temperatur
is below the critical point.
When paraffin chips from trimming blocks are reclaimed, or if there
is any sign of dirt in fresh embedding paraffin, the paraffin must be
filtered before use. Various heated funnels are available for this purpo .
The paraffin may be filtered by setting up a funnel with coarse filter
paper in the oven, over a container, heating the paraffin and pouring it
into the heated funnel and leaving it in the oven .
Infiltration and embedding are greatly simplified if the infiltrating
paraffins are placed in flat dishes (such as Stender dishes) or wide-
mouthed vials which are fitted into holes in a wooden block. The wooden
block will retain enough heat to prevent the paraffin from solidifying
during short periods outside the oven.
Paraffin Ovens
The best type of oven for paraffin infiltration is a convection oven,
which is warmed by a mO\'ing supply of hot air drivcn by a fan. The
temperature will be uniform throughout and the oven reheats almo t
immediately when the door is opened and closed. A water-jacketed oven
is also excellent, since again the temperature tends to be uniform and the
oven temperature will not drop rnuch when the door is opened and
closed. Temperature recovery is somewhat slower than in the convec-
tion oven. Both of these types are quite expensive and may not be
available in some laboratories.
Ovens heated by a coil at the bottom of the oven must be carefully
cheeked for temperature variation in different areas. The e ovens are
generally referred to as "gravity convection" ovens. Reheating of such
ovens is very slow and great car must be taken to keep the door closed
as much as possible. A fairly effective oven can be constructed in an
insulated box, provided with a thermostat, and heated with an electric
light hulb. These have the same disadvantages as gravity convection
ovens. Individual embedding, ovens arc readily constructed out of metal
bread boxes supplied with a light socket and low wattage bulb, which
trial shows will provide temperature adequate to just melt. the paraffin
being used. A very simple method of infiltration i to place a goose-
necked lamp over a pan of paraffin so that the paraffin is melted by the
heat of the lamp. The distance of thc light from the paraffin and the
wattage of the bulb should be adjusted to that there is a layer of solid
paraffin in the bottom of the paraffin container and a surface layer of

molten paraffin deep enough to cover the tissue. The light bulb should
not be in contact with the paraffin.
Exercise 24
Paraffin Processing of Tissue Blocks
1. Fixation. The tissues should be fixed by a method appropriate to
the material and the specific structures to be demonstrated (Chapters
4 and 5). Most of the fixatives give satisfactory results for paraffin
embedding, although formalin-fixed material may not be sufficiently
hardened and alcohol-fixed material may be over-hardened. Reminder:
a working-label should be prepared at the time the tissue is fixed and
this label is processed with the tissue.
2. Postfixation Treatment. After fixation the tissues should be
washed as required by the particular fixative used. These procedures
are dependent upon the fixative and independent of the tissue used or
later processing.
3. Initial Dehydration. Partial dehydration through 30, 50 and into
70 or 80 per cent alcohols is frequently included as a part of t he post-
fixation treatment and may proceed the actual processing of the tissue
into paraffin by a considerable period. In the case of picric acid- or
mercuric chloride-fixed materials, the tissue will be in 70 or 80 per cent
alcohol at the end of the postfixation treatment. Formalin- and bi-
chromate-fixed tissues should be washed and then processed as far as
70 or 80 per cent alcohol. Bichromate-fixed tissues may then be stored
or processed immediately. It is usually desirable to store form alin-fixed
tissue for a week or more in 80 per cent alcohol to furth er harden it
before processing into paraffin .
•. Prepare a series of solution s~including: 95 per cent alcohol ; 100
per cent alcohol I (first change) ; 100 per cent alcohol II ; 100 per cent
alcohol and toluol, 1: 1; toluol I (first change); toluol II ; and toluol
saturated with paraffin (at room temperature) . These solutions must
be placed in clean and perfectly dry containers, which should be ap-
propriately labeled. The size of the container should be such that the
tissues can be covered with a volume of solution ten or more times that
of the tissue. Do not use rubber stoppers for containers of dealcoholiz-
ing agents, since the solutions will attack the rubber. Screw-capped jars
or covered Stender dishes are convenient for processing the tissues.
5. Transfer the tissue 'from 70 or 80 per cent alcohol into 95 per cent
alcohol and leave for 20 to 60 minutes.
6. ptional. When objects are colorless it is desirable to stain them
PARAFFIN KETHOD. 1 123
slightly so that they will be visible in the paraffin. Thi can be ac-
complished by treating the tissue with 0.2 per cent eo in in 95 p r c nt
alcohol. Leave for 2 to 5 minutes, rin e in 95 p r cent alcohol and tran -
fer into 100 per cent alcohol.
7. Complete Dehydration. Process the tissue through two changes
of 100 per cent alcohol, leaving for 20 to 30 minutes in each. (Large
hlocks must be left for longer periods.) Transfer the blocks with clean
dry forceps. If the material is fragile, transfer it on a piece of stiff
paper or with a spatula. It is advisable to agitate each container at least
once about midway through treatment with each change of solution.
8. Dealcoholize. Process the tissue through toluol-alcohol and two
changes of toluol, leaving blocks of 6 mm. square or less for 20 to 30
minutes in each change.
9. Infiltration. Transfer the tissue from the second change of toluol
into toluol saturated with paraffin. The tissue may be left overnight
at room temperature at this point, or may be processed through the
melted paraffin and embedded. Loosen the cap of the container and
place it in the paraffin oven. Leave until the solution has been raised
to the temperature of the oven (20 minutes or more depending upon the
volume).
10. Have on hand a lighted alcohol lamp and clean dry forceps. Re-
move the toluol-paraffin and the infiltrating paraffin from the oven
(shut the oven door) . Warm the forceps in the flame of the lamp (do
not overheat them) and quickly transfer the object from toluol-paraf-
fin into the first change of paraffin and return them to the oven. Nate.
Keep the alcohol lamp away from the toluol-paraffin , which is highly
inflammable. If a number of blocks are being transferred and the
forceps are reheated , the toluol-paraffin on the forceps will ignite.
This will burn off harmlessly, but be sure it i out before putting the
forceps back into the toluol-paraffin I Toluol-paraffin should not be
stored in the oven, since it will contaminate the embedding paraffin.
1 J. Lea,'c the tissue in the first change of paraffin for 20 to 30 min-
utes. Remove from the oven, and with heated forceps transfer the
tissue into the second change of paraffin and return it to the oven a
quickly as possible. If the paraffin solidifies, the infiltration time should
not be counted until thc paraffin has mclted again. Leave for 20 to 30
minutes in the second change of paraffin.
12. Embedding. Before you embed your first tissue block, read
through the following discussion and that regarding trimming the
blocks, attaching them to the microtome peg and orienting the blocks
in the microtome. You will then have a clearer concept of the orienta-
124 GENERAL ZooLOGlCAL MICROTECHNIQUES
tion required during embedding and the size of the embedding container
that you should use.
I. Embedding Containers
a. Glass dishes may be used for embedding. One inch diameter
Stender dishes are suitable for average blocks. One inch diameter
syracuse watch glasses are excellent for very small tissue blocks. Petri
dishes can be used for multiple embedding of a number of blocks. In
any case the glass containers used for embedding must be somewhat
wider at the top than at the bottom so that the paraffin can pop out
when it is cool. The glass should be coated with a thin layer of glycerine
before embedding so that the paraffin can be readily removed.
b. Metal "L's" placed on a metal base may be adjusted to accommo-
date various sizes of blocks and are available in different heights and
lengths from biological supply houses.
c. Paper "boats" make excellent embedding containers and are some-
what more favorable for cooling the paraffin mass than are thick
walled glass containers. They can be made in any size required by the
block, but in general should never be more than twice as long as they
are high. The boats can be prepared from stiff paper, heavy aluminum
foil or the foil-backed paper that is used in packaging cigarettes.
Some workers prefer to use graph paper, since the squares facilitate
orientation of the block in the paraffin. The foil-backed paper is very
useful since it is stiff enough to produce a sturdy boat; the foil (placed
inside) can be readily separated from the paraffin mass, and the paper
(placed outside) can be labeled directly with India ink. This avoids
the difficulties frequ ently encountered when labels are inserted into
the paraffin mass and the label is essentially inseparable from the ma-
terial. With plain foil, or the paper-backed foil, the label may be im-
pressed into the end flap with a sharp, hard pencil. Plain paper should
be coated with glycerine beforc the boat is filled with paraffin.
The boats are most readily prepared by folding a strip of paper
around a wooden block of appropriate size. The paper is cut so that
it is longer than wide. The length of the paper should equal the length
of the boat plus twice the height, plus 8 to 10 mm. for the end flaps.
The width should equal the width of the boat plus twice the height.
Thus for a boat 22 by 11 mm. and 22 mm. high, the paper should be
33 by 52 mm. Place the wooden block in the center of the paper (foil
side up in the case of paper-backed foil as shown in figure 10, A).
Fold the sides of the paper up, creasing the bottom edges sharply
against the form . Fold one end up in the same way (fig. 10, B) and
PARAFFIN METHOD. J 125
A B
----'~/--____1~.~' ~
_ llIllIlll
r- n
·r-4
~----____.
c o
'fIIIIllWllii ~
:rI:r ~15
E F
FlO . 10. Preparation of a papcr hoat for embeddinp:. If papel'-uockrd foil i~ ul i-
Iized. the foil surface (indicated here by lines) should b on the in ide of the boat .
The form is placed in the center of the paper (A) and the pap r is creased sharply
against the long edges of the form at 1. One end is then creased sharpl~' againsl th
end of the (orm as at t in B. One comer is then pinched together Ilnd {oldc'd hack
over the end of the block as at S in C. This is repeated at the opposite ~orner. fold-
ing the paper as at 4 in D ; and the end is locked in place by folding the nd Allp
down as at 5 in E. The process is then repeated at the opposi te end nnd fh e finish d
boat (F) is removed from the form .
pinch each of the corners together. Fold the corners back over th
ends of the block (fig. 10, C and D) and fold the end fl ap down to lock
the boat together (fig. 10, E). Repeat at the opposite end. The label
may now be written on the outside of the boat before it is withdrawn
from the block. Use waterproof ink. With some practice boats may b
folded freehand by creasing the sides, ends and corners, in that order,
and folding the corners back over the ends and the ends down as if
on a form.
II. Orientation of the Block in the Paraffin

The tissue must be placed in the paraffin so that its position is known
and it is surrounded by enough paraffin to permit trimming the block
and attaching it to a microtome peg. The tissue must, of course, be
oriented so that the desired plane of section may be obtained later.
When orienting the tissue in the embedding paraffin the following re-
quirements must be met.
a. The side to be attached to the peg must have enough paraffin
(at least 4 mm.) so that some of it may be melted down to form a firm at-
tachment to the microtome peg.
b. The face of the block (which will be sectioned first) should have
at least 1 mm. of paraffin between it and the surface.
c. The sides of the block should be surrounded by 2 or 3 mm. of paraf-
fin so that when the block is trimmed square, at least 1 mm. can be
left on each side.
d. Extra paraffin (4 to 5 mm.) must be left toward the upper surface
of the block, since some depression of the surface occurs when the block
is cooled. The larger the paraffin mass, the deeper this depression may
become.
e. Within certain limits all dimensions should be kept as small as
possible to facilitate cooling of the block. Small tissue blocks should
be embedded in boats which are essentially square. In general it is
best to put the face of the block toward the bottom of the boat. The
upper paraffin mass will then be attached to the microtome peg. (fig.
11, G). Long blocks, however, must be embedded flat in long boats
which should be at least one-half as high as they are long (fig. 11, A-C).
In figure 11, A-C, showing the orientation of salamander larvae for
different planes of sections, note that all are embedded in boats of the
same size and that the orientation of the tissues within the boats ful-
fills the requirements outlined abo·ve.
III. Embedding Procedure
a. Have the embedding dishes or boats prepared. Label the paper
boats on the outside with India ink. The label may be impressed into
the end flap of aluminum foil boats with a sharp, hard pencil. If metal
"L's" or glass dishes are used for embedding, prepare a small label
on a strip of bond paper, dip it in hot paraffin and place it in the dish
of paraffin with the tissue. If the working-label is small enough it
may be used for the embedding label. Coat the inside of paper boats or
gla s dishes with glycerine.
b. Have avai lable a supply of melted embedding paraffin which is
E
B I~ I
c I ;~I F
o H
FIG. 11. Orientation of tissue blocks in the paraffin mass. A, Band C show th po-
sitioning of three uniform blocks in boats of the same size to provide transv rse
(A ), sagittal (B) and frontal (C) sections. The views to the left are from above ; the
views to the right are from the side . Figures D - H show vario-.m ethods of mul-
tiple embedding. In D a series of paper boats is lined up on a sliFe or glass plate to
facilitate embedding of several tissues in separate boats at one time. In E a series
of small objects (such as root tips) is embedded in a row in a single boat 80 that
they may be sectioned in sequence after trimming, and attaching to the peg as in-
djcated in figure F . In G a series of tissue blocks from one animal has been em-
bedded for simultaneous sectioning (see discussion in text). In H a series of blocks
from one animal is embedded in a single block for later separation and individual
sectioning.
free of impurities (dirt, oot, etc.). Filter the paraffin if necessary.

If a large number of blocks is to be embedded at one time it is advisable
to have several containers of embedding paraffin 0 that when one
begins to solidify, another is available. Th e embedding paraffin should
not be more than a few degrees above melting point. Very hot paraffin
will cook the tissue and excessive shrinkage of the block will occur if
the container is filled with very hot paraffin. If fairly large embedding
containers are u cd, the paraffin should be in some container from
which it can be easily poured. If small boats are to be filled, have on
hand several clean dry pipettes with which the paraffin can be trans-
ferred into the dishes.
c. Prepare a dish or pan of cold water, using about one ice cube for
every two cups of water.
d. Have a lighted alcohol lamp and clean, dry forceps ready for use.
e. Remove the embedding paraffin from the oven and fill the em-
bedding dish. If a pipette is used to transfer the paraffin, warm it in
the lamp flame. The dish should be as full as possible. Paper boats
should be placed on a small glass plate, such as a slide, to facilitate
handling.
f. Insert the label against one side of the container. The writing
should be placed outward so that it can be read through the paraffin. If
the label is completely submerged it is less apt to cause embedding
difficulties. Alternately, the label may be placed in the paraffin by one
end with the writing exposed above the paraffin surface.
g. Permit a layer of paraffin to solidify on the hottom of the con-
tainer, keeping the surface melted by touching it occasionally with
heated forceps . The process may be hastened by touching the bottom of
the container to cool water.
h. Remove the tissue in the infiltrating paraffin from the oven. Warm
the forceps, quickly melt off any surface film from the dish of em-
bedding paraffin and immediately transfer the tissue from the infiltrat-
ing paraffin into the embedding boat.
i. Reheat the forceps and orient the tissue in the paraffin, carefully
removing any ~ bubbles which may be trapped in the paraffin.
j. Carefully blow on the paraffin surface. If any bubbles appear in
the surface film, melt them out with heated forceps and again cool.
Transfer the embedding dish (or paper boat on the slide) to the cool
water and lower it until just the surface is above water. Let a film
of wat r flow over the surface to thicken the upper layer of paraffin.
Then let the glass or metal blocks sink to the bottom of the pan. Paper
boats may be inverted.
Leave the blocks in water for 15 to 60 minutes depending upon size.
The paraffin block will usually pop out of glass or metal containers ..
If nece sary work them loose carefully around the edges. Blocks em-
PARAFFIN METHOD. 1 129
bedded in paper boats are left in the boats until they are to be mounted
on the microtome peg.
The tissue may be stored in paraffin indefinitely or may be pro d
as soon as the block is completely cool. It is best to store block in
a refrigerator or other cool place. torage of blocks in paraffin i the
best procedure when tissues are to be held for long periods befor
staining. The staining properties of tissues are retained better in par-
affin than in alcohol.
IV. Multiple Embedding

After some experience with embedding, considerable time can be
saved by embedding several blocks at once. They may be embedded in
separate small dishes or a number of blocks may be placed in one larger
dish. If paper boats are used, lin e up 2 to 5 on a slide (fig. 11 , D); fill
them all with embedding paraffin; place a block of tissue in each ; orient
the blocks, and cool them as a unit, transferring them on a slide to
the cool water. The number of blocks which can be processed at one
time will depend upon the speed with which you can work. If severa l
tissues are placed in one dish, they must be carefully arranged to permit
later separation while retaining proper orientation of the ti ue and
adequate paraffin around each block to permit separation , trimming,
and attachment to the peg (fig. 11 , H).
Occasionally it is practical to embed a group of tissues in a single
block and section them together. This is very useful when severnl
small tissues are being processed from each of a large series of animal!'.
Careful orientation is necessary so that the required section will be
obtained simultaneously from eaeh tissue. In thi type of mbedding
the distance between the tissue blocks shou ld be minimized (fig. 11 , (,')
so that the composite block which is sectioned will be as mall AS
possible.
V. Embedding Difficulties
Most embedding problems are apparent when the block i ' ca t 01'
when it is trimmed; these will be con idered here. A few will not be
apparent until the material is sectioned, and these are di 'cu ed 1st r.
In most instances the means of avoiding the difficulty will be appar nt
in the discussion of the cause.
When embedding faults occur the block sMuld be re-embedd ed as
follows. Trim the paraffin down close to the t issue and place it in fresh
infiltrating paraffin in the oven. Leave the tissue in the paraffin for 20
minutes after all of the superficial paraffin i completely melted and

then re-embed. Do not attempt to section imperfect blocks!
a. White areas appear in the paraffin, which should have a uniform,
semitransparent appearance. This may be due to slow cooling of the
block which results in coarse crystallization of the paraffin mass. Most
commonly, however, such white areas indicate that the embedding
paraffin is contaminated with the dealcoholizing agent. If a quantity
of the dealcoholizing agent is present" it may be possible to detect its
odor and the hloek lIIay he 'oft or ('\'('n cl'1I1l1hle. Such material should
be run through two changes of fresh paraffin before re-embedding.
b. The block fractures. This is not usually a complete break, but a
distinct fracture line is visible in the paraffin. If the fracture line is
at right angles to the upper and lower surfaces of the block, the block
was cooled too rapidly. If the paraffin was poured into the embedding
dish in two layers, a fracture line will occur in the same plane as the
top and bottom of the block. The dish should be filled with paraffin and
then the bottom layer hardened while the top i · kept fluid with heated
forceps. Never pour a bottom layer, harden it, and then introduce a
second layer. Extra paraffin may be added to the embedding dishes only
while the upper layer of paraffin is still melted.
c. The block surface is raised into a sharp peak. This is usually the
result of dropping the embedding dish too suddenly into the water and
before an adequate surface film was formed. Usually water has poured
into the center of the paraffin.
d. A depression in the top center of the block extends all the way to
the tissue. If the depression is shallow but still touches the tissue, the
boat was not deep enough or the tissue was embedded too high in the
paraffin mass. If the depression is very deep and extends to the tissue
several millimeters below the surface, the paraffin used for embedding
was probably too hot so that exce; sive contraction occurred on cooling.
Contraction of the upper surfaces can be minimized in paper boats if
these are inverted for final cooling. This tends to equalize contraction
to all sides.
e. Water in the block. Refer to c, above. Water may invade through
weak areas in the surface film , such as those produced by small bubbles
in the paraffin. These bubbles should be removed with heated forceps
and the paraffin surface re-cooled before the block is submerged. Labels
which protrude through the paraffin surface will often produce weak
points through which water invades the block.
f . Dry air space in the center of the block. This usually results when
the water is so cold that the paraffin walls harden before sufficient con-
traction occurs at the surface, and the paraffin contracts away from the
center. This is most apt to happen when very cold water i combined
with very hot embedding paraffin.
13. Trimming the block. The paraffin block mu t be carefully
trimmed before it is attached to the microtome peg. The following points
houJd be kept in mind:
a. The block should be at room temperature when it is trimmed, or it
may fracture.
b. Do not remove more than 1 mm. of paraffin with a single cut, or
the paraffin may fracture.
c. Whenever possible the sides of the paraffin block should correspond
to the sides of the tissue block. Thus, in the case of the larva used in
the illustrations (fig. 12, C) the sides of the block correspond to the
right, left, dorsal and ventral sides of the larva. In the case of a square
block of excised tissue the paraffin should ht, tl"imtn'd to correspond
to the shape of the tissue. If the tis ue block is irr gular, however, the
paraffin is trimmed into a square or rectangle, irrespective of the shape
of the tissue (fig. 12, A) .
CORRECT A
B
CORRECT OFF TILTED WEDGED POORLY EXCESS

CENTER TRIMMED WAX
C
FlO. 12. Trimming the paraffin block . The face (and sides) of the paraffin block
should be square, even though the tissue is irregular in shape (A) . B hows the
method of separating tissues embedded in a. mass of paraffin by scratching out
a. line from between the blocks with the back of a thin scalpel blade. C shows the
faces of a series of blocks; one is correctly trimmed so that the block is square and
the sides of the block correspond to the sides of the animal; the others show
various errors in trimming the block.
d. Opposite sides of the paraffin block should be parallel to one

another and at right angles to adjacent sides.
e. When the tissue tapers along its length (as in the case of the larva
used in the illustrations) the paraffin block should not be tapered but
should be maintained at the same width from front to base.
f. About 1 mm. of paraffin should be left on the face of the block.
g. Enough paraffin must be left on the base of the block so that some
of it can be melted down when the block is attached to the microtome
peg; about 3 to 4 mm. will suffice.
h. Trim the block with a clean, sharp !'ingle-:edged razor blade. The
paraffin should be removed with straight cuts so that the sides are
smooth . If the block is whittled down the irregular 'urfaces produced
may not adhere to form a ribbon when the mat~rial is sectioned.
i. When a number of tissues have been embedded in a single block
they must first be separated. Scratch out a thin trough of paraffin from
between the tissues with the back of a thin seapcl blade (fig. 12, B).
Attempting to cut between the tissu~s with a razor will usually result
in a fractured block.
j . The best sectioning results are usually obtained if an equal amount
of paraffin is left beyond the tissue on opposite side. of the block.
Except in the case of material to be cut in serial H'ctions, this outer
layer may be about 2 mm. and should not be less than 1 mm. Very
long blocks (20 mm. or more) should be left fairly thick, so that the
block will be rigid enough to with tand sectioning without bending or
vibrating.
k. Whenever possible it is desirable to trim thc block so that the
face is slightly rectangular rather than square.
14. Attaching the Ti88ue Block to the Microtome Peg. The paraffin
block must b' attached to a base or peg which can be clamped into
the microtome. •
I. The Microtome Peg

a. Metal pegs are provided with most microtomes but are difficult to
work with and are recommended only when very large blocks are being
processed. The advantage of these metal pegs is that they provide a
large base with a small neck which may be inserted into the microtome
peg clamp of the microtome. In general the surface of the peg should
be as large as, or larger than, the diameter of the paraffin block. When
such metal pegs are used the surface must be thoroughly cleaned and
then coated with a layer of hot paraffin which will flow into the sur-
face grooves and provide a base to which the tis ue block may be at-
tached. The block is attached a described below.
b. Wooden pegs can be easily prepared from dowling, and provide
an excellent base on which to mount the paraffin blocks. Round p gs
are desirable since no special placement of the paraffin block is nec-
essary on these. If square blocks are used the paraffin block must be
placed on the peg so that the block and peg sides coincide.
Dowling of appropriate diameter is cut into 40 to 50 mm. lengths
and placed in a container of melted paraffin in an oven. The pegs are
thus infiltrated with paraffin. Only one such treatment is usually nec-
essary and the pegs may be used indefinitely. With small pegs (up to
12 mm in diameter) no grooving of the peg surface is necessary to
obtain a firm attachment of block to peg. With larger peg it is de-
sirable to groove or cross groove the upper end to increas the strength
of attachment. The diameter of the peg which can be u 'cd is limited
by the size of the peg clamp on the microtome.
II . Mounting Procedure
Place the pegs in a holder (such as a drilled wooden block) 0 that
they are held upright and both hands are freed for work. Wooden pegs
may be stuck down to a glass plate with a mall amount of melted
paraffin. Place a chip of paraffin on top of the wooden peg and with
a hot copper spatula, melt the chip down into the peg surface (fig. 13, A) .
It is not necessary, nor even desirable, to have a mound of paraffin on
the peg surface. The metal peg is coated with hot paraffin as m ntioned
earlier. Reheat the spatula and place it on top of the peg. J mmediately
place the bottom of the paraffin block on top of the hot spatula so that
some of the paraffin on the bottom of the block is melted . Imm diately
withdraw the spatula and gently but firmly press the paraffin block
down against the peg. Excessive pressure must be avoided 0 that the
block is not broken or badly squashed at the top. Apply the pressure
with your forefinger placed on top of the block. While the pressure
is being applied, check that the paraffin block is perfectly perpendicular
to the peg (fig. 13, A). Slight tipping can be corrected with g ntle
directional pressure applied from the top as soon as the block is mounted
on the peg.
15. Label the Preparation. Attach the label to the face of the metal
peg or the side of the wooden peg with paraffin and a heated spatula.
It is best to cover the label with a piece of cellophane tape which should
completely encircle the peg and label. The tape alone i not ad ·quat ,
----- PEG-------
TAPERED TILTED EXCESSIVE BASE SMALL PEG
B
FIG . 13. Attaching the paraffin block to the mi crotome peg. A shows the sequence
of attaching the block. B shows variolls errors in trimming and attaching the block .
since it might be lost if the block is soaked in water before sectioning.

The blocks may be stored for an indefinite period before sectioning.
Exercise 25
Alternate Paraffin Methods
A. Dioxane
The ideal situation for processing tissue through paraffin is to de-
hydrate with some solvent which is directly miscible with both water
and paraffin. Dioxane fulfills this requirement but its use should be
limited since it is extremely toxic. It cannot be recommended .for class
work and should not be used by persons who are continuously or re-
peatedly engaged in technique work, since it is accumulative. It is
extremely useful for occasional projects but should always be used
with maximal ventilation, preferably under a hood.
1. Fix the tissues (Chapter 5).
2. (a) Wash formalin-, bichromate-, etc., fixed tissues in running
water and transfer into 50 per cent dioxane (dioxane and distilled water,
1: 1). (b) Picric acid-fixed tissues are transferred from the fixative
into 50 per cent dioxane and washed in several changes over a 24-hour
period. (c) Tissues fixed in aqueous mercuric chloride fluid hould be
washed in running water, dehydrated through the lower ethyl alcohol
series into iodine-alcohol and then into fresh 70 per cent alcohol (p. 35).
(d) Materials which are stored in 70 per cent or 80 per cent alcohol are
ready for step 3.
3. Complete dehydration through 2 or 3 changes of pure dioxane over
a 24- to 48-hour period.
4. Transfer to dioxane saturated with paraffin and place in a paraffin
oven.
5. When the temperature of the dioxane-paraffin has been raised to
that of the oven, transfer the tissue into infiltrating paraffin and com-
plete processing as directed in Exercise 24, beginning with step 9.
It is advisable to use three changes of infiltrating paraffin.
B. Tertiary Butyl Alcohol

Tertiary butyl alcohol is extremely useful for preparing plant tissues
and fragile or yolky animal tissues for paraffin infiltration and em-
bedding. It does not harden the material to the degree produced by
100 per cent ethyl alcohol-tuluol processing. In effect the tertiary butyl
alcohol (TBA) is substituted for 100 per cent ethyl alcohol and toluol
in the process outlined in Exercise 24. The procedure is outlined below;
times in each solution should be comparable to or slightly longer than
those in the ethyl alcohol-toluol process.
1. Fix the tissue, wash as required and process into 70 per cent ethyl
alcohol by the usual methods. Plant material fixed in aqueous fluids
should be dehydrated through an extended series of solutions in 10 per
cent grades from 10 to 70 per cent alcohols and then into the first TBA
solution. F.A.A.-fixed tissues are washed in 50 or 70 per cent alcohol and
then transferred into the first TBA solution.
2. From 70 per cent alcohol transfer the tissues into a mixture of:
60 cc. of 95 per cent ethyl alcohol; 20 cc. of TEA, and 20 cc. of distilled
water.
3. Transfer into a mixture of 50 cc. of 95 per cent ethyl alcohol; 40
cc. of TBA, and 10 cc. of distilled water.
4. Transfer into a mixture of 45 cc. of 95 per cent ethyl alcohol and
55 cc. of TBA.
5. Transfer into a mixture of 25 cc. of 100 per cent ethyl alcohol and
75 cc. of TEA.
6. Run through two changes of pure tertiary butyl alcohol.
7. Leave overnight in a third change of TEA.
8. Transfer into a 1: 1 mixture of TEA and paraffin oil.
9. Transfer into a 1: 1 mixture of TEA and paraffin oil saturated with
paraffin, and place in a paraffin infiltrating oven.
10. When the TEA-paraffin oil-paraffin mixture has warmed to the
temperature of the oven, transfer into the first change of infiltrating
paraffin. Use three changes of infiltrating paraffin and follow the pro-
cedures outlined in Exercise 24, beginning with step 9.
Exercise 26
Processing Minute Organisms in Paraffin
The general procedure for embedding minute objects in paraffin is
the same as for blocks of tissue. The differences lie in the necessity of
concentrating the material and handling it in such a way that it will
not be lost in processing. There are innumerable methods for accom-
plishing this and those presented here are representative of three general
methods. The first involves concentration by gravity settling or centrif-
ugation ; the second involves enclosing the material in a sac of animal
tissue; and the third involves "embedding" the material in a substrate
which is then processed as though it were a block of ti ssue. These pro-
cedures are applicable to any small objects such as protozoa, devrlop-
mental stages of molluscs or echinoderms, etc.
In each instance the material should be fixed and given the necessary
postfixation treatment, as determined by the fixative used. It is best
to color minute objects with a preliminary stain (such as eosin in 95 per
cent alcohol) during the dehydration series so that they can be located
in the paraffin.
A. Centrifugation or Gravity Settling
If possible, process the material in a pointed centrifuge tube (Pyrex)
and settle the material by gentle centrifugation before changing each
solution. If a centrifuge is not available the material may be processed
in narrow vials, and the material allowed to settle to the bottom of the
vial before changing each solution. For most objects Y'2-dram shell
vials are suitable containers for processing. The tubes or vials should be
supported in some type of rack to prevent tipping.
Remove as much of the fluid as possible without disturbing the ma-
terial. Do not attempt to pour the solutions off. An ordinary glass
pipet.te wit? a rubber b.ulb and a fine tip is useful for removing the
solutIOns. Smce some flUid must be left on the material at each chang ,
it is advisable to extend the series of solutions to the following:
1. One change each of 20, 30, 40, 50, 60, 70, 80, and 95 per cent alcohol.
2. 0.2 per cent eosin Y in 95 per cent alcohol.
3. 95 per cent alcohol.
4. Three changes of 100 per cent alcohol.
5. Two changes of toluol-alcohol , 1; 1.
6. Three changes of toluol.
7. One change of toluol saturated with paraffin.
S. Three changes of paraffin kept above melting point.
Minute objects need be left for only about 5 minutes in each of the
alcohols and dealcoholizing solutions, or as much longer as is required
for gravity settling. If the objects are relatively heavy it is not necessary
to attempt centrifugation while they are in paraffin. If the material is
very minute (such as small protozoa), centrifuge very quickly to setti
the material before the paraffin cools. Leave about 15 minutes in each
change of paraffin.
After the material has settled to the bottom of the last change of
paraffin, remove the tube from the oven, being careful not to disturb the
objects. Dip the bottom of the tube or vial into a dish of cold water. In
the case of centrifuge tubes, insert a handle into the paraffin mass to
facilitate removal. The handle may be made of wire provided with a
small hook or loop at one end to grip the paraffin ma.ss, and a larger
loop at the opposite end to keep it from slipping into the tube (fig. 14, A) .
The distance between the two 'loops should be adjusted so that the wire
will not extend into the area of paraffin which will be sectioned. If suffi-
cient contraction of the paraffin occurs at cooling, it may be possibl e to
withdraw the paraffin from the tube by pulling on the handle. If the mass
will not come free, submerge the tube, for a distance equal to the height
of the paraffin mass, in a dish of warm water a.nd exert a continuous pull
on the handle so that the paraffin mass is removed as quickly as possible.
The material may be removed from the small shell vials by carefully
breaking the vials. (This procedure should not be used with centrifuge
tubes which are not expendable.)
The resulting paraffin mass should have the objects concentrated at
one end, with enough paraffin at the opposite end to permit attachment
to the microtome peg. Since the tubes and vials are round, the block must
be trimmed to obtain a ribbon of sections. This will result in the los8 of
some material. The loss may be minimized by only trimming two faces
A B c o
FIC . 14. Methods of embedding minute objects in paraffin. A showe the material
embedded in the tip of a centrifuge tube . A wire handle has been inserted into the
paraffin mass to facilitate removal of the hardened paraffin. B-D show the method
of embedding in a sac of animal tissue, in this instance a length of small intestine .
In B the intestine has been closed off at one end and tied to a short pipette at the
other end. C shows a cross section of the intestine and attached pipette as the
minute material is transferred and permitted to settle into the intestine. In D the
intestine, now filled with the minute material, has been tied off just below the tip
of the attached pipette and cut free .
of the block so that they are parallel to one another and aligning these
so that th ey will be parallel to the knife edge.
'Vhen it is essential that none of the material is lost by trimming the
block , the cylinder of paraffin containing the material may be embedded
in n second mass of paraffin. The second embedding paraffin should be
just above it melting point, and should be cooled immediately after the
introduction of the core of paraffin containing the material. The disad-
vantage of this method lies in the tendency of the two paraffin masses to
sepHrHte from one another during sectioning.
B. Embedding in a Sac of Animal Tissue
Many parasitic or symbiotic organisms may be most easily processed
in situ. Thus termite protozoa may be processed within the hind gut
where they occur, by the simple process of fixing and embedding the gut.
Free living forms may be introduced into a sac formed by fixing some
animal membrane (such a mesentery or bladder) into a sa -like con-
tainer, or by placing them in some tissue lumen, such a the lumen of

the gut, and processing the tissue and enclosed organi ms as a block
of tissue. One relatively simple method is to employ a length of small
intestine from some small animal. The intestine should be fixed in a di -
tended condition by inflating it with the fixing fluid . The process may be
briefly outlined as follows:
1. Remove a length of small intestine from some small animal.
2. Flush the intestine thoroughly with a stream of saline solution in-
troduced into one end with a small pipette.
S. Tie off one end of the intestine with a fine thrcad.
4. Insert a short, Bouin's-filled pipctte into the open end of the intes-
tine and fasten this in place with a fine thread tied around the gut over-
laying the tip of the pipette (fig. 14, B).
5. Gently distend the intestine by compressing the rubber bulb
on the pipette so that the intestine is filled with fixative . K cping the
intestine distended, submerge it in a dish of warm Bouin's fluid.
6. When the intestine has been somewhat hardened by fixation, lip
the rubber bulb off of the pipette and drop pipette and attachcd intestine
into the fixing fluid .
7. After fixation (several hours or overnight) tran fer the tissue to
70 per cent alcohol. Tip the pipette so that most or all of the fluid is
drained out and then tip it in the fresh solution so that the pipette will
be refilled. Wash in several changes of 70 per cent alcohol.
8. The objects to be embedded are fixed and washed as necessary and
processed in narrow vials to 80 pCI' cent alcohol. Pennit them to settle
to the bottom of the container between each change of solutions (see
Section A, above).
9. Drain off most of the alcohol in the pipette inserted into the intes-
tine. By means of a second pipette, transfer somc of the minute material
from the bottom of the container of specimens into the pipette which is
attached to the intestine. Keep the latter upright so the material will
settle into the intestine (fig. 14, C). Decant off the fluid left in the at-
tached pipette, introduce more material, let it settle, and repeat thc
process until the material has been transferred into the length of intes-
tine.
10. Tie off the upper end of the length of intestine ju t below the tip
of the attached pipette and cut the gut free (fig. 14, D).
11. Complete dehydration , dealcoholize, infiltrate and rmbed the
tissue and enclosed organisms in paraffin as directed for tiRRlle hlork.
(Exercise 24, beginning with step 4) .
12 Orient the tissue in the paraffin 0 that one tied end is out nnd
140 GENERAL ZOOLOGICAL MlCROTECHNIQUE '
the other next to the peg. Trim the face of the block down with a razor
to remove the thread before sectioning. Stop sectioning before you con-
tact the second thread. It is advisable to use colored thread to facilitate
observation, and the ends of the threads should be cut as short as pos-
sible.
C. Embedding in Agar-Agar
Minute objects may be progessed by concentrating the fixed and
washed material in a depression slide and mixing with egg albumen or
horse serum which is then coagulated with alcohol. The mass produced
is then processed through paraffin in the manner described for tissue
blocks. Alternately, the objects may be mixed into a drop of warm 3
per cent agar-agar which is then allowed to cool and solidify and is proc-
essed into paraffin. The method outlined below is somewhat more com-
plicated than the "drop" method, but produces a good concentration of
numbers of organisms in a uniform block. It is desirable to color the
material with eosin before processing.
1. Prepare a 3 per cent solution of agar-agar in water, heating care-
fully to avoid scorching.
2. Pour out some of the agar-agar to produce a block deep enough to
accommodate the material and at least one inch square. The agar-agar
may be poured into a dish or "boat" of paper such as those used for par-
affin embedding. Keep the rest of the agar-agar fluid over a hot water
bath.
3. Cool the agar-agar mass, either at room temperature or in a re-
frigerator.
4. From the center of the solidified mass, remove a core of material
with a small cork borer. If possible, leave a layer of agar-agar at the
bottom of the cavity; otherwise,. plug the bottom with a few drops of
warm agar-agar.
5. Transfer the material to be embedded into the opening in the block
with a small pipette (the material should be in water following fixation
and washing). Let the organisms settle, decant off the fluid, refill the
cavity with additional material and repeat until all of the material has
be.en transferred or the cavity is about one-half full. Remove as much
fluid as you can without disturbing the material. Introduce fluid agar-
agar into the cavity and stir into the material with a fine needle. Cool
the mass and trim out the material-filled core, leaving 1 to 2 mm. of
substrate on each side of the core. Dehydrate, dealcoholize, infiltrate
and embed the agar-agar block as directed for tissue blocks.
12
The
Paraffin
Method
II. Sectioning
Once the tissue has been embedded, trimmed and attached to the
microtome peg it is ready for sectioning. The sections should be of a
known and uniform thickness, free from scratches, breaks or fold s.
This chapter is concerned with the process of cutting the sections and
with the instruments which make this preparation relatively simple.
A considerable degree of patience and practice is required to master the
process of cutting uniformly good sections. The beginning technician
should take comfort in the thought that it is through encountering
difficulties that one really learns how to adjust and handle the micro-
tome.
The Cutting Blade
Before proceeding to a discussion of the rotary microtome and the
process of sectioning, some consideration must be given to the blade
which will be used to cut the material.
The Microtome Knife
The microtome knife is undoubtedly the best instrument for c-
tioning and is almost a necessity for obtaining sections under 5 microns
(1 micron equals 1/ 1000 mm.) . The blade is useful only if it is per-
fectly sharp and correctly ground. If these conditions cannot be met,
much better results will be obtained with the razor blade method
discussed later. The ability to grind the knife on a stone and strop
I'll
requires careful and persistent practice. Even for the experienced

technician the process of blade sharpening is slow and laborious. The
results achieved, however, justify the effort involved. There are a
number of automatic and semiautomatic knife sharpeners available and
if any amount of technique work is undertaken in a department, such
a grinder will soon pay for itself. Since these sharpeners vary greatly
in design it is best to refer to the manufacturers' directions for in-
structions regarding their use.
The microtome knife should be a heavy blade of moderately hard
steel. The blades may be wedge-shaped or hollow-ground on one or
both faces. The wedged blade, or those which are hollow-ground on one
face, are to be preferred for paraffin sectioning. With the latter type
the fiat surface of the blade should face the tissue block in the micro-
tome. Doubly hollow-ground blades may spring under the impact
exerted when sections are cut. These blades, however, are easiest to
sharpen.
The cutting edge of the knife is not ground at the same angle as the
blade surface, but is inclined at a sharper angle to form the cutting
facet (fig. 15, A). In order to obtain the cutting facet, the back of the
knife must be elevated slightly off of the stone during sharpening and
polishing. The higher the back is raised, the shorter and more acute
the cutting facet becomes. The knife is elevated by means of a rounded
metal sleeve which is slipped over the knife back. These sleeves are
provided with the knife and are designed for use with the specific
blade. The cutting facet produced will vary with the diameter of the
sleeve and the depth of the blade. Some automatic grinders are de-
pendent upon the sleeves for setting the angle of the cutting facet. In
others the knife is inclined against a vertical or horizontal wheel and
the cutting facet may be varied by shifting the position of the knife
relative to the position of the wheel. The advantage of the latter system
is that the angle may be 'readily adjusted to accommodate the re-
quirements of the material and the preferences of the technician. It
also permits grinding at one angle and polishing at a second, more
acute angle to produce a short cutting facet on a beveled edge. One
end of the knife (the base or heel) is drilled to receive a handle
which is placed on the knife when the latter is to be hand ground or
stropped and is removed during sectioning. The sleeve should always
be placed on the knife back with the same end toward the base.
The knife should be handled with extreme care at all times and every
effort must be made to avoid any contact with the edge which will
result in dulling or nicking. It must be free from moisture when stored
THE PARAFFIN METHOD. n 14.3
34- CUTTING FACET
34- CUTTING FACET OF

WORN KNIFE
PROFILE OF MICROTOME KNIFE
A
- - -PARAFFIN--
BLOCK
·, ..
.
•
•
I
I
•
,
I
, '
.
I
'
~.J (
~ .~
" 5~CLEARANCE • .... _ 5·,
, 'ANGLE
'-2'3
0- - " : 45.----- J
KNIFE
ANGLE
B c o
FlO. 15. The microtome knife. A, profile of a wedged knife blade showing the
cutting facet. The blade is sharpened at an angle to the sides of the knife; the
bevel formed by sharpening is known as the cutting facet. In the illustration (A)
the cutting facet is set at 34·. whereas the angle form ed by the faces of the knife is
only 16·. Note that as the knife is worn the depth of the cutting facet increases while
the angle is maintained at 34·. Figures B, C and D illustrate the differ nce between
the knife angle and the clearance angle. The clearance angle is that formed be-
tween the cutting fa cet and the face of the block and is usually set at about 5·, If
the kn ife is 11 simple wedge, as in B. the clearance angle of 5· is formed between the
face of the block and the fare of the blade. The angle from the face of the block to
t he midline of the blade would be about 15· . In C the cutting facet is about 30 to 34·,
the clearance angle is 5· and the angle between the midpoint of the knife and the
face of the block is about 23·; this is the usual situation . Figure D shows the situa-
tion which would occur with a cutting facet of 70·; a clearance angle of 5· would re-
quire positioning of the knife so that the midpoint of the knife WOllin be at an angle
of about 45· to the face of the block .
and should be coated with oil when not in frequent use. Some type of
box should be provided for storage. It is preferable that this box is
designed so that the blade, edge down, is suspended by slotted blocks
at the two ends. The knife should never be laid on a table or other
surface, but should be in the microtome or in the storage box. It should
not be left in the microtome when not in use. Paraffin should be removed
by washing the blade with xylol. When using the microtome do not
forget the presence of the knife, nor that its entire cutting edge is
sharp. At first you will find it requires a conscious effort to avoid
contact with the knife edge. Any moment of carelessness can result in
severe injury to both technician and knife.
Grinding the knife. Place the knife on a clean glass plate on the
stage of the microscope. When moving the knife to examine the edge
move the plate and the knife, holding them firmly with both hands.
Examine the edge with the 16-mm. objective, using reflected light. Note
the general condition of the edge and the location of any nicks. A
dull blade will have an irregularly serrated margin. If the blade needs
sharpening, attach the knife handle to the base of the knife and then
slip the sleeve on the back of the blade. The sleeve should be marked
so that it is always placed on the blade in the same position. Flood the
grinding stone with the appropriate lubricant (oil or water, depending
upon the type of stone used). The stone should be a fine finishing
stone 2 to 3 inches wide and 8 to 12 inches long, and must be perfectly
flat. Place the knife across the stone at one end with the cutting edlZe
leading and the knife base (next to the handle) resting on the stone.
Keep the back of the knife at right angles to the length of the stone and
draw the knife forward diagonally along the length of the stone. The
angle of the stroke should be adjusted so that the entire knife edge
will have crossed the surface of the stone by the end of the stroke.
Rotate the blade on the sleeve s'O that the opposite face of the knife
lies on the stone, with the edge leading and the knife base on the stone
surface. R epeat the diagonal stroke to the opposite end. The only pres-
sure applied should be that provided by the weight of the kniff>. Con-
t.inue to grind the knife edge with successive strok s on alternate faces
of the knife, until the edge appears even under the 16-mm. objective.
The edge may have a series of very fine and regular serrations. If
you find that these persist after prolonged grinding the stone i not
fine enough to produce a perfect edge, but if the serrations are fine
enough they may be removed by stropping. If irregular serrations or
even nicks appear in the edge, the stone may be dirty or you may be
hitting the knife edge against the stone edge as you turn the blade.
THE PARAFFI,S METHOD. n 145
The stone lubricant should he renewed as frequently as nec ary and

if the grinding is prolonged, the stone should be cleaned 0 ca ionallv
during the process. If deep nicks are present in the knife edg and
it is possible to avoid these areas and still cut the sections, it will
save wear on the knife to leave them in. They will eventually he
removed during the process of grinding the general knife edg . Badly
nicked blades may first be ground on a coarser stone, then on the fine
stone.
Poli8hing or Stropping. When microscopic examination indicate
that the knife edge is sharpened, the edge may be improved by light
stropping to polish the cutting facet and the edge. The amount of
stropping required will depend upon the fineness of the edge produc d
with grinding. This in turn will be limited by the fineness of the stone
employed. With a very fine clean stone the knife edge may be p rfect
enough to use without stropping.
The strop should be mounted perfectly flat on a wooden block. The
stropping action is the reverse of that used in grinding, with the knife
edge following or trailing the back of the knife on the stroke. It is
extremely important that no pressure is applied during the stropping,
or the edge of the blade may be turned. Strop about twenty times on
each side, with successive strokes on opposite faces of the blade. Ex-
amine under the microscope. The surface of the cutting facet should
have a smooth almost mirror-like appearance when viewed with re-
ffected light and the edge should be smooth. Repeat the stropping as
necessary.
The Razor Blade Holder
The use of a microtome knife is very impractical in large classes,
since the time and expense involved is usually prohibitive. For general
class work, and indeed for many research projects, a razor blade in a
special holder (fig. 16) provides excellent results with section above 9
microns. For thick sections (20 or more microns) the razor may
give better results than a knife. Sections of less than 9 microns are much
easier to prepare with a knife and below 5 microns a knife is almost
a prerequisite. The use of a razor is to be recommended whcn ctioning
any material which might contain hard particle that would damage
a knife. Thus, material containing spicules or occa ional and grain ,
or tissues from animals collected with a shot gun, should be ctioned
with a razor if at all possible.
The razor provides an excellent cutting edge but lacks the rigidity
necessary for sectioning. There are available, however, special holders
146 GENERAL ZOOLOGICAL .MlCBOTECHNIQUES
Flo. 16. The razor blade holder. CAO model 966; photograph courtesy of the
Americal Optical Company.)
designed to securely clamp double-edged razor blades in the micro-

tome. For good results, the razor blade holder must be perfectly ma-
chined and clean, the blade must be of the correct weight, carefully
placed in the holder and securely held. Some companies market special
microtome blade razors which are somewhat heavier than the average
double-edged razor blade. These are relatively expensive. It has been
my experience that the double-edged razor blades produced by the
Marlin Gun Company give excellent results and the Gillette blue
blade is also satisfactory. No "thin" type blades should be used, as they
tend to vibrate. Failure to obtain satisfactory sections with a razor
is most frequently due to the type of blade being used or incorrect
placement of the blade in the holder.
The razor blade is slipped intQ the jaws of the razor holder after
first loosening the set screw. Align the blade very carefully so that
the upper edge is prefectly straight with the upper lip of the ·holder. In
the case of the blades mentioned above, the blade bevel and a very
thin line of the blueing of the blade face should be visible above the
upper flange of the holder when the set screw is securely fastened.
Never attempt to use a broken blade since it will vibrate and produce
irregular sections. The blade will also vibrate if it is set out too
far . If the blade is set too far into the holder, the paraffin block will
be crushed against the upper front lip of the razor blade holder. Be
sure the inner faces of the holder jaws are perfectly clean before insert-
ing the blade. If any paraffin is present, clean the holder thoroughly
with xylol or toluol.
THE PARAFFIN METHOD. n 147
Flo. 17. The Spencer rotary microtome (AO model 815; photograph courtesy of
the American Optical Company) . !, the drive or advance wheel; t, the return wheel;
S, the micron advance indicator; 4, the swivel clamp; 5, the drive wheel lock .
. Exercise 27
The Rotary Microtome
This exercise is designed to familiarize you with the rotary microtome
(figs. 2 and 17) before you actually attempt to prepare sections. When-
ever possible have someone who is familiar with the microtome demon-
strate how to use it. Although paraffin blocks are most readily sec-
tioned on the rotary microtome they may also be prepared on microtomes
of the sliding or clinical type (Exercises 36; and 40, Section A) .
1.' General Rules. The rotary microtome is a precision instrument
and must be handled with intelligence and care.
a. Be sure you understand the function of each part before you
attempt to use the machine.
b. Never force any adjustment.
c: Leave repairs to qualified personnel.
d. Keep the machine protected from moisture and dust.
e. Keep the machine properly lubricated according to the manufac-

turers' directions.
f. Always lock the knife carriage back from the block holder when
the machine is not in use.
g. Lock the advance or drive wheel when you are not actually using
it.
h. The knife or razor should always be removed from the machine
when not in use.
2. Locate the following parts on the microtome (see figs. 2 and 12) :
the "advance" wheel and lock; the "return" wheel; the block holder and
swivel ciamPi the knife carriage and loCki and the thickness scale and
adj ustment knob.
3. The Advance Wheel. Move the knife carriage back from the face
of the block holder and lock it in place. Open the top of the machine
(if it has a lid), release the advance wheel lock and turn the advance
wheel slowly in a clockwise direction.
a. Notice that as the advance wheel is turned, a ratchet contacts and
turns a toothcd drive or ratchet wheel. Set the thickness adjustment
scale at 10 microns and note the distance the ratchet wheel is turned
with each revolution of the advance wheel. Change the setting to 20
microns. The ratchet wheel should now turn twice as far with each revo-
lution of the advance wheel.
b. Notice that with each revolution of the advance wheel, the block
holder moves downward and upward. At what point during this cycle
are the ratchet wheel and ratchet engaged? This is the point at which
the paraffin block will be advanced the distance indicated on the thick-
ness scale. With the knife in a fixed position, a section of known thick-
ness will be removed from the face of the block each time the advance
wheel is turned once.
The microtome cannot be advanced indefinitely. When the machine
has advanced as far as it will go, it disengages automatically and, al-
though the advance wheel is turned, n'o further sections will be obtained.
Reversing the direction of movement of the advance wheel will not re-
verse the direction of movement of the block or ratchet wheel. The ma-
chine is returned to its starting position by using the "return" wheel.
4. The Retnrn Wheel. The return wheel permits rapid movement of
the block holder and attached mechanisms, forward or backward within
the working distance of the microtome. Note that the rate of movement
is not controlled by the setting of the thickness scale.
a. Rotate the advance wheel so that the ratchet wheel and ratchet
are disengaged. The block holder will be down from the top of the cycle.
Usually the handle of the advance wheel will be down.
b. Rotate the return wheel, first in one direction and then in the op-
posite direction and notice that the block holder is moved rapidly for-
ward or backward without moving up or down. Caution. The return
wheel should move readily and soundlessly. If you hear a grinding sound
it means that you have not disengaged the ratchet wheel. If the ratchet
wheel is not disengaged it will be badly worn when the return wheel is
moved in either direction.
c. Determine the working distance of the microtome by moving the
mechanism from one end of its adjustment range to the other by means
of the return wheel. Rotate the wheel at a moderate speed. If you hit
the limits of the adjustment range with any force you may jam the
mechanism. The machine does not need to be reset each time a block
is sectioned. You should always determine H the working distance avail-
able is sufficient to permit you to section the tissue block so that you
will not have to readjust the machine halfway through a tissue.
5. The Block Holder. The actual block holder is a simple clamp
which is tightened against the peg holding the tissue. This clamp is sus-
pended in a swivel clamp which provides a means of aligning the tissue
with the knife. The clamp may be pivoted from top to bottom by loosen-
ing the two top set screws and tightening the bottom one or vice versa.
The block clamp may be pivoted from side to side by loosening one top
set screw and tightening the opposite one. Only minor adjustments
should be made with the swivel clamp. Be sure that all screws are set
firmly against the swivel head when alignment has been completed. Ac-
tual alignment of the block with the knife is discussed in Exercise 28.
6. The Knife Carriage. The knife carriage moves forward or back-
ward on a stationary track upon which it must be securely locked
during sectioning. The carriage consists of a rigId base having two up-
right arms whose upper surfaces embrace two half-cylinders which are
the actual knife clamps. In many types of holders the two knife clamps
move independently of one another. In others, such as that shown in
figures 17 and 18, the two knife clamps are connected by a crossbar so
that they move as a unit within the arms of the knife carriage.
Each knife clamp has three screws or levers. The angle screws or levers
bear upon the knife clamps and hold them rigid in the arms of the car-
riage. The elevator screws bear upon the bottom of the knife, and may
be used to adjust the height and level of the knife. They also serve to
hold the knife firmly against the jaws of the knife clamp. The clamp
FIG . 18. The knife carriage (AO model 826; photograph courtesy of the American
Optical Company). This holder is shown in position in the microtomes in figures 2
and 17. The knife clamp screws (1) bear upon the back face of the blade; the
elevator screws (!) force the knife upward into the jaws of the clamp. (In other
models the elevator screws lie below the knife and bear directly against its back .)
The angle levers (3) , when loosened, permit adjustment of the knife angle; wheu
tightened, they lock the knife clamp firmly in the knife carriage. (In other models
the levers are replaced by angle screws which bear directly against two independent
knife clamps.) The carriage lock (4) holds the knife carriage firmly in the micro-
tom e.
screws bear upon the back face of the blade and hold it rigidly in place
in the clamps.
a. Release the knife carriage lock and move the carriage to the outer
end of the track and lock it in place.
b. Loosen the clamp screws and insert a blade holder or knife into the
clamps, being careful to handle the knife by its back margin.
c. Test the adjustments made possible by the elevator screws. They
must be adjusted with the knife in position so that it is kept perfectly
level. The knife should be elevated so that its upper edge will clear the
clamps. The upper edges of the arms of the razor blade holder may be
level with the top of the clamps.
d. Tighten the clamp screws down on the knife just enough so that
THE PARAFFIN lItETHOD. II 151
both clamps move together with the knife when the angle screws are
loosened. In holders having independent knife clamps the knife cannot
be moved if the clamp screws are too tight, and if they are too loose the
clamps may not be set at the same angle when the angle screws are
tightened.
e. With the angle screws loosened, pivot the knife and clamps to test
the angular adjustment available. Setting the correct angle is a matter
of trial and experience. In general the knife should be as perpendicular
as possible but must be angled forward enough so that only the cutting
edge of the blade will come into contact with the paraffin block. The
angle with which we are concerned is that which is formed between the
block face and the cutting facet of the knife (fig. 15, B-D). An angle of
50 is usually required to avoid contact between the shoulder of the knife
(where the cutting facet joins the face of the blade) and the paraffin
block. When the knife angle has been adjusted the angle screws are
tightened firmly against the knife clamp.
f. Tighten the clamp screws firmly against the knife.
Before you begin sectioning you should always check each screw on
the block holder and knife carriage and clamp to be sure that they are
firmly set. If you encounter difficulties when you begin sectioning (Exer-
cise 28) refer back to these directions to be sure that you have set up the
machine and knife as required.
Exercise 28
Paraffin Sectioning
Although the following directions may be painfully detailed, they
represent, step by step, the procedures which must be followed each
time a block is sectioned. With experience they are completed very
quickly and almost automatically.
1. Familiarize yourself with the actions and adjustments of the rotary
microtome as described in Exercise 27.
2. Have available some type of tray to receive the sections as they
are cut. The tray should be provided with a stiff lid and should be lined
with a clean paper, cut so it will lie smoothly in the bottom . Durable
section trays can be made by using sides of 5- to 9-mm. wooden slats
glued and tacked to a stiff cardboard with a sheet of stiff cardboard as a
cover. Flat, covered boxes may be used but when the lid overlaps the
sides it must be removed with extreme care or the sections will be dis-
rupted. Such trays protect the sections from drafts and dirt and permit
storage between sectioning and affixing.
3. Have available two clean "camel's hair" brushes, Nos. 3 to 5. These

should have tapered points.
4. Position the microtome so that the advance wheel is toward you
and the knife and block holder are to your left. You may prefer to work
with the advance wheel to the right and the knife toward you.
5. Recheck the paraffin block to be sectioned. Be sure that all sides
are cleanly cut and that opposite sides are parallel. Retrim the block if
necessary.
6. Set the advance mechanism to the required thickness.
The thickness at which the material should be sectioned varies with
the tissue and the obj ective of the study. The best thickness for a particu-
lar purpose and tissue is a matter of experience, but the following re-
marks may be helpful as an initial guide. As a general rule thickness
should be regulated by cell size. If sections are so thick that numerous
cells are superimposed upon one another it becomes difficult to study the
individual cells. On the other hand, extremely thin sections which do
not include any complete ce)]s may be difficult to interpret. Amphibian
tissues usually have relatively large cells and for general histological
w~rk may be sectioned at 10 to 12 microns. Most inv.ertebrate, reptile,
bird and mammal tissues have relatively small cells and for general
histology 7- to 10-micron sections are desirable. Within any group of
animals the cell sizes will vary and within a given animal cells of dif-
ferent tissues will be larger or smaller than the average.
When sections are prepared for studies of cytoplasmic structures, such
as mitochondria, which are extremely small and numerous, the sections
should be cut at 3 to 5 microns.
Relatively thick sections are prepared for studies of gross structural
features of tissues and organs; for a consideration of embryological
development; or for the observation of general morphology of small en-
tire organisms. Such preparation~ are sectioned from 12 to 25 microns,
depending upon the material. In general the larger the object the thicker
the sections.
7. Using the return wheel, adjust the microtome to provide enough
working distance to section the entire block of tissue. Be sure to disen-
gage the ratchet wheel before rotating the return wheel.
8. Lock the advance wheel.
9. Insert the microtome peg with attached tissue block into the block
clamp so that it will approximate the correct orientation for sectioning.
The long edges of a rectangular block should be parallel to the knife
edge as viewed from the front. When the face of the block is square,
either pair of edges may be parallel to the knife edge. Entire organisms
THE PARAFFIN METHOD. II 153
should be oriented so that the resulting section can be readily inter-

preted when examined under the compound microscope ( ee "serial sec-
tions," p. 171) . Orientation of excised tissue blocks for the required
plane of section is possible only if the tissue was properly subdivided at
the time of fixation (p. 47) and correctly oriented at the time of em-
bedding (p. 126) and attachment to the microtome peg (p. 133) .
10. Insert a double-edge razor blade into the blade holder as directed
earlier (p. 146) .
11. Grasp the blade holder (or knife) by the back edge and care-
fully insert it into the knife clamp. A knife having one hollow-ground
and one straight face should be placed in the holder with the straight
face toward the paraffin block. Adjust the elevator, angle and clamp
screws so that the knife is correctly positioned and held firmly in place
(Exercise 27, step 6).
12. Release the advance wheel lock and rotate the wheel so that the
paraffin block is lowered somewhat but not far enough for the knife to
hit it as the latter is moved forward.
13. Release the knife carriage lock and slide the carriage forward
until the knife is just in front of the paraffin block. Lock the knife car-
riage in place.
14. Carefully align the block with the knife, utilizing the three adjust-
ment screws in the swivel clamp which supports the block clamp. The
adjustments made here should be minor in magnitude and assume that
the block was properly attached to the peg and placed in the microtome
in the correct position. Note that the first two alignments are concerned
with the actual tissue block and the third is concerned only with the
paraffin block surrounding the tissue. Make the adjustments in the order
indicated. Be careful not to contact the knife edge.
a. Align the block with the knife while viewing it from the side (fig.
19, A). The face of an excised tissue block should be parallel to the knife
edge. The plane of section of an entire organism should be parallel to
the knife edge. This alignment is the most difficult to make since the
point of reference, the knife edge, is difficult to visualize. (Do not con-
fuse the knife edge with the face of the knife.) This alignment requires
considerable practice and a further suggestion for obtaining it is given
later. The bloek is adjusted by loosening the two top swivel clamp screws
and tightening the bottom one or vice versa. Lock the drive wheel while
adjusting the screws.
b. Align the tissue while viewing from above. The face of the tissue
block should be exactly parallel to the knife edge. The plane of section
of an entire organism should be parallel to the knife edge (fig. 19, B) .
PLANE OF
SECT I ON -SWIVEL CLAMP
FROM THE
KNIFE
EDGE FROM ABOVE
FROM THE FRONT

KNIFE EDGE
c
FlO. 19. Orientation of the paraffin block in the microtome . Alignment of the
block from the side (A), from above (8), and from the front (C) . In each instance
the block is out of alignment on the left and correctly positioned on the right
Screw 1 bears upon the microtome peg. Screws g and S are the upper swivel clamp
screws and are opposed by the lower swivel clamp screw, 4.
This adjustment is made by tightening one of the top swivel clamp

screws and loosening the opposite one.
c. Align the bottom edge of the paraffin block so that it is perfectly
parallel to the knife edge as viewea from the front (fig. 19, C). This ad-
justment may be made by loosening one of the upper swivel clamp
screws and turning the block holder (rather than just the microtome
peg). When the block is straight, be sure to tighten the same screw you
loosened or you will throw the block out of alignment again.
15. Recheck all set screws on the block and blade holders.
16. Move the block toward the knife by turning the advance wheel
clockwise. As the block passes the knife, estimate the distance between
them. Remember that to advance the block 1 mm. 'will require 100 turns
of the advance wheel with the mechanism set at 10 microns. Unless the
knife is very close to the block it will save time, as well as wear and
tear, to advance the block toward the knife with the return wheel. Of
course tht: knife may be moved closer to the block by releasing the car-
riage and pushing it forward, and this should be done if the distance is
2 or 3 mm. or more. This adjustment is difficult to control, however, and
it is best to advance the block on to the knife for the final distance. If
you use the return wheel, have the tissue block just above the knife
edge (this should be low enough to disengage the ratchet wheel) ; make a
quarter turn with the return wheel (in a forward direction) and lower
the tissue block across the knife to test for contact. If no paraffin is re-
moved from the block, raise the block just above the knife and rotate the
return wheel another quarter turn. Continue to advance the block until
the knife removes some paraffin from the surface of the block. Never
advance the return wheel with the block below the knife and do not turn
it more than a quarter turn without testing the position of the block
against the knife.
17. The first sections which are removed from the face of the block
a.re usually incomplete. Turn the advance wheel until you can see tha.t
a full section is removed from the face of the block each time it passes
over the knife edge.
18. When the face of the block has been cut even by the knife, recheck
the orientation of the tissue as viewed from the side (step 16, a). The
surface of the block now indicates the exact plane of section you will
obtain. This provides an excellent point of reference for perfect align-
ment of the block. If it is necessary to readjust the position of the
block recheck the alignment from the top and front.
19. As soon as complete sections are being removed from the face of
the block, successive sections should adhere together to form a smooth,
straight ribbon. If the ribbon does not form or appears irregular in any
way every effort should be maGe to correct the difficulty before the tis-
sue is reached. This will not only avoid loss of tissue sections but will
help in distinguishing those faults which are inherent in the microtome
adjustments or the paraffin rather than in the tissue. It is not advisable
to leave a great mass of paraffin in front of the tissue or you will waste
time and may dull the knife edge before you begin sectioning the tissue.
When you become experienced with the microtome adjustments, the
paraffin on the face of the block should be kept at a minimum so that
the tissue is reached within about 30 sections of the surface. Some com-
mon faults of sectioning which do not actually involve the tissue block
are listed below with suggestions for possible causes. In many instances
corrective measures will be apparent from the cau e; otherwise sug-
gestions are given for eliminating the difficulty.
Problem8 Encountered in Sectioning

a. Successive sections will not adhere to form a ribbon. Are opposite
sides of the block trimmed parallel to one another; are they aligned so
as to be perfectly parallel to the upper edge of the knife; was the block
trimmed with a clean, sharp, single-edged razor blade? Retrim and re-
align as necessary. Are you cutting across the shortest dimension of a
rectangular block; if not, realign. The block may be too cold; permit it
to warm to room temperature. The room may be too cold for high melt-
ing point paraffin to form a ribbon; work in a warmer room or increase
the temperature in the working area with a lamp, hot water, etc. If fail-
ure to form a ribbon is apparently due to curling of the individual sec-
tions refer to b, below.
b. The sections curl into a tight tube as they are cut. This can some-
times be corrected by cutting seyeral sections and then carefully hold-
ing these down with a brush while cutting seYeral more sections. If this
is effective, the weight of the ribbon will prevent further curling. If the
sections persist in curling and breaking one of the following may be re-
sponsible: the knife may be inclined too far forward; the room may be
too cold for the melting point of the paraffin used in embedding; the knife
may be dull; with thick sections, the paraffin used for embedding may
have too high a melting point; you may be cutting across the long di-
mension of a rectangular block.
c. The ribbon curves sharply to one side (fig. 20, E and F). Is the
block square? If it is even slightly wedge-shaped the rihbon will cun'e
in the direction of the short side. Retrim if necessary. Never try to com-
pensate for curvature of the ribbon by trimming a square block into a
wedge, since this will lead to difficulties at the time the sections are
affixed to slides. If the ribbon curves even when the block is perfectly
square the knife edge may be irregtIlar; try a different area of the hlade.
d. The ribbon splits longitudinally or shows surface scratches (fig.
20, B). This is most frequently cause by a bit of dirt or paraffin on the
knife edge. Clean the edge as follows. Leave the block belou' the knife
edge. Place a small amount of xylol or toluol on a finger tip and care-
fully wipe up on the blade, over the knife edge on the upper surface bf
the knife. Repeat with the front face of the knife. Be sure any xylol or
toluol left on the blade is wiped off (with an upward stroke of the finger)
or is permitted to evaporate off before you section again. The blade may
be cleaned with a brush moistened with xylol, but this is usually not as
effective as using a finger. Be sure to brush upwards, since a bru&h
drawn down over the knife edge will dull or even badly nick the blade.
~--- .LOCK----~----TISSUE---f2J
-
---RIBBON- - -
-w
A B c
o
o
o
o
o E F
FIG. 20. Common difficulties encountered in sectioning. A, a partial scratch or
split occurs in the ribbon. B, the ribbon is scratched and splits longitudinally. C,
the sections are excessively compressed. D, the sections vary in thickness ; alternate
sections are usually thick and thin. E, the ribbon curves to one side even though
the block is squarely trimmed. F, the ribbon curves to one aide because of a wedge-
shaped block . See the text for remarks regarding causes and corrections of theee
and other difficulties.
Test the effectiveness of the cleaning by cutting a section. If the

scratching or splitting persists in the same area it indicates that the
knife edge is damaged at that point. Move the knife laterally so that
a new area will be used. Whenever you move the blade laterally it is
best to take it back from the face of the block and then realign the block
and knife. If necessary sharpen the knife or use a new razor blade.
Persistent splitting of the ribbon in the same place after the knife has
been moved indicates that some hard material is present in the block.
Frequent reoccurrence in different areas indicaws that you are damag-
ing the knife edge (perhaps with a brush) or that the paraffin in which
the material is embedded is contaminated with dust, dirt or soot. Such
blocks must be re-embedded in clean paraffin.
e. The sections are greatly compressed, so that the individual section
is only a fraction of the size of the original block (fig. 20, C) . Some com-
pression of the section usually occurs and will tend to increase with thin
sections. This can be minimized by cooling the block in ice water and
cutting very thin sections in a cold room. Excessive compression may in-
dicate that the melting point of the paraffin used is too low for the section
required. Use high melting point paraffin for very thin sections (5 mi-
crons or less). The knife may be inclined too far forward; try reducing
the tilt slightly. If compression of the sections is combined with damage
to the face of the block refer to f, below.
f. The sections are compressed or irregular and shattered or mashed;
the face of the block appears white or smashed, especially at the edges.
The knife is not adequately inclined and the block is hitting the face of
the blade. Increase the angle of the knife. If a r!j.zor blade is being used
it may not be far enough out of the holder and the block is hitting the
front lip of the razor blade holder.
g. The sections crumble and the block feels soft . The paraffin in
which the material is embedded is contaminated with the dealcoholizing
agent. Re-embed the material, running it through two changes of fresh
paraffin.
h. The machine is not removing a section each time the advance
wheel is rotated, or successive sections are not uniform in appearance;
frequently , alternate sections are thick and thin and appear wide and
narrow (fig. 20, D) . Something ma'y be loose; recheck all the set screws
on the block and blade holder; check that the razor is firmly held in
the razor blade holder ; check that the block is firmly attached to the peg.
The knife may not be inclined sufficiently. The razor may be too far
out of the holder.
i. The sections are badly wrinkled across, so that they may appear
greatly compressed. The room is too warm for the melting point of the
paraffin or the thinnes of the sections. The knife is inclined too much.
j. The sections adhere to the paraffin block on the upstroke, instead
of remaining on the knife. The knife edge may be dirty; clean as directed, ·
under d. This may also occur if the knife is not tilted enough; increase
the angle.
k. The sections have a washboard appearance, with thick and thin

areas in the individual sections. The razor blade may be out too far. The
angle of the knife may be excessive. One of the set screws on the block
or knife holder may be loose.
l. The sections cut well but the ribbon whips toward any object
brought close to it and sticks to the object. This is cau ed by static elec-
tricity, which may be reduced in one of several ways: ground the micro-
tome; boil some water in an open container near the microtome; obtain
one of the "static eliminators" now available on the market. In extreme
cases it is advisable to wait for better atmospheric conditions.
m. Each section has a visible fold line across the section at about the
same point. This is a typical problem when using a razor blade holder.
The fold is formed at the point where the razor blade face emerges from
the holder. The fold is formed as the section rests on the razor and holder
faces for the moment until the next section displaces it. In most in-
stances this fold is readily removed at the time the section is affixed to
the slide. It may be minimized by cooling the block and by supporting
the ribbon up off of the blade by suspending it on a brush.
Further problems encountered while sectioning are discussed under
step 21.
20. Assuming that you have been able to obtain smooth, uniform sec-
tions, continue to cut the block until you are obtaining sections of the
tissue. As the paraffin ribbon lengthens, it should be supported on a soft
brush held in the left hand. The smoothness of the ribbon can sometimes
be improved by holding the entire ribbon up off of the knife. Cut a rib-
bon about as long as your section tray. Remove the ribbon from the knife
by lifting the sections away from the knife edge with a second brush
slipped under the ribbon. If you are having difficulty starting a ribbon
it is advisable to leave some sections on the knife to serve as the begin-
ing of the next ribbon length. In this case cut the ribbon against the
knife face with a scapel blade, being careful to cut between two sec-
tions. Use a rounded blade and cut the ribbon with a rocking motion of
the blade.
21. Although the paraffin may section perfectly, further problems may
be encountered when you begin cutting the actual tissue. These are
listed below with suggestions for avoiding or correcting them.
Problems Encountered in Sectioning (Continued)
n. The sections are split or scratched Longitudinally from section to
section. See step 19, d. If scratches reappear at the same point in the
sections after the knife has been moved, some grit is present in the tissue
itself. Frequently it is possible to see that the scratch originates within

the tissue. This is a common occurrence when sectioning small entire
organisms with sand in the gut. Every effort should be made to avoid
this by starving the animals before fixation. If necessary, sand grains
lllay be picked out of the face of the block with a dissecting needle. This
procedure may also be required to remove bird shot. In either case a
space will be left which may cause difficulties in obtaining good sec-
tions. Such ribbon splitting may also occur when calcified objects have
been imperfectly decalcified. This may be corrected by running the tis-
sue back down through the series of solutions and completing decalcifi-
cation and reprocessing.
o. The tissue shatters, falls out of the paraffin and has an opaque or
chalky appearance. The tissue was not properly infiltrated and prob-
ably not perfectly dehydrated. uch tissue is usually damaged beyond
redemption. If the material is irreplaceable you may attempt to reclaim
it by soaking it in several changes of toluol and returning it through
toluol-alcohol and into 100 per cent alcohol. The tissue is then reproc-
esssed through the dealcoholization and infiltration series.
p. Th e block shatters but is not chalky in appearance. The block has
been over-hardened by the dealcoholizing agent or by excessive tem-
peratures, or by the fixative 01' alcohols, 01' may be naturally inclined
to hardness when processed in paraffin. Such blocks should be processed
in celloidin. If the block is irreplaceable remove the paraffin in toluol,
process through toluol-alcohol and into 100 pel' cent alcohol and embed
in celloidin as directed in Exercise 35.
Hard blocks may often be successfully sectioned in paraffin if they
are soaked for 24 to 48 hours 01' more in water. This treatment is effective
only if the face of the tissue block is exposed. The surface of the block
must be trimmed down until a portion of the tissue has been removed.
This procedure i recommended for most plant tissues and for liver,
spleen and similar animal tissues. The material must be sectioned as
soon a it is removed from the water and the blocks cannot be success-
fully Rtored after such treatment.
q. The tisS1Je falls out of the paraffin, although it does not shatter and
is not chalky in appearance. This is usually the result of poor embedding.
A layer of paraffin solidified on the surface of the tissue block when it
was heing tran ferred from the infiltration into the embedding paraffin.
The two containers should be close together and the transfer made as
rapidly a. pos ible. Such tissues should be re-embedded.
r. The tissu e bulges out of the paraffin ribbon and may break loose at
one or more edges. This is most frequently encountered when cutting
very thin sections. This results from excessive compression of tM pa.r-

a.ffin as compared with the paraffin-infiltrated tissue. It may be mini-
mized by reducing the amount of paraffin around the tissue, especially at
the sides of the block. It may also be reduced by cooling the block in ice
water before sectioning. It may be necessary to re-embed the tissue in
higher melting point paraffin.
s. The ribbon curves sharply to one side. If this did not happen when
only the paraffin was cut (see step 19, c) but occurs when pa.raffin and
tissue are sectioned, it indicates that the tissue is off center in the block.
Since the infiltrated tissue compresses slightly less than the paraffin
alone, the ribbon will tend to curve toward the side having the greatest
amount of paraffin. If the tissue block is square this can be corrected
by trimming the paraffin so that its width is equal on opposite sides of
the tissue. In the case of wedge-shaped or irregular tissues, this type of
curving is difficult to avoid or correct. It may be minimized by cooling
the block or cutting the sections more slowly. Do not trim the block out
of square to compensate for curvature. If the block if! square the ribbon
can be straightened when the sections are affixed to the slides.
t. The sections vary in thickness, and sectioning is accompanied by a
clunking or ringing sound. Something may be loose (see step 19, h) ; the
tissue may be too hard (see step 21 , p) ; the knife may be inclined too
far forward (reduce the angle). If the face of the block is visibly dam-
aged the knife is too perpendicular ; increase the angle. The razor blade
is too far into the holder; reset it according to the directions given earlier.
u. The tissue cuts well and appears uniform except for occasional
sections, which are slightly, or much, thicker than the other sections.
This may not be apparent until the sections are affixed or even stained. It
usually results when ·a very cold block is being sectioned, and sectioning
is interrupted for a few minutes or even a few seconds (to the remove
the ribbon, for example). During the interval the block warms slightly
and expands and the first section removed from the face of the block is
thicker than the other sections. Chilled blocks should be cut with as
little interruption as possible, and the block may be recooled by holding
an ice cube against it if any delay occurs during sectioning.
22. Storage of Sections. The lengths of ribbon should be placed in
the section tray in the order in which they are cut. The first section
should be in the upper, lef~hand corner of the tray and successive ec-
tions should be in order to the rigbt. Subsequent lengths of ribbon are
placed below the first, like the printing on a page. Be careful not to in-
vert the ribbons. Note that one surface of the ribbon is dull (the upper
surface as the sections lie on the knife) and one is shiny. See furth er re-
162 GENERAL ZOOLOGICAL MICROTECHNIQ'{(ES
marks regarding arrangement of serial sections in Exercise 30. A nota-

tion should be made on the paper lining the tray regarding the tissue, ac-
cession number, plane and thickness of sections, etc. If the entire block
of tissue was ectioned, the working-label may be removed from the peg
and placed in the tray.
Be careful to keep the tray away from any heat source (such as a
lamp) while you are cutting the sections and later. When sections are
stored before affixation it is best to keep them in a refrigerator or other
cool place. If the tray is warmed the sections will stick to the tray. The
section tray should be kept covered at all times when you are not adding
or removing sections, since the slightest draft will blow them about.
23. Storage of Blocks. When only a few sections are required from a
block of ti . ue it i adyisable to retain the remainder of the block until
the sections are processed and found to be satisfactory. If possible, leave
the block attached to the microtome peg along with the label. Otherwise,
remove the block and label and store in a vial in a cool place. Blocks
which have been soaked for a period in water (step 21 , p) cannot be
stored successfully.
13
The
Paraffin
Method
III. Affixing and P rocessing
the Sections
Before further processlDg, the paraffin sections are attached to glass
microscope slides. This process is referred to as affixation. An adhesive
or affixative is usually used to attach the sections to the slides. Before
affixation the sectIOns must be flattened or "spread." This not only as-
sures adherence of the material to the slides but improves observation
under the microscope. Flattening is achieved by floating the sections on
some medium which is warmed just enough to spread the sections. This
is usually accomplished on the slide, but may be carried out in a water
bath before the sections are placed on the slides. It is important that the
temperature of the floating medium is kept well below the melting point
of the paraffin. The paraffin must not be melted nor the tissue distorted.
Once the sections are affixed, the slides are dried thoroughly. It is ad
visable to affix sections at least one day before further processing. The
sections may be stored for a long period after affixation provided they
are protected from dust and dirt.
Affixative8
Two of the most widely used affixatives are Haupt's and Mayer's.
Haupt's is the most readily prepared in the laboratory. Mayer's may be
purchased from biological supply houses and the commercial product is
recommended. A very small amount of either affixative will provide
medium for a large number of slides.
163
Haupt's Affixative
Distilled water . . . ... . .. ... .... ... . ..... .................. .. 100 cc.
Gelatin ... _... _. . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . 1 gm.
Phenol crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 gm.
Glycerine . . . .. ..... ............. . . .. . . •. . ... ... . ......... 15 cc.
Dissolve the gelatin in warmed distilled water, add the phenol and glyc-
erine, and filter. This affixative is used with 2 per cent neutral formalin
as the floating medium for the sections. The formalin denatures the gela-
tin so the film (and the sections) will not be washed from the slides
during processing. When a large number of slides are being processed, or
if the technician must work for many hours spreading serial sections, the
fumes from the warm formalin may irritate the eyes and throat and
Mayer's affixative should be used instead of Haupt's.
Mayer's Affixative
Egg albumen .... . . . . . . . . . . . . .. . . . . . . . . . . . . . . . ... . .. ... . . ... 5 cc.
Glycerine. . . . . . . . . . . . .• . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 50 cc.
Sodium salicylate ....... . . ........... .. . . . .. • . ..•....... •_ 1 gm.
Shake together and filter. Filtering is very slow. Mayer's affixative is

used with distilled water as the floating medium for' the sections. It is
advisable to boil the distilled water before using. This drives out exc,es-
sive air which may otherwise lead to the formation of bubbles under the
sections. Cool the water to room temperature or to the temperature of
the spreading table, before using and store in a closed container.
The most common error in affixation is using an excessive amount of
affixative. The film of affixative should be so thin that it is difficult to
tell it is on the slide. Both Haupt's and Mayer's affixatives tend to break
down in strong alkaline solutions. If such a solution is to be used during
processing it is best to float the sections on perfectly clean slides without
any affixative.
Slide Warmers
Thermostatically controlled slide warmers are a good investment in
any laboratory where quantities of slides are prepared. The slide warmer
is usually provided with a copper top plate with a uniform heat source.
The temperature of the warming plate should be set well below the melt-
ing point of the paraffin. A temperature 10 to 15 or even 20 degrees below
THE PARAFFIN METHOD. m 165
the melting point of the paraffin is sufficient. Warmer temperatures will

spread the sections more rapidly but may result in bubbles, distortion
of the tissue, or the sections may collapse into folds before they can
spread.
When thermostatically controlled warmers are not available, or when
it is desirable to have a number of small spreaders for use by individual
students, effective warmers may be prepared from small metal boxe
heated by an electric lamp. The box should have an open end, so that
the light bulb may be inserted entirely or partly within the box depend-
ing upon the temperature required. The box should be constructed 0
that the lamp will not be in contact with the box. A box 3 inches high, 3
wide and 6 long will permit preparation of 6 slides at one time and may
be heated by a 15- or 20-watt bulb. The box may be constructed of gal-
vanized iron or, preferably, may combine a bottom box of sheet iron with
a copper top plate. The lamp should be held in a swivel clamp to permit
adjustment of its position. Both the box and lamp should be mounted on
a heavy wooden block or attached directly to the work table. Large
spreading boxes may be made on the same principle, using several lamp
bulbs as a heat source. A cruder, but still functional, spreader can be
made by inverting a cut-down No. 10 can over a lamp socket. It is ad-
visable to leave flanges on the bottom of the can, which are bent out and
attached to a wooden block to prevent tipping. In this case an opening
made in the side of the can permits changing the bulb.
Alcohol lamps are frequently indicated for purposes of warming the
slide to flatten the sections. Temperature control is achieved by holding
the slide the proper distance above the open flame . Although this method
may be successful with individual sections it is extremely difficult to use
with serial sections and the danger of overheating is always present. A
simple heating box is to be preferred to the use of an open alcohol lamp
or other flame.
Water baths which are thermostatically controlled may greatly ac-
celerate the preparation of numbers of slides of sample sections. They
are not recommended for serial preparations. A simple water bath may
be set up by filling a flat pan with water of the correct temperature and
placing this on a controlled heat source such as a slide warmer. When
only a few sections are to be spread and no spreader is available, a pan
of warm water may provide the safest and simplest method of spreading
sections. Water temperature can be controlled by adding warm (or cold)
water. The water bath should be filled with boiled distilled water and
the slides coated with Mayer's albumen.
Exercise 29
Affixing Paraffin Sections
The following discussion is based upon the mounting of individual or
several representative sections on each slide. Affixing serial sections is
discussed in Exercise 30.
1. Clean the required number of slides (see Chapter 9).
2. Mark the slides with the proper initials and accession numbers (see
Chapter 3). :t3e careful not to bring the fingers into contact with the slide
surface. The marked surface of the slide is referred to hereafter as the
upper surface.
3. Place a small drop of affixative on the upper center of one slide.
4. Bring the upper, center surface of a second slide into contact with
the drop of affixative on the first slide. Hold the slides by the end edges
between the thumbs and index fingers, crossing the slides at right angles
to one another. Repeat the transfer to successive slides until no further
transfer of affixative occurs. A small drop is usually sufficient for 10 to 12
slides. See fig. 21, A .
5. With a clean finger tip spread the small amount of affixative over
the area where the section will be affixed (the center third of the slide).
Hold the finger flat, being careful not to rub it along the edge of the
slide.
6. Place the slides on a flat surface (with the marked side up) and
add a few drops of floating medium to the center of each slide (2 per cent
formalin with Haupt's and boiled distilled water with Mayer's).
7. Subdivide the paraffin ribbon into the desired lengths (lor more
sections) being careful to cut between the sections. The ribbon is most
readily subdivided with a rather large, rounded scalpel blade, the cut
being made with a rocking motion of the blade.
8. Transfer the sections to the drop of floating medium on the slides,
being careful to place them shiny side down. (This is the position in
which they were removed from the microtome knife. The upper surface
of the ribbon is dull.) The transfer may be made by touching the section
with the tip of a small brush which has been slightly moistened, or with
the tip of a dissecting needle. With large sections, it is best to pick them
up on a spatula (section lifter) or a piece of stiff paper. Be careful not
to damage the tissue.
9. If the section is not floating completely free of the slide add addi-
tional floating medium before step 10. Add the fluid to one side of the
section. Do not use so much fluid that it will tend to run off of the slide.
The section should be positioned in the center of the slide.
10. Transfer the slides to the warmer and check that the temperature
is high enough to spread the section but not so warm as to melt the par-
affin. If the sections are very slow to.flatten it may be necessary to add
additional floating medium. This should be placed to one side and al-
lowed to flow under the section. Let the sections spread until they are
completely flattened.
11. When the sections appear perfectly flat, remove the slides from the
spreader and drain away any excessive floating medium. This should be
done by tipping the slide carefully against an absorbant surface. If the
section tends to flow with the fluid, hold it in position with the tip of a
needle. A thin film of moisture will remain on the slide surface and the
section can now be positioned exactly to the center of the slide with a
needle tip. (It is helpful to have a guide for positioning the sections on
the slides. Draw an outline of a slide on a piece of paper and connect op-
posite corners with diagonal lines. The center point will indicate the po-
sition for the section. You may prefer to mount the section oft' center to
the right. This will a.llow a larger label space to the left. Such positioning
may be inconvenient if stage clips are used on the microscope. All slides
in a series or set should have the sections positioned on the same area of
the slide, since this simplifies observation on a microscope equipped with
a mechanical stage.)
12. Return the drained slides to the spreader to complete drying. With
thermostatically controlled warmers it may be convenient to leave the
slides overnight. On small spreaders which may overheat, the slides
should be removed after 20 to 30 minutes or when they appear perfectly
dry. Store the slides in a dust-free place until they are processed.
Common Difficultiel$ Encountered in Affixation
a. The section collapses instead of spreading. The warmer may' be too
hot so that the section does not have a chance to flatten; reduce the tem-
perature. The paraffin used for embedding may have been too soft for
the thinness of the sections or the hardness of the tissue. This problem
may be corrected by applying a slight, steady pull at opposite ends of the
section (fig. 21, B) with a pair of dissecting needles (placed in the paraf-
fin) .
b. The tissue buckles up off of the slide when the paraffin is expanded.
This. indicates excessive compression of the paraffin, as compared with
the paraffin and tissue at the time the material was sectioned. It usually
indicates that the melting point of the paraffin was not high enough for
the thinness of the sections prepared, or that the block was not cold
enough at the time of sectioning. Sometimes this can be corrected by
FLOATING MEDIUM
A B
Fro . 21 A, method of transferring a drop·of affixative from one slide to a second
slide . B, method of applying tension to the spreading ribbon in order to straighten
the sections.
applying a pull at opposite ends of the ribbon with a pair of dissecting

needles. In extreme cases it may be necessary to cut through the paraffin
above and below each section. This is very laborious but may be the
means of saving irreplacable material. The paraffin must be stretched or
cut before the section dries onto the slide.
c. Tissues appear to be flat but, upon complete drying, are found to be
up off of the slide tn whole or in part. When viewed from below, areAS
which are raised off of the slide produce a mirror-like effect when tipped
before a light. This occurs when the section dries from the edges inward,
trapping some water under all or part of the tissue. This should not oc-
cur if the section is first spread on an excess of floating medium and then
drained before final drying. It uiually occurs if excessive water is left
on the slide to evaporate away, or if the slides are blotted dry to the edge
of the sections instead of being drained.
d. Bubbles appear when the sections are placed on the floating me-
dium, or when the medium is added to the preparation. Ail' has been
trapped under the sections. This may be avoided by lowering the sections
from one side to the other. Trapped air bubbles under areas of paraffin
may be removed by pricking with a needle. Air trapped under the tissue
may be "fished" out from one side with the tip of a curved needle, or the
section may be gently raised from the slide surface and the bubble al-
lowed to escape Do not warm the slide until such bubbles are removed
since they will expand when heated
THE PARAFFIN METHOD. III 169
e. Numerous bubbles develop under the section as it is heating. If

the bubbles develop rapidly the spreader may be too hot. If they
appear after a period on the warmer, they may be due to air in the float-
ing medium The distilled water used with Mayer's albumen should be
boiled shortly before use. If the slides are drained after the section are
spread and before final drying, bubbles are not apt to appear even with
unboiled water. If the bubbles are noticed before the sections have dried
onto the slide, it may be possible to save the tissue by rolling the ertion
as directed under alternate and supplementary method.
f. The sections flow off of the slides onto the slide warmer. The
spreader may be tipped; it should be perfectly flat. Excessive floating
medium has probably been used. The medium should be enough to float
the sections but not be excessive. With luck you may be able to save the
sections by adding cold water quickly, gently pulling the sections back
onto the slide with the tip of a dissecting needle. Remove the slide from
the warmer while holding the sections on the slide; remove the excessive
floating medium and return the slide to the warmer.
g. A deposit or ring is visible around the edge of the spread section.
This usually indicates excessive affixative; the amount of affixative
should be kept to a minimum so that it is difficult to detect its pre ence
on the slide. A ring may also be formed around the tissue if exce sive
fixative has been left in the tissue. If you are still able to process the
slides the ring may be wiped away with a clean cloth after the slides have
been processed into water.
h. The section is visibly distorted and the paraffin melts. Thi is most
commonly due to an overheated spreader. If, however, you are sure that
the spreader is not too hot, it may indicate imperfect removal of the de-
alcoholizing agent (toluol or xylol) during the infiltration of the tissul:'.
Such sections must be discarded. If some of the tissue block remain un-
sectioned, it may be re-embedded in fresh paraffin and new sections pre-
pared.
i. Certain areas of the tissue refuse to flatten. This often occurs when
tissues contain a band of circular muscle, as in transverse sections of the
gut. It will probably be necessary to flatten the tissues mechanically by
rolling them as described under alternate and supplementary methods.
j. Sections are lost during processing. This may be due to anyone of
the above difficulties. It may result if too much affixative is used; if the
slides were not clean; if the slides were not dried thoroughly before
processing; if the sections are processed through strong alkaline solu-
tions; if the slides were scratched; if excessive convection currents de-
170 GENERAL ZOOLOGICAL IIUCROTECHNIQUES
velop due to rapid hydration and dehydration procedures; if the affixa-

tive was spread with a dirty finger; or if the paraffin was overheated and
melted during spreading.
Alternate and Supplementary Methods
The Water Bath

When sections are spread on a water bath they are floated on to the
surface of the water, shiny side down. After they have flattened, an
albumen-coated slide is brought up under the section. The section is held
in place on the slide with the tip of a ~eedle as the slide is withdrawn
from the bath. The slide is then drained, the section placed in the center
of the slide, and the slide placed on a warmer to complete drying. Drying
may be accomplished without further warming, in which case it may be
advisable to roll the sections as described below. The water bath may
give good results with tissues which are otherwise difficult to flatten.
Boiled distilled water should be used in the bath and must be kept clean.
Rolling Sections
Sections containing masses of circular muscle or large masses of con-
nective tissue may be very difficult to flatten by floating alone. In order
to attach them firmly to the slide it may be necessary to use some me-
chanical pressure after initial flattening by floating. Initial efforts at
rolling sections may be unsuccessful and the technique should be prac-
ticed before being used on irreplacable material.
Roll a piece of fine surfaced filter paper on to a small shell vial about
12 mm. in diameter and 75 mm.long. Moisten the paper slightly. Spread
the sections on the slide and drain off the excess water as described ear-
lier. Position the section on the center of the slide and place the slide on
the table (not on the warmer). Carefully roll the paper-covered vial
over the section from·one end of the slide to the other. If the section tends
to move ahead of the roller, there is too much fluid under the section. If
the section sticks to the paper, the section is too warm or the paper too
dry If the tissue is torn, too much pressure was applied or the vial was
pushed instead of being rolled over the material. The slide may be' re-
turned to the warmer or stored in a box to complete drying.
Affixation Without Affixative8
I
If you are employing a technique which breaks down the affixative
(such as a strong alkaline solution) you may have better success by
mounting the sections on slides without any affixative. In this case the
'!'HE PARAFFIN METHOD. III 171
slides must be scrupulously clean, and it is desirable to wash them with

a chemical cleaning solution (see Chapter 9), rinse them very thoroughly
and be extremely careful to keep them clean while the sections are being
spread. The process is otherwise identical with that described in Exerci e
29, the material being spread on boiled distilled water.
Floating Sections on Dilute Affixative
Some workers prefer to dilute Mayer's affixative and use this dilute
solution as a floating medium. This eliminates the necessity of coating
the slides with the affixative. Dilute 1 part of affixative with 100 part
of water (boiled and cooled), and use as a floating medium. The main
objection to this technique is that a ring of floating medium is al-
most always left around the edge of the section, producing an un-
sightly preparation. Be sure to drain the slide after the section has
flattened, or an excessive amount of affixative may be retained under
the section.
Exercise 30
Affixing Serial Sections
The preparation of serial sections involves several separate con-
siderations: (1) proper orientation of the sections on the slide, which
necessitates proper orientation of the block in the microtome ; (2)
mounting the sections in a minimal space; (3) obtaining true serialljec-
tions; (4) actual affixation of the sections to the slides.
Orientation of the Tissue
Serial sections of excised tissues do not generally require any special
orientation. The block should be placed in the microtome so that the
sections are cut across the smallest dimension of the face of the block.
Thus the long axis of the block face is parallel to. the knife edge. This
assumes that the block has been properly subdivided and embedded
in order to obtain the plane of section required.
Whenever possible transver.se sections of entire organisms should
be oriented so that the sections will be viewed "head on" under the
compound microscope. Dorsal should appear up, ventral down, and
the left hand side to the viewer's right. Since the image is "inverted"
when viewed with the compound microscope, the sections must be
mounted in an inverted position. Furthermore, since the sections are
obtained and mounted as a ribbon, the material must be cut from
side to side in order to obtain the proper orientation. If the most an-
172 GENERAL ZOOLOGICAL MICROO'ECHNIQUES
terior section is placed in the upper, left hand corner of the slide (as
is the usual procedure) the block must be placed in the microtome with
the left hand side of the object down (next to the knife) when the
object is cut from anterior to posterior (fig. 22) . The sections must be
,(!!. !.r.lo'OTolo'o'o'o ISLo~o'oro!o~oio~

ItIil.!!!.Y'9f91910J 019~OO to Jij'9lQ1O'9 '0'0 §: 0\
"@!Ii ,,,,-.,.i ro §!J (!!{!CiI
lJ'r-I02.
@~
i---
SECTION TRAY
KNIFE c
~ ::~ SECTIONS r--Tl'-~--~--S

-2--~-~--i;--i-i--~-~--~-~-:m
: ~ ; 0 ~ ~I :' ~ ~ ~ ~ =:~
a
~ : " ' 0'0'00101

: ______
L
oooo,i:~":
: O ' O'OICjJ' O'O I_Q_'OT(7ro 0
0 0 1 0 10-" 0_______
0 0 0 0 0 __
0 J:
I
A COVER GLASS AREA

o
-:-- --- --t-
DORSAL
:~l
~ RIGHT~}EFT
,-------r
I I
~ J
E VENTRAL
'/7'/'77 /--r-a-r-J'
B F
FlO. 22. A , positioning of the block in the microtome to obtain proper orientation
for sections for a serial, transverse preparation. The animal is being sectioned from
anterior to posterior. Note that the sections adhere from side to side in forming
the ribbon. B, reduction of block size after removal of the sections for the first
slide (see discussion in text). C, orderly arrangement of sections in a section tray .
Note that the paper liner in the tray has been labeled. D, arrangement of the sec-
tions on a slide so that they will lie within the cover glass area; note that the slide
has been labeled . E, appearance of the individual section on the slide as viewed
with the unaided eye or with a simple microscope. F, appearance of the section
under the compound microscope.
THE PARAFFIN METHOD. ill 173
regularly arranged in the section tray in the order in which they are
cut and a note should be made on the tray when sections ar 10 i.
Transverse sections of very thin, dorso-ventrallv flattened objects
(such as chick embryos, flatworms, etc.) are usually cut from dor al
to ventral surface, across the smalle t dimension of the block face ,
from anterior to posterior.
Utilization of Space
The usefulness of serial preparations is greatly improved if as many
sections as possible are placed on a single slide. Not only does thi re-
duce the number of slides which must be processed. and stored and
reduce the cost of preparation, but it simplifies the examination of the
material. The largest cover glass which is practical with a standard
slide (24 by 75 mm.) is 22 by 60 mm. A I-mm. margin should be left
between the tissue and the cover glass edge, since sections at the edge
of the cover glass tend to fade very rapidly. This means that the maximal
working area of the slide is 20 by 58 mm. When estimating the number
of sections which can be fitted into this area; it must be remembered
that the sections are compressed to some degree during sectioning and
an allowance must be made for expansion at the timE' the sections
are spread. A fairly accurate estimate can be made on the ba is of
the block size. With large blocks the number of sectIOns can be de-
termined very readily. With small blocks the safest procedure is to
take a test strip from the face of the block (before the tissue is reached)
and actually spread them on a slide. If such a test strip is not available
the first row of sections may be spread before mounting subsequent
rows If you find that too many sections were used. remove the slide
from the warmer, cut the extra sections free from the right hand end
of the row and reposition these at the beginning of the second row.
This must be done while the sections are still floating free of the slide
and while the slide is cool Once you have determined the number
of sectIOns which can be accommodated in one row, subdivide the ribbon
into strips of the necessary length. It is advisable to mount the same
number of sections per row on a given slide. This fa.cilitates both mount-
ing and observation of the slide.
In order to conserve space, the paraffin block should be trimmed as
close to the tissue as is practical. Furthermore, when the ti ssue tapers,
as in the salamander larvae used in the illustrations (fig. 13), it is
desirable to reduce the size of the block as the tissue narrows. The block
should not be trimmed into a wedge, however (fig. 13, B), since this
weakens the block and each row of sections would vary in number.
The block is initially trimmed into a uniform block of para.ffin with

parallel sides (fig. 13, A) and a.ll of the sections are removed for
the first slide. The size of the block is then reduced while the block
is in the microtome (fig. 21, B). The technician is cautioned to avoid
any conta{lt with the edge of the knife and to lock the advance wheel in
place. This type of reduction is practical when a large number of
preparations are being made of similar tissue blocks and one can
make a reasonable estimate of the number of sections per row and
the number of rows per slide beforehand; or by carefully measuring
the block and allowing for some expansion. It is not desirable to re-
duce the size of the block for sections on a given slide, since it c~mpli
cates mounting of the sections. If, however, only a small portion of
the block remains, it may be most practical to reduce the size of the
block for one or two rows on the last slide to eliminate the preparation
of an extra slide with only a few sections.
Obtaining True Serial ections
The term "serial sections" refers to a preparation which includes
every section from one end of the tissue block to the other. The term
is frequently applied to preparations which approximate this; that is,
where occasional sections are lost. Thus, class preparations of embryos
for gross microanatomical study frequently are lacking occasional sec-
tions. This usually goes undetected, but if a critical section is missing it
may lead to considerable confusion. In the case of serial sections for
research purposes it is extremely important that every section is ac-
counted for. If, as often happens, an occasional section is lost, the
exact position of the missing sections should be recorded. If large
sections are involved so that only a few are mounted on each slide, a
note may be made on the appropril!,te labels regarding missing sections.
If the sections are small and numerous, the missing sections can be most
readily indicated by leaving blank spaces on the slides (such as would
occur if the sections were lost during processing). Such a system de-
pends upon uniform mounting of the same number of sections in each
row with the rows carefully aligned one below the other. During sec-
tioning a note should be made on the paper liner in the section tray
regarding the position and number of the missing sections.
Affixing the Sections
Before attempting to affix serial sections the student is advised to
mount individual sections as directed in Exercise 28. The general pro-
cedures and problems are the same, and the directions given here are
supplementary to those given earlier. It should be kept in mind tbat
the difficulties of preparing good serial sections are compounded with
small sections.
1. Draw on a card or paper an outline of a slide and superimpose on
this an outline of the cover glass to be used (22 by 40 mm., 22 by 60
mm., etc.). Leave a 3- to 4-mm. margin at the right hand end of the
slide and as much room as possible at the left end.
2. Clean and mark the slides and coat the entire upper surface with
a very thin film of affixative. Since the entire area must be coated,
slightly more affixative will be needed than for single sections. Mark
tbe slides in the area which will not be covered by the cover glass.
3. Determine the number of sections which will fit under the cover
glass to be used. Use a test strip from the same block or make a con-
servative estimate on the basis of block size. Be sure to allow for ex-
pansion of the sections.
4. Divide the ribbon of sections into strips of the correct length
by counting the sections. If you rely upon length of the ribbon you may
find that the rows will vary in size (when small sections are involved),
since the amount of compression or folding is not usually constant
throughout the length of the ribbon. Cut between sections with a rocking
motion with a rounded scalpel blade.
5. Lift the strip by supporting it at each end with a brush tip or
dissecting needle, or by using a section lifter. Place the first row of
sections, shiny side down, at the top of the slide with the most an-
terior section at the left. Successive rows of sections art! placed as
closely as possible to their final position below the first. Be careful
not to overlap the edges of adjacent rows, nor to press the sections
onto the slide. Do not place the slides on the warming table until the
floating medium has been added. Avoid accidental beating of the slide.
6. When all of the sections are in place, add the floating medium
to one end of the slide so that it will flow under the sections. Add enough
fluid to float all of the sections free of the slide. Do not, however, add
so much fluid that it will flow off of the slide, pulling the sections with it.
If any air bubbles are trapped under the sections remove them before
warming the slide (see correction d in Exercise 28) .
7. Carefully transfer the slide to the slide warmer, being careful
not to bring your fingers into contact with the floating medium. Hold
the slide by the edges at the left end.
8. Permit the sections to flatten, adding additional fluid if necessary.
If the ribbon strips tend to curve toward the edge of the slide they must
be straightened at this time. Each row of sections should be perfectly
parallel to the edge of the slide so that the finished preparation can be
effectively examined with the aid of a mechanical stage. The time ex-
pended in straightening the sections will be saved many times over
during examination of the material. When the sections have warmed
and while they are spreading, a slight tension is applied to the ends of
each row with clean, sharp dissecting needles (fig. 21, B). The needles
are inserted into the paraffin at either end of the row at the edge which
forms the inside of the curve. It is advisable to rest the hands upon the
spreading table. Just enough tension is applied to straighten the ribbon
and bring the sections into alignment with the edge of the slide. If
the needles are not clean and sharp they may stick to the paraffin with
disastrous results. In general it is best to straighten the bottom row
first if the sections curve toward the top of the slide and vice versa.
This avoids contacts between successive rows. If, however, the upper
(or lower) row is curving so badly that there is danger of its flowing off
of the slide, it must be straightened first. If the amount of curvature is
extreme it may be necessary to place the rows on the slide one at a
time, straighten one, remove the slide from the warmer, add the next
row, and so on. Straightening curved ribbons is effective only if the
sections originated from a properly trimmed block. If the sections
curve because the block was wedgeshaped, the sections cannot be
straightened without danger of distorting the tissue.
9. During the flattening process the slide should be kept well flooded
with the floating medium. When the sections are flattened and straight~
ened the slide is removed from the warmer and the excess fluid is drained
away. Be careful not to lose the sections! A thin film of fluid should
remain on the slide so that it is possible to move the sections into perfect
alignment before final drying. Position the sections so that they lie
within the cover glass area and sq. that successive sections are aligned
with comparable sections in the rows above and below (fig. 22, D).
This positioning is carried out on the cooled slide, off of the warming
table. If you find that the sections are not perfectly flattened , reflood
the slide, rewarm, and repeat steps 8 and 9 as required.
10. Return the slide to the warmer to complete drying. Refer to Ex-
ercise 28 for common difficulties encountered in affixing sections. When
processing serial sections it is best to spread, drain and position the
sections on one slide before begining work on the next slide.
Processing Paraffin Sections
Processing the affixed sections involves seven successive steps:
1. Deparaffination. Most stains are in aqueous solution and a few
are in alcohol. Neither will have any effect on tissues permiated with
paraffin. It is necessary, therefore, to remove the paraffin before further
processing. This is accomplished by submerging the slides in a paraffin
solvent such as xylol. Two changes of the solution should be used, leav-
ing the slides in each change for about 5 minutes.
2. Removal of the Deparaffining Solution. Since the deparaffining
solution is not miscible with water or dilute alcohol it must be re.-
moved from the tissue. This may be accomplished by processing the
slides through two changes of 100 per cent alcohol, leaving the slides for
about 5 minutes in each change.
3. Hydration. If the staining solutions are in water, as is usually
the case, the slides must be hydrated before they are stained. If the
stains are in alcohol the slides should be processed into alcohol of com-
parable strength. The slides are processed through a series of alcohols
(usually 95, 70, 50 and 30 per cent) and into water. Rapid hydration
from 95 per cent alcohol directly into water may distort the tissue, and
the convection currents produced will tend to strip the sections from
the slide. The slides are left 2 to 3 minutes in each solution.
4. Staining. As has been indicated in Chapter 7, staining procedures
vary greatly with different stains and usually involve staining, de-
staining, and washing of the tissue. A separate mordanting solution
may be required, as in the iron hematoxylin technique, or a series of
staining solutions may be employed, as in Mallory's triple stain. De-
tails of these procedures are discussed in the exercises which follow.
The primary or nuclear stain is usually in aqueous solution, whereas
the cytoplasmic or counterstain is frequently in alcoholic solution and
is introduced during the dehydration process.
5. Dehydration. In order to obtain a permanent preparation, having
optical properties which are optimum for examination by transmitted
light, the material should be mounted in a medium having a high
refractive index which approximates that of the tissue (Chapter 1) .
The resins utilized as mountants are not miscible with water and
the material must be dehydrated before the cover glass is mounted. This
is accomplished by running the slides up through a graded seri s of
alcohols (usually 30,50, 70, 95 and two changes of 100 per cent) . lide
are left 2 to 3 minutes in each of the lower alcohols and about 5 minutes
in 95 and each change of 100 per cent alcohols.
6. Dealcoholization and Clearing. In the case of Euparal, the slides
may be mounted directly from 100 per cent or even from 95 per cent
alcohol. With other resins, however, the alcohol must be re,moved with
xylol , toluol or some solvent which is miscible with the moun ting
178 GENERAL ZOOLOGICAL )(ICBOTECHNIQUES
medium. Xylol is recommended for this process since it is less volatile

than toluol and the problem of mounting the cover glass without drying
the material is thereby reduced. Xylol may be employed even when
the resin is dissolved in toluol. The slides should be left for 5 minutes
in each of two changes of the dealcoholizing agent. The dealcoholizing
agent also clears the tissue, since it has a high refractive index. If
the material is opaque in appearance it indicates that the object was
not properly dehydrated, and it must be returned to fresh 100 per cent
alcohol.
7. Mounting the Cover Glass. The procedure for mounting the
cover glass has been described in Chapter 9. An alternate method for
mounting long cover glasses on serial sections is given here. The long
cover glass is difficult to handle with forceps and is difficult to lower
on to the slide without trapping bubbles in the preparation. If the cover
glass is held in the finger tips, however, and flexed slightly as it is
lowered, the procedure may be simplified. Remove the slide from the
second change of xylol, grasping it in the left hand from below at
the left hand side of the slide. The tips of the index finger and thumb
should extend up at the sides of the slide at the level of the first sections.
Place a drop of mountant at the left end of the slide at the level of the
first sections. Grasp a long cover glass in the right hand, holding it by
the edges and from above at one end. The thumb and index finger should
extend downward over the edges. Place the free edge of the cover glass
on the slide in contact with the drop of mountant ami grasp it firmly
between the finger tips holding the slide. Flex the cover glass slightly
and slowly lower it until it is fiat on the slide. The finger tips holding
the cover glass are extended downward over the edges of the slide as
the cover gla.ss is lowered.
Setting up ihe Solutions
The solutions used in processing the sections should be placed in
clean, perfectly dry conta.iners. This is particularly critical with the
deparaffinating-dealcoholizing solutions and the 100 per cent ·alcohols.
All containers should be provided with lids and should be kept covered
as much of the time as is practical. The containers should be plainly
labeled (on the jar, not just on the lid) and should be set out in an
orderly fashion. The types of containers useful for processing slides
and the mechanics of handling the slides are discussed in Chapter 9.
In considering the general procedure of processing sections you will
notice that the solutions utilized are comparable to those employed
for dehydration and clearing of wet smears. In gener&l, a single set-up
of solutions may be employed for running slides "down" (deparaffina-

tion, etc.) and "up" (dehydration, etc.) the series. The two changes
of xylol should be marked "I" and "II" as should the two changes of
100 per cent alcohol. These must always be used in the same order when
running slides down, and in the reverse order when running slides up.
It is best to use a separate xylol for final clearing before mounting
unless a very s~all number of slides is being processed. This change
might be labeled "xylol M" (mounting). The sequence of use for the
standard series would be: xylol I and II; 100 per cent alcohol I and II;
95, 70, 50 and 30 per cent alcohols. When running up the series the
order is reversed (30, 50, 70, 95, 100-II, 100-1, xylol II) and xylol M
is substituted for xylol 1.
Some xylol will be introduced into 100 per cent alcohol I and some
alcohol into xylol II. These solutions should be watched very carefully
for signs of contamination. If, upon placing the slides in 100 per cent
alcohol I from xylol, a distinct cloudiness occurs and persists for more
than a few seconds, the alcohol is no longer sufficiently water-free to re-
move the xylol. If a flocculant precipitate appears in the alcohol, it
indicates that the xylol is not removing all of the paraffin. Similarly,
if a white film forms on the slides when they are passed from 100 per
cent alcohol I into xylol II, it indicates that the material is not suf-
ficiently dehydrated and the solutions must be changed. The frequency
with which solutions must be changed is a matter of experience. If
the xylol and alcohol are watched they serve as good indicator of when
olutions need changing. Lower alcohols shOUld be changed fairly
frequently, especially if they are ObVIOusly discolored with certain
stains. A considerable amount of eosin may be tolerated in both the
lower and higher alcohols when processing slides through the alum
hematoxylin and eosin techniques. If a film of eosin is visible on the
slide surface, however) the solutions need changing. Do not attempt to
down-grade solutions (that is, do not use 100 per cent alcohol I as
'II or 100 per cent alcohol II as 95 per cent) . This is not practical
since the contamination is not only one of water passing up the
series, but of xylol passing down the series. Xylol M may be used to
replace xylol I when the latter becomes dirty or contains considerable
paraffin. A fresh solution is then used for xylol M.
Exercise 31
Heidenhains's Iron Hematoxylin
Suggested Materials. Onion root tips fixed in Karpechenko'8; rat,
mouse or other testes fixed in Bouin's fluid.
Prior Treatment. Fix as recommended in Chapter 5. Wash the ma-

terial as required by the fixative; dehydrate, dealcoholize and embed
in paraffin (Exercise 24) ; section at 8 to 10 microns (Exercise 28), and
affix the sections to slides (Exercise 29). The sections should be affixed at
least one day before processing.
1. Remove the paraffin in xylol I and II and remove the xylol in
100 per cent alcohol I and II; leave for 5 minutes (or. more) in each
solution.
2. Hydrate the material by running through 95, 70, 50 and 30 per cent
alcohols and into distilled water; leave for 2 minutee (or more) in each.
3. Mordant the slides in 2 per cent iron alum (ferric ammonium
sulfate) for 1 hour.
4. Rinse the slides, one at a time, through three changes of dis-
tilled water. Leave about 20 seconds in each change, agitating the
slide in each solution.
5. Place in ripened 0.5 per cent hematoxylin (p. 81). Leave for 1
hour to several days. Overnight staining is recommended.
The hematoxylin solution should be a light brown color. It will
darken gradually to a blue or black color as more and more slides are
processed. If the solution darkens suddenly and a flocculent precipitate
forms, leaving a straw colored solution, it means that an excessive
amount of iron alum has been carried into the stain and the solution
will no longer stain. This occurs if slides are not washed adequately
before being placed in the stain. Properly washed sections will noL
noticeably discolor the staining solution and the sections thems~lves
will begin to darken after a few minutes in the stain. If they do not
darken it indicates that they were washed too well and the stain is not
being picked up by the tissue. In this case rinse in distilled water, re-
turn to the mordant, repeat the wa.shing and return to the stain.
6. Rinse the slides in distilled water and store in a container of fresh
distilled water until they are destained.
7. Working with one slide at a time, destain the slides in 2 to 4
per cent iron alum. The stronger the solution the more rapidly the
destaining will proceed and the less control you will have over the
process. Place the first slide in the destaining solution for about 20
seconds, agitating it slightly; remove and rinse well in distilled water.
Place the slide on a glass plate on the microscope stage and carefully
examine the material. Be careful not to let it dry out and do nQt let
the mioroscope lens contact the fluid on the slide. See Exercise 21 for a
detailed description of the appearance of the material while it is being
destained. Remember that areas having stain will be blue or black:
THE PARAFFIN METHOD. ill 181
areas which are destained will be yellow or brown. The preparation

should show details of nuclear structure and the cytoplasm should be
as free of the stain as possible.
S. Rinse the destained slide in clean distilled water and place in a
. container such as a Coplin jar, under a gently running water tap.
9. Destain the rest of the slides one-by-one.
10. Wash the slides for 30 minutes to 1 hour in running tap water; the
longer period is preferable. This will remove the iron alum, clearing
the cytoplasm of the brown color and will avoid later fading of the
finished preparation.
11. Dehydrate through 30, 50, 70 and 95 per cent alcohols; leave for 2
or 3 minutes in each solution.
12. Optional. Counterstain in 0.2 per cent fast green in 95 per cent
alcohol. Stain for 10 to 15 seconds, rinse in 95 per cent alcohol and
examine on a dry glass plate under the microscope. If necessary re-
turn to the stain.
13. Complete dehydration through 100 per cent alcohols II and I,
in that order; leave for 5 minutes in each change.
14. Dealcoholize and clear the slides through xylol II and M, in that
order; leave for 5 minutes in each change.
15. Mount the cover glass (see Chapter 9) being careful not to let
the preparation dry. Store the slides fiat until the mountant hardens.
Common Faults
a. The ·cytoplasm in the finished preparation is brown or yellow al-
though the chromatin is blu'e or black. Material was not washed long
enough in tap water; old iron alum used for mordanting or destaining;
destaining solution too weak so that an excessive period was required
for destaining.
b. Chromatin not blue or black; frequently the nucleoli are black
but chromosomes brown, yellow or colorless. Material was over-de-
stained.
c. Chromatin black but no details of structure are visible. The
material was not destained enough or the preparation was permitted
to dry.
Exerci8e 32
Ehrlich's Acid Hematoxylin
Sll88ested Materials. AlmOst any histological preparation may be
processed by this method.
Prior TreatmenL Fix as recommended in Chapter 5. Wash the ma-

terial as required by the fixative; dehydrate, de alcoholize and embed
in paraffin (Exercise 24) ; section at 7 to 10 microns (Exercise 28), and
affix the sections to slides (Exercise 29). The sections should be affixed
at least one day before processing.
1. Remove the paraffin in xylols I and II and the xylol in 100 per
cent alcohols I and II; leave for 5 minutes (or more) in each solution.
2. Hydrate the material by running through 95, 70, 50 and 30 per
cent alcohols and into distilled water; leave 2 minutes (or·more) in each
solution.
3. Stain the slides progressively in Ehrlich's acid hematoxylin (p.
SO) . The material may be stained for 1 minute (more or less) in the
undiluted stain; for 1 to 5 minutes in the stain diluted 1: 1 with
saturated aqueous aluminum ammonium sulfate; or for a longer period
in a more dilute solution of the stock. The best combination of stain
concentration and staining period must be determined by trial with
the particular tissue. The use of the saturated aluminum ammonium
sulfate as a diluent, instead of distilled water, aids in restricting the
stain to the chromatin.
4. Rinse the slide .in distilled water and examine. The chromatin
should be blue and the cytoplasm colorless or yellow, but not blue. If
necessary, return to the staining solution. If the material is over-
stained it may be differentiated as described for Harri~' alum hema-
toxy lin in Exercise 33.
5. Wash in running tap water for 30 to 60 minutes. The longer
period is preferred. This assures complete removal of the aluminum am-
monium sulfate and prevents rapid fading of the finished prepara-
tion. It also intensifies the blue color of the chromatin stain, provided
the tap water is slightly basic a is usually the case. If the water is
not basic, treat the slides, after washing, with a weak soda solution.
6. Begin dehydration by running the slides through 30, 50 and 70
per cent alcohols for 2 minutes (or more) in each.
7. Counterstain in 0.2 per cent eosin Y or B in 95 per cent alcohol.
Leave the slides for 10 to 15 seconds, agitating the slides while in
the stain. Remove and rinse quickly through two changes of 95 per
cent alcohol and place the slides in 100 per cent alcohol II. The slides
should not be left for more than about 10 seconds in each wash of 95 per
cent alcohol. From 100 per cent alcohol, examine one of the slides under
the microscope. Place the slide on a clean, perfectly dry glass plate
on the microscope stage, examine quickly and return to the alcohol. Do
not let the slide dry. The cytoplasm should be light pink; the color
THE PARAFFIN METHOD. m 1 3
will be intensified when the slides are cleared. If nece ary restain in
eosin.
8. Complete dehydration through 100 per cent alcohol I, leaving the
slides for 5 minutes.
9. Dealcoholize and clear the slides through xylol II and M, in that
order, leaving for 5 minutes in each change.
10. Mount the cover glass (see Chapter 9), being careful not to let
the slide dry. Store the slides flat until \he mountant hardens.
Common Faults
a. The chromatin is a pinkish purple instead of a bright blue. The
slides may not have been stained long enough; the water wash (step
5) may not have been basic; the solutions used for processing may
have been acid; most commonly, the slides have been overstained in
eosin.
b. The nuclei are too dark, showing little detail of structure and/or
the cytoplasm is blue. The material was overstained in hematoxylin.
c. The cytoplasm is not pink. The material was not left long enough
in the eosin; most commonly the slide was left in 95 per cent alcohol
after staining and the dye was extracted. Do not skip the 95 per cent
alcohol washes or a film of eosin will be left over the slide surface.
Exercise 33
Harris' Alum Hematoxylin

Suggested Materials. Almost any histological preparation may be
processed by this method. Amphibian tissues are recommended for
initial work since the cells aTe relatively large and it is easier to
judge the destaining process than in materials with small cells.
Prior Treatment. Fix the tissues as recommended in Chapter 5. Wash
the material as required by the fixative; dehydrate, dealcoholize and
embed in paraffin; section at 7 to 10 microns, and affix the section
to slides. The sections should be affixed at least one day before proc-
essing.
1. Remove the paraffin in xylols I and II and the xylol in 100 per
cent alcohols I and II; leave for 5 minutes in each solution.
cent alcohols and into distilled water; leave 2 minutes (or more) in
each solution.
3. Stain in' Harris' alum hematoxylin (p. 80) for 30 to 45 minutes.
Ehrlich's a.cid hematoxylin may also be used as the staining solution.
184 GENERAL ZOOLOGICAL MICROTECHNlQUES
4. Rinse the slides in distilled water and store in distilled water

while destaining one slide at a time as directed in step 5.
5. Working with one slide at a time, destain in 0.5 to 1.0 per cent
hydrochloric acid in water. Leave the first slide for 20 to 30 seconds.
Agitate the slide once or twice.
6. Rinse the slide in distilled water and place in tap or alkaline
(0.25 per cent sodium bicarbonate) water. Leave in the alkaline wash
for at least 1 minute before attempting to judge the extent of the de-
staining. The hematoxylin is pinkish until it is blued in the alkaline
wash. If the slides are not blued you will find it extremely difficult to
distinguish stained from destained areas.
Examine the slide under the microscope, protecting the stage with
a clean glass plate. There are two things to watch for: (1) the chromatin
should be blue and should be sharply differentiated from the nucleo-
plasm; (2) the cytoplasm should not be blue. You may find that the
details of nuclear structure are clearly visible before the cytoplasm is
destained. If so, continue to destain until the cytoplasm is free of the
dye, unless in so doing you remove the stain from the chromatin. You
should find that the hematoxylin has a greater tendency to stay in the
chromatin than in the cytoplasm. Destained areas of the cytoplasm
may be whitish, grayish or even pink, but should not be blue. Certain
cytoplasmic areas will retain the hematoxylin, especially after certain
fixatives. Thus, connective tissue areas may retain the dye when the
general cytoplasm is destained and the nuclei properly differentiated.
The short, initial destaining period will probably not be enough to
differentiate the tissue. If necessary, rinse the slide in distilled water,
return to the destaining solution and repeat the destaining, rinsing,
blueing and examining until the correct effect is obtained.
7. Destain the remainder of the slides.
8. Blue the slides in a weak alkaline solution for 10 to 20 minutes.
A strong alkaline solution will blue the slides more rapidly but may
break down the affixative holding the section on the slide.
9. Re-examine the slides after blueing to be sure they were properly
differentiated. Destain further if necessary, rewash and reblue.
10. Begin dehydration by running through 30, 50 and 70 per cent
alcohols, leaving for 2 minutes in each.
11. Counterstain in 0.2 per cent eosin Y or B in 95 per cent alcohol,
leaving the slides for 10 to 15 seconds. Wash quickly through two
changes of 95 per cent alcohol and place in 100 per cent alcohol. See
Exercise 32, step 7, for further remarks regarding the staining and rins-
ing.
THE PARAFFIN METHOD. ill 1 5
12. Complete dehydration through 100 per cent alcohol I, leaving

the slides for 5 minutes.
13. Dealcoholize and clear the slides in xylol II and xylol M, in that
order; leave for 5 minutes in each change.
14. Mount the cover glass (see Chapter 9). Store the slides flat
until the mountant drys.
Common Faults
The faults described for Ehrlich's acid hematoxylin (Exercise 32)
apply here as well. If you find that you cannot destain the cytoplasm
without losing the stain from the chromatin, try varying the length
of the staining period; in general, the best procedure is to increase the
time in the stain by 15 minutes.
Exercise 34
Mallory's Triple Stain
Suggested Materials. Most effective with materials containing dif-
ferent tissue elements including masses of connective tissue.
Prior Treatment. Fix as recommended in Chapter 5. Mercuric
chloride fixatives are generally recommended before Mallory's; freshly
fixed Bouin's-treated tissues also give good results. Wash the material
as required by the fixatives; dehydrate, dealcoholize and embed in paraf-
fin; section at 7 to 10 microns, and affix the sections to slides. The sec-
tions should be affixed at least one day before processing.
1. Remove the paraffin in xylols I and II and the xylol in 100 per cent
alcohols I and II; leave for 5 minutes (or more) in each solution.
cent alcohols and into distilled water ; leave 2 minutes (or more) in
each solution.
3. Place in 0.2 per cent acid fuchsin for 5 to 10 minutes. The entire
tissue will take up a bright red stain.
4. Transfer to 1 per cent phosphomolybdic or phosphotungstic acid;
leave for 10 minutes to overnight. The chromatin should remain a bright
red; the cytopillsm, varying shades of pink and the connective tissue
should be free of the stain. This is a selective destaining-mordanting
solution and will not over-destain. In general the longer the tissue is
treated with phosphomolybdic acid the longer it may be stained in
Mallory'S II (giving a deeper blue to the connective tissue), without
obtaining an unrestricted stain.
5. Transfer the slides directly from phosphomolybdic acid into Mal-
lory's II (aniline blue and orange G, see p. 86). Stain for 5 to 30

minutes.
6. Rinse the slides in phosphomolybdic acid and examine under the
microscope. The chromatin should still be red; the general cytoplasm,
pink; connective tissue should be blue, and erythrocytes orange. If
necessary return to the stain.
7. Dehydrate rapidly to 95 per cent alcohol. It may be necessary to
skip 30 per cent or even 30 and 50 per cent alcohols. Reduce treatment
from the usual 2 minutes to about 20 seconds in each change, agitating
the slides in the solution. Leave in 95 per cent alcohol for 2 minutes.
Examine the slide under the microscope; if the aniline blue has been lost
return down the series to phosphomolybdic acid and reprocess through
the stain; obtain a deeper stain and/or dehydrate more rapidly.
8. Complete dehydration through 100 per cent alcohols II and I (in
that order) leaving for 3 minutes in each change. If the fuchsin appears
too dark it may be lightened by a longer treatment in 100 per cent
alcohol. The alcohol should not remove the aniline blue, but may ex-
tract the orange G.
9. Dealcoholize and clear in xylol II and M; leave for 5 minutes in
each solution.
10. Mount the cover glass and store the slide Bat until the mountant
has hardened.
Common Faults
a. The aniline blue is very weak or absent. It was probably washed
out during the dehydration series. Increase the initial staining period;
this will be most effective if combined with a long period in phos-
phomolybdic acid before staining.
b. The aniline blue is unrestricted, replacing the acid fuchsin in the
cytoplasm. The acid fuchsin stain may have been too weak or the ex-
posure to phosphomolybdic acid too short.
c. The acid fuchsin is very pale. The initial stain may not have been
dark enough; the slide may have been left in 100 per cent alcohol for
a long period; the slide may have been exposed to a basic solution.
d. The acid fuchsin is very dark. The initial staining period may .have
been too long; the staining solution may be too concentrated; the treat-
ment with phosphomolybdic acid may have been too short. This latter
is indicated if the connective tissue is pink or red. The acid fuchsin
may be lightened by leaving the slides for a long period in 100 per
cent alcohol. It may be preferable to destain the acid fuchsin in a
ba ic water (such as tap water or 0.25 per cent sodium bicarbonate)
THE PARAFFIN METHOD. In 17
after treating with phosphomolybdic acid. The slides should be re-

turned for a further treatment with the acid before staining in the
second Mallory's solution. Destaining in basic water must be watched
very closely, since the dye will be removed from the chromatin a
well as from the cytoplasm.
e. The orange G is unrestricted, staining all of the nuclei. The stain
may be differentiated in 95 and 100 per cent alcohols. If possible re-
duce the time in the second staining solution.
/. The orange G is absent. Stain longer in Mallory's II; leave a
minimal time in 100 per cent alcohol; treat longer in phosphomolybdic
acid before staining. If the tissue will not take up orange G using this
procedure, try using Cason's Mallory stain, as described for wet smears
in Exercise 23. The sections are first processed through xylol, alcohol
and into water, stained as directed for smears and completed as di-
rected in the present exercise.
14
The
Celloidin
Method
The celloidin technique provides a method for embedding and sec-

tioning large, dense, hard, secretion-filled or very delicate objects.
It is extremely useful in botanical technique. Celloidin provides a
tough, resilient embedding mass which affords maximal support to the
tissue. The procedure is carried out at room temperature, and thereby
avoids the hardening and distortion which always occurs to some de-
gree with the heat employed in paraffin embedding. Its primary disad-
vantage is the production of individual sections as compared with the
adhering ribbon of sections obtained from paraffin blocks. It is also
somewhat slower than the paraffin method, although not so much so
as to discourage its use for this reason. The sliding microtome used to
obtain the sections may not be available and this would preclude the
use of the technique.
The terminology associated with this technique is somewhat con-
fusing. As used here, celloidin refers to the general procedure. Nitro-
cellulose refers to low viscosity compounds and Parlodion refers to
high viscosity compounds. Nitrocellulose is used in 10, 20 and 30 per
cent infiltrating solutions and Parlodion in 4, 8 and 12 per cent solu-
tions. Either material is dissolved in a 1: 1 mixture of anhydrous ether
and 100 per cent alcohol. Parlodion (available from most supply
houses) may be obtained in solution form or in dry slabs from which
the solutions are prepared. Nitrocellulose (available from the Hercules
Powder Company) may be obtained in cotton form wetted with
188
TBl!: CELLOIDIN METHOD 189
alcohol or in solution in ether-alcohol. The latter is recommended.

Either product is highly inflammable, burns rapidly and is explosive if
ignited in a confined space. The solvent is also highly inflammable and
very volatile as well, and the materials must never be handled near
open flames, cigarettes, sparking machinery, etc.
The procedure outlined here is based upon the use of Hercules nitro-
cellulose, but Pariodion or other celloidin compounds may be sub-
situted directly, using an appropriate percentage series depending upon
the viscosity of the compound.
Exercise 35
Infiltrating and Embedding in Nitrocellulose
1. Fix, wash and dehydrate the tissues to 70 or 80 per cent alcohol as
described in earlier exercises.
2. Complete dehydration through 95 per cent alcohol and two changes
of 100 per cent alcohol. With small tissues (up to 4 mm. square) 30
to 60 minutes in each solution is adequate. Large blocks of tissues
should be left 12 to 24 hours in each solution depending upon size and
density.
3. Transfer to a 1; 1 mixture of 100 per cent alcohol and anhydrous
ether. Leave for a period comparable to that used in step 2. This and
subsequent solutions should be in stout, tightly stoppered containers.
Do not use rubber stoppers but corks which are covered with foil or
which have been dipped in nitrocellulose. Keep the containers away
from any heat source or open flames.
4. Transfer to a 10 per cent solution of nitrocellulose in ether-alcohol.
The exact percentage is not critical but the solution should be thin.
Leave small blocks for a day or more, large blocks for a week or more.
5. Transfer to 20 per cent nitrocellulose in ether-alcohol for a com-
parable period.
6. Transfer to thick nitrocellulose (about 30 per cent) for a com-
parable period.
7. Pour fresh, thick nitrocellulose into a Stender or similar dish of
appropriate size. The depth of the nitrocellulose should be about twice
that of the tissue. The diameter of the dish should be at least 10 to
15 mm. greater than that of the tissue. Pour a very thin film of ether-
alcohol on to the surface of the thick nitrocellulose. Transfer the tissue
from the thick infiltrating nitrocellulose into the embedding nitro-
cellulose. The cut surface of the block, .which is to be s.ectioned ~rst,
is placed flat on the bottom of the contamer. Cover the dlsh but do not
seal the lid i.n place. Invert a beaker or similar container over the
dish to form a small evaporation chamber. In areas with a high hu-

midity it is advisable to place the dish in a desiccator.
The hardening of the block should be gradual and uniform. If a
hard glassy layer develops on the surface of the nitrocellulose mass
the rate of evaporation must be reduced. If no visible thickening has
occurred in 24 hours the rate may be accelerated by propping up the
covering beaker or even the lid of the container. As the mass begins to
solidify, the upper margin of the nitrocellulose should be freed from
the surface of the glass dish by running a sharp instrument around
the margin ; usually this needs to be done only once. Several days to
a week will be required to air harden the block to the consistency of
firm rubber. The material should be soft enough to be readily trimmed
with a razor blade.
Small blocks of ti 'sue may be embedded in vials whose diameter
should approximatc thc height of the nitrocellulose mass in the vial.
The cork is insertcd loosely and the vial placed under an inverted beaker.
The rate of evaporation may be controlled by adjusting the tightness of
t he stopper in t he vial.
8. Trim the block with a razor blade. Leave at least 5 mm. of
nitrocellulose under the tissue to serve as a base for attachment to
the peg. Leave 3 to 4 mm. of nitrocellulose on each side of the block.
Blocks which have been embedded in vials may be left in cylindrical
form if the amount of nitrocellulose is not excessive. The block may be
labelled by scratching the accession number in the nitrocellulose.
9. Soak a hardwood or fiber peg in ether-alcohol. The peg should
have a surface area approximating, or larger than, the base area of
the block to be moun~ed . It is best to cross-groove the upper face of
the peg to improve attachment of block to peg. Square pegs are usually
employed for mounting celloidin blocks.
10. Pour some thi ck nitrocellulose into a flat dish and add a thin film
of eth er- alcohol to the surface. Remove the peg from eth~r-alcohol and
place the grooycd end in the nitrocellulose. Dip the base end of the
tissue block in ether-alcohol for a few moments and then hold it
in th thi ck nitrocellulose. Remove block and peg from the nitro-
cellulo e simultaneously and press the two nitrocellulose-coated sur-
faces together. This must be done very quickly or the peg and block
will separate during sectioning. A paper label may be attached to
t he peg ""ith thick nitrocellulose.
11. Place the peg and attached block in a container of chloroform or
chloroform vapors (the latter is obtained by having a generous supply
of chloroform in the bottom of a tightly stoppered bottle). The chloro-
THE CELLOIDIN METHOD 1 1
form will harden the nitrocellulose attaching block to p g, and will com-
plete hardening of the tissue block itself. The compl d block i much
firmer than the rubberlike mass produced by initial hardening by
evaporation. If evaporation is allowed to continue beyond the rubbery
stage the block becomes glasslike and will shatter. The final harden-
ing, therefore, is one of displacing the remaining alcohol with chloro-
form instead of permitting the last of the solvent to evaporate off.
lt is desirable to change the chloroform at least once before attempting
to section the block. The material may be stored in chloroform or chloro-
form vapors for an indefinite period before sectioning. uch blocks
will provide very thin sections. The material may also be stored in
80 per cent alcohol or in glycerine-alcohol, but such material does
not usually provide a hard enough block for thin sections (under 12
microns) and is only recommended when thick sections of 15 or more
microns are required.
If an adequate mass of nitrocellulose is left below the tissue block
and the nitrocellulose is well hardened in chloroform, the tissue may be
clamped directly into the microtome, using the nitrocellulose itself as a
microtome peg. This procedure is recommended when a large number of
blocks are to be prepared and stored.
Exercise 36
Cutting Celloidin SectiODS
Celloidin is extremely tough and resilient as compared with paraf-
fin. It is almost impossible to cut celloidin blocks on a rotary or other
microtome where the knife edge lies at an angle of 90° to the cutting
direction. Indeed, the resistance is so great that damage may result
not only to the block but to the knife and the microtome. If, however,
the knife edge is slanted between about 20 and 45° to the direction of
the cutting action, the material may be sectioned without difficulty.
The exact slant angle of the knife will vary with the hardness of the
material. The slant of the knife should not be confused with the tilt
of the knife. The tilt angle is that formed between the cutting facet
of the knife edge and the face of the block (fig. 15, B- D). The slant
angle is that formed between the entire cutting edge of the knife and
the direction of movement of the block or knife during sectioning
(fig. 23, A and B).
The sliding microtome (fig. 24) differs from the rotary type in
both structure and action. As mentioned earlier, the knife on the rotary
microtome is held at an angle of 90° to the direction of cut, whereas
on the sliding microtome it may be varied from 0 to 90°. The knife is

positioned vertically on the rotary microtome and horizontally on the
sliding microtome. The knife is stationary during sectioning on the
rotary type and in motion on the sliding type. The tissue block is ad-
vanced upon the knife and brought down over the surface of the knife
on the rotary microtome. On the sliding microtome the block is ad-
vanced between the cutting of each section, but is stationary at the
time of sectioning. Both types of machines are equipped with adjust-
ments for aligning the block with the knife and for adjusting the tilt
of the knife.
In addition to the differences in action inherent in the microtomes,
the process of cutting celloidin sections differs from that for paraffin
sections in that the knife and block must be kept wet with alcohol
CUTTING
BLOCK DIRECTION
I
@J I
I
I
190 0
~ SLANT
~I
'KNIFE
A - KNIFE
~ I
BLOCK
C
FIG. 23. The sla.nt of the knife on the rotary microtome (A) compared with the
slant of the knife on the sliding microtome (8). The b'haded areas on the edges
of the knives indicate the areas which are utilized in cutting the sections. C, the
alignment of sections on the knife blade ror the preparation of serial celloidin sec-
tions (see discussion in text) .
THE CELLOIDIN KETROD 193
during sectioning. Furthermore, the celloidin sections must be kept

wet during storage and processing even though the celloidin i still
present in the tissue.
1. Familiarize yourself with the sliding microtome. Locate the block
clamp and the vertical and pivot adjustments that p rmit alignment
of the block with the knife. Locate the knife clamp and the screws that
permit adjustment of the knife slant and the knife tilt. Locate 8llQ
understand the action of the manual and automatic advance mecha-
nisms Note. Although the knife carriage slides backward and forward
on the knife track, its vertical position remains the same. Check that
the knife track is clean and oiled.
2. Remove the celloidin block from the storage chloroform and
place it in 80 per cent alcohol about 10 minutes before you b gin sec-
tioning.
3. Have on hand a dish of 80 per cent alcohol for flooding the block
and wetting the sections; a second dish of 80 per cent alcohol, to re-
ceive the sections; a wad of clean cotton; and a soft, medium (No.
4) brush. Prepare a label which may be kept in the dish with the sec-
tions.
4. Push the knife carriage to the back of the knife track and place the
knife in the clamp.
5. Set the slant of the knife at about 40° to the direction of motion
of the knife (fig. 23, B).
6. Adjust the tilt of the knife until it is as fiat as possible but angled
enough so that only the cutting edge will strike the block. As in the
case of the rotary microtome the clearance angle should be about
5° and the actual angle of the knife will vary, depending upon the angle
at which the cutting facet has been set (fig. 15).
. 7. Remove the ceJloidin block from the alcohol and clamp it securely
in the peg holder. Do not tighten the clamp to the extent that the
peg will be compressed or the celloidin block may pop off of the peg.
Flood the block with alcohol and be sure not to allow it to dry at any
time. If sectioning is interrupted, cover the block with a wad of cotton
saturated with 80 per cent alcohol.
8. Lower the block vertically until it is well below the level of the
knife and move the knife forward until it lies above the block.
9. Slowly elevate the block until it is just below the level of the
knife. Adjust the block holder so that the tissue is properly aligned
with the knife. This must be done from two positions: from the end
of the microtome, and while viewing the block from the side. Be careful
not to contact the knife edge.
Fro . 24. The slIding microtome. (AO model 860 with knife holder 861; photograph
courtesy of the Americal Optical Company .)
10. Pu h the knife to th e back of the track and flood the knife and
block with alcohol. Elevate the block about 50 microns with the hand
advance mechanism and test its elevation by bringing the knife forward.
Repeat the proce s, being sure that the knife is behind the tissue before
elevating t he block, until the knife contacts the celloidin. Keep block
and knife wet with alcohol. Remove the upper layer of the block with
fairly thick cuts (up to 30 microns) until you are obtaining complete
sections of the tissue.
11. et t he automatic advance mechanism for the thickness of sec-
tion required. Flood the knife and block with alcohol.
12. Pull the knife forward with a smooth steady motion. If the
section rolls as it is cut, cut through the tissue and stop the forward
progress of the knife before it has cut through the celloidin mass
beyond the tissue. Unroll the section upon the blade with a wet
brush; the bru h should cross the knife edge only where it is covered
by the section. Pull the knife through the corner of celloidin attaching
the section to the block and move the section up on to the face of
THE CELLOIDIN METHOD 195
the knife away from the edge. If the sections do not roB as they are cut,
the knife may be drawn through the block with a single stroke.
Although the rolling of individual sections may seem a disadvantage,
it indicates that the block is adequately hardened for the thickness of
section being obtained. UsuaUy these sections are much more perfect
than those which cut without rolling. If the individual sections are un-
rolled as they are cut and allowed to remain, flattened, on the face of
the knife for a few minutes they will remain flat as they are being pro-
cessed. If they are removed without flattening they may be very dif-
ficult to unroll in the processing solutions. You can avoid the rolling
by soaking the block in alcohol to soften it before sectioning (leave
several hours), but you may t hen have difficulty obtaining sections of
the thinness required.
One method of cutting a hard block and preventing rolling is to place
the wetting brush on top of t he block as the sections are being cut. You
must be very careful that the brush does not overlap the edge of the
block, or the knife will be damaged. Furthermore, if you should forget
to activate the advance mechanism by pushing the knife all the way
to the back of the knife track, the knife will contact the brush in-
stead of cutting the tissue, with disastrous results to the knife edge.
This procedure is not recommended until you become very proficient in
the use of the machine.
13. After you have removed the section, push the knife all the way
back and activate the advance mechanism. Pull the knife forward to
clear the advance mechanism before stopping to remove sections, rewet
the block or knife, etc. The sections are not removed from the knife
one at a time but are collected on the blade until a number of sections
have been obtained. Be sure to keep them wet with alcohol.
14. Continue to cut the sections until you have all that are re-
quired or the knife face becomes crowded. The sections are then removed
to a dish of 80 per cent alcohol. The sections may be removed by brush-
ing them over the edge with a brush, or by flooding them over the edge
with a quantity of alcohol. Pushing the sections over the edge may
result in damage to the knife, and pushing or flooding them over the
edge may cause som~ of the sections to slip onto the bottom face of
the blade, where they are difficult to remove. A better method is to
lift the sections by brlnging a wet brush into contact with one edge of
the section and then pushing gently toward the section while turning
the brush in a counterclockwise direction.
The sections may be stored in alcohol before processing, but long
storage will tend to soften the tissue and make it difficult to handle
without damaging the material.
15. When you have finished sectioning, remove and carefully dry
and oil the knife before putting it away. Remove the knife carriage from
the track and carefully clean the riding surfaces on both carriage and
track and coat them with a thin oil.
Common Difficulties Encountered in Sectioning
a. The tissue sections appear mushy and broken. The block is not
hard enough for the thinness of sections being cut. Return to fresh
chloroform. The knife may be generally dull; the slant of the knife
may be wrong; the tilt of the knife may be wrong; you may not have
enough alcohol on the block and knife. ~
b. The tissue is badly scratched or torn. The knife is nicked; move
the knife in the carriage so that a different area of the blade is utilized.
Sharpen the knife.
c. The tissue separates or falls out of the surrounding nitrocellulose.
The block is not adequately hardened; the tissue is not properly in-
filtrated; the slant or tilt of the knife are not adjusted correctly; or the
knife is dull.
Exercise 37
Processing Free Celloidin Sections
The celloidin sections may be stained, dehydrated, and mounted with
the celloidin intact. The sections are processed in fiat dishes and a
large number may be handled at one time. The material may be
moved from solution to solution on a brush, spatula or strip of stiff
paper; or the solutions may be removed and fresh solutions added to
the original container. Use the first method for a few sections, the latter
method for a large number of sections. In general the sections may be
processed by any of the methods described for paraffin sections or by
those presented in Chapter 16, or, for that matter, by most of the other
procedures used in histology. Stains which color the celloidin should
be avoided, or such stains must be carefully removed from the celloidin
during processing. Two examples are given here to provide a general
outline for adapting other staining techniques to processing free cel-
loidin sections. It should be noted that: the dehydration series may
be shortened, since the sections are not readily distorted while sup-
ported by the celloidin; if the celloidin is to be retained through
mounting, absolute alcohol must be avoided in the dehydration series;
the length of time required for mordanting, staining, destaining, etc. is
generally shorter than for attached sections; some mechanical pre sure
must be applied to the finished mount in order to obtain a Bat prepara-
tion.
A. Mallory s Triple Slain

1. Transfer the sections from 80 per cent alcohol into 50 per cent
alcohol for 1 minute.
2. Hydrate in distilled water for 1 minute.
3. Stain in Mallory 's I (aCld fuchsin) for 2 to 5 minutes.
4. Rinse in distilled water and place in 1 per cent phosphomolybdic or
phosphotungstic acid for 5 to 30 minutes, or until the connective tissue
and the celloidin are free of the acid fuchsin.
5. Stain for a few seconds in Mallory's II (aniline blue and orange G)
and remove to phosphomolybdic acid and examine. If the connective
tissue is not blue, return the section to the stain. If staining is dark
enough proceed to step 6.
6. Transfer to 70 per cent alcohol and leave until all of the aniline
blue has been extracted from the celloidin. Agitate the sections slightly.
7. As soon as the aniline blue is removed from the celloidin, transfer
the tissue to 95 per cent alcohol and leave for 3 minutes.
8. Submerge a piece of stiff paper in the alcohol containing the
sections and pull a section onto the paper. Flatten the section carefully
with a clean, water-free brush. Do not let the material dry.
9. Invert the paper and submerge the Battened section into a dish of
carbol-xylol. The tissue must be perfectly flattened when placed in
the carbol-xylol; any wrinkles which are present at this point will be es-
sentially impossible to remove since the carbol-xylol and xylol will
harden the material. The tissue should become transparent almost
as soon at it is placed in the carbol-xyol. Opaque areas indicate the
presence of water which usually is introduced with a wet brush or by
placing the carbol-xylol in a wet container. If necessary transfer to
fresh carbol-xylol.
10. After a few minutes transfer the sections to xylol and leave for
3 to 5 minutes before mounting. The sections may be left for a longer
period before being mounted. Thus, a number of sections may be col-
lected in xylol before mounting procedures are initiated.
11 Have a number of clean slides and cover glasses available. The
slides should be marked with the accession number or other appropriate
information (see Chapter 3) .
12. Place a drop of mountant on the center of a clean slide. Pick
a section up on a piece of thin paper (cigarette papers are excellent
198 GENERAL ZOOLOGICAL MICROTECHNlQUES
for this purpose) ; invert the paper and place the section in the drop
of mountant. Roll a finger tip over the section and remove the paper;
if the section tends to stay with the paper hold one corner down with
a dissecting needle.
13. Immediately place another small drop of mountant on top of
the section and mount the cover glass.
14. Cut a disk of cork from a cork stopper and place this on the
center of the cover glass over the section. Clamp the cork in place
with an ordinary snap type clothes pin and set the slide aside to dry.
B. Safranin 0 and Fast Green
Suggested Materials. Plant tissues fixed in F.A.A. or Karpechenko's.
1. Hydrate the sections by passing through 50 per cent alcohol and
into distilled water; leave 1 minute or more in each solution.
2. Stain in 1 per cent safranin 0 for 10 to 30 minutes.
3. Rinse well in distilled water to remove superficial stain.
4. Transfer to 70 per cent alcohol for 1 minute.
5. Destain in 0.25 per cent hydrochloric acid in 95 per cent alcohol
until the cytoplasm is almost free of the safranin and the chromatin is
slightly darker than finally required.
6. Stop the destaining process by placing the sections in fresh 95
per cent alcohol; leave for 1 to 2 minutes.
7. Counterstain in 0.2 per cent fast green in 95 per cent alcohol; a few
seconds should suffice. Rinse the sections in 95 per cent alcohol and
examine. If necessary, return to the stain. If staining is too dark the
fast green should be diluted further for subsequent sections.
8. Dehydrate in carbol-xylol, complete clearing in xylol and mount
as directed in Section A.
In animal tissues the chromatin should be red, the cytoplasm green.
In plant tissues the chromatin, cutinized, and lignified material will
be red; other areas, green.
C. Removing the Celloidin before Mounting
If you find that the stains are retained in the celloidin it is necessary
to remove the matrix after staining and before mounting. This is
practical with dense, solid structures such as spleen, liver, etc., but
is impractical with materials which tend to separate. The celloidin
may be removed by running the sections through two or more changes
of fresh 100 per cent alcohol after· staining and dehydration through 95
per cent alcohol. They should be left until all of the nitrocellulose is
dissolved. The sections are then processed through xylol and mounted
as described in Section A. Unless the tissue is very tough it is inad-
visable to apply a clothes pin for flattening, since without the support of
the celloidin the tissue may be crushed. Some pressure may be applied
by ' placing a small lead weight on the cover glass.
Exercise 38
Affixing Celloidin SectiODS
It may be desirable to affix the celloidin sections to slides and re-
move the celloidin before processing. This is indicated when the stains
employed tend to color the celloidin itself, or when serial sections are
required from tissues which cannot be sectioned in paraffin. This affixa-
tion procedure is much more difficult to master than the affixation of
paraffin sections and in most instances it is desirable to leave the cel-
loidin intact and process the sections as described in Exercise 37.
1. The sections are cut as directed in Exercise 36. As each section is
cut, align it carefully on the upper, back edge of the knife blade so that
successive sections are in serial order (fig. 23, C). The sections should
be arranged as they will be positioned on the slide, with the most anterior
section at the upper left-hand corner. Care must be taken to orient each
section correctly. When a row approximating 58 mm. has been cut and
aligned, start a second row below the first, and so on, until the area
covered approximates, or is less than, 58 by 20 mm. (assuming that
a 60 by 22 mm. cover glass will be used).
2. Lay a piece of thin paper, such as cigarette paper, over the sec-
tions and moisten thoroughly with 80 per cent alcohol. Roll a finger tip
firmly over the paper so that the sections and paper are brought into
close contact with one another.
3. Coat a slide with Mayer's albumen affixative and mark it with an
appropriate number (see Chapter 3).
4. Carefully slip the paper, with the adhering sections, off of the back
of the knife and place it, sections down, on the albumenized slide. Flood
the paper with alcohol and press the sections firmly onto the slide with
a finger tip which is drawn over the paper-backed sections from one
end to the other.
5. Withdraw the paper carefully. Watch to see that the sections stay
on the slide and not on the paper. If they tend to stay on the paper,
reflood the paper and repeat the pressing procedure. Be careful not
to let the sections dry.
6. Let any excessive alcohol evaporate off of thp sections but do not
let them dry.
7. Place the slide in clove oil; this will remove the celloidin and
coagulate the affixative.
8. When the cellOIdin has been removed (this may require an hour
or more) wash the slide through three or more changes of 100 per cent
alcohol to remove the clove oil and any traces of celloidin which re-
main. Leave at least 5 minutes in each change.
9. Transfer to 95 per cent alcohol and process the material exactly
as though it was a paraffin section from which the paraffin and de-
paraffinating solution had been removed.
Individual sections may be processed in the same way by transferring
them from the dish of 80 per cent alcohol onto the albumenized slide on
a piece of paper.
Exercise 39
Double Embedding
It is possible to combine the supporting qualities of celloidin with the
ribboning properties of paraffin by double embedding the tissue. This
technique may be very useful for processing small arthropods when
serial sections are required and paraffin alone is not providing adequate
tissue support. Large blocks of material are difficult to process with
this method.
1. Fix, wash and process the material into thick nitrocelhllose as
described in steps 2 through 7 in Exercise 35.
2. Trim the air hardened block (of soft rubbery consistency) . close
to the tissue. Leave overnight in chloroform or until the block sinks t.o
the bottom of the container.
3. Transfer to fresh chloroform for about 1 hour.
4. Transfer to chloroform saturated with paraffin.
5. Warm the chloroform-paraffin to the temperature of the infiltrat-
ing paraffin and infiltrate and embed as directed in Exercise 24, begin-
ning with step 9. Use three to five changes of infiltrating paraffin, em-
bed, trim, attach to the microtome peg, section and affix the sections
as directed for paraffin sections. When spreading the material it will
probably be necessary to roll the sections as described on page 170.
Chloroform, like terpineol, is rather difficult to remove from the
tissue and may be replaced by toluol (or xylol) and t~luol-paraffin (or
xylol-paraffin) after the first change of chloroform. If there is any
indication that the material is not perfectly dehydrated, treat with
carbol-xylol before final clearing.
15
The
Freezing
Method
The freezing technique provides a method for sectioning: (1) fre h

tissues immediately upon dissection or biopsy; (2) fixed material
without exposing the tissue to the reagents necessitated by the paraffin
and celloidin techniques. In zoological procedures, the sectioning of
fresh tissue may be useful in providing thin sections of material for
fixation in low penetrating fixatives such as osmic acid. The sectioning
or' fixed tissues is useful for preparing material for histochemical
tests and for the demonstration of fats.
Exercise 40
Cutting and Staining Frozen Sections
A. Sectioning
1. Fix the material in 10 per cent formalin . If the tissue is fixed
considerably in advance of processing store in 5 per cent formalin. Wash
the tissue overnight in running tap water before sectioning.
2. Familiarize yourself with the operation of the microtome to be
used in sectioning. The sliding microtome, discussed under celloidin
sections, is readily adaptable to cutting frozen sections. The machine
must be provided with a block holder having a dry ice chamber or
with an attachment for connection with a tank of carbon dioxide gas.
The clinical microtome (fig. 25) is satisfactory for preparing frozen
sections. In machines of this type the knife is held in a horizontal posi-
201
FlO. 25. The clinical microtome equipped with a CO. freezing platform . (AD
model 888 ; photograph courtesy of the American Optical Company .)
tion as on the sliding microtome but swings, instead of slides, through

the tissue. The motion of the knife is controlled by a rotating wheel
to the side (as in the rotary microtome) or by an arm at the top of
the machine. In either case the rotation of the wheel moves the knife
through the plane of the tissue, returns the knife back over the tissue
to the starting position, and then activates the adv~nce mechanism
which elevates the block into the path of the knife. The thickness of
the section is determined by the setting of a micron scale, which varies
THE FREEZING METHOD 203
in its intervals on different machines. Frozen ection are u uaily ut

between 12 and 15 microns.
The -tissue block holders are metal and consist of a flat, grooved
platform which rides upward toward the level of the knife. No adju t-
ments are provided for aligning the ti ue and th exact plane of sec-
tion is dependent upon the positioning of the ti sue on the freezing
platform. Holders adapted for use with carbon dioxide gas have a gas
chamber below the tissue platform and a circle of escape holes around
the sides below the platform (fig. 25 ). The tissue is frozen with short,
intermittent blasts of carbon dioxide. Holders adapted for use with dry
ice are of two general types. In one the dry lce i introduced into a
chamber immediately below the tissue platform and is provided with
a lever which presses the ice again t the bottom of the platform as it
is needed. In the second type the dry ice is held in an open cup to on
side of the tissue platform. The two are connected by a metal bridg
and the dry ice is introdu ced into the cup and covered with alcohol.
The amount and proportions of the mixture are used to control the
hardness of the material.
The tilt of the knife may be regulated as in both rotary and liding
microtomes. The slant of the knife is adju table on some microtomes and
not on others. On the sliding microtonle it i well to position the knife at
a slant of about 55° to the direction of movement. The rcquired posi-
tion will vary considerably with the hardness of the tis ue and must
be determined by trial.
3. Optional. Infiltrate the fixed and washed tissue with 10 per cent
gelatin kept fluid in an incubator for about 12 hours. olidify the
gelatin mass by cooling and cut the tissue out of the mass. Harden
the resulting block in 10 per cent formalin for a few hours before
sectioning. This method is advisable with tissues which tend to separate
upon sectioning, such as testes. Solid tissues need not be embedded.
The use of gum arabic and sugar solutions to reduce the size of the ice
crystals formed when freezing the tissue is not generally necessary when
carbon dioxide is employed for freezing.
4. Place a few drops of water on the freezing Platform and solidify
by adding dry ice to the freezing chamber, or with short blasts of
carbon dioxide gas. Add a few more drops of water and position the
tissue in this as it is frozen. If necessary add additional water to
secure the tissue firmly to the base. It is not desirable to have a large
mass of ice around the tissue itself. Continue to chill until the tissue ap-
pears to be completely frozen . Test the cutting consistency by attempt-
ing to cut a section. If the tissue crumbles it i too hard; if it com-
204 GENICIlAL ZOOLOGICAL MICBOTECHNIQUES
presses or distorts it is not hard enough. Permit it to soften in the first

instance; harden it further if necessary. When you obtain a good
section, indicating that the tissue is at the correct degree of hardness,
cut a number of sections as rapidly as possible. The procedure of
refreezing, thawing to the correct point and cutting a number of sec-
tions is then repeated.
A number of sections may be allowed to accumulate on the knife
but should not be allowed to dry. They may be removed from the
knife and transferred to water with a wet brush; by flooding them
off of the knife with a stream of water; or by rolling them onto a finger
tip which was first dipped in water. This latter technique, once it is
mastered, is the most satisfactory If the material was not fixed before
sectioning place it directly into 10 per cent formalin or other suitable
fixative.
5. Frozen sections may be processed through the usual staining tech-
niques. In general progressive methods are recommended, since they
require a minimum of tissue handling and the sections are quite fragil e.
The sections may be handled and processed as directed for free cel-
loidin sections, although the normal series of dehydration alcohols
should be used (30, 50, 70, 95 and two changes of 100 per cent). Alter-
nately the sections. may be affixed to slides in the manner described
for celloidin sections (Exercise 38) and, after removal of the clove oil
in alcohol, are hydrated, stained , dehydrated and mounted.
Materials to be stained for the demonstration of fats are processed
without attachment to slides and are mounted in glycerine or glycerine
jelly. The procedure for staining fat in sudan II is explained in Section
B of this exercise.
B. Surian II jor Fat
Suggested Materials. Blocks of tissue containing fatty areas, fixed
in formalin and sectioned on the freezing microtome. Ii frozen sections
are not available this technique may be applied to such preparations as
mesenteric spread fixed in formalin (Exercise 5) .
1. Sections fixed after freezing, or mesenteric spreads fixed in for-
malin, should be washed in water before processing.
2. Replace the water with 50 per cent alcohol and leave for about 1
minute.
3. Replace the alcohol with a saturated solution of sudan II in 50 per
cent alcohol. Leave the material in the stain until the fatty areas are red.
About 5 minutes will suffice.
THE FREEZING ~OD 205
4. Wash in several changes of 50 per cent alcohol until all of the

superficial stain has been removed from the tissue.
5. Transfer to distilled water.
6. Optional. Stain in hematoxylin (Exercises 32 and 33) .
7. Mount in glycerine or glycerine jelly using the procedurt'f; outlined
in Exercises 9 and 10. The tissue is transferred to the slide on a strip of
paper and every effort should be made to mount it as flatly as possibl
Seal the finished preparation.
16
Miscellaneous Techniques
The procedures presented in this chapter represent special techniques

which require considerable practice and technical judgement. In a
general course they may be utilized as special projects or supplementary
work for students who have achieved a degree of excellence in the stand-
ard procedures.
Exercise 41
Bulk Staining
In certain instances it may be practical to stain tissues in bulk be-
fore they are sectioned. Usually the material is stained with the primary
stain, either carmine or hematoxylin, before embedding and is then
counterstained after sectioning, ;ffixation and removal of the paraffin.
This is very useful for serial preparations of small embryos, etc. Blocks
which are to be embedded in celloidin may also be processed'in this man-
ner. The primary problem is in obtaining a uniform stain through the
block of tissue, and materials which are bulk stained should not exceed
3 or 4 mm. in thickness. When in doubt, leave the material somewhat
darker than is finally required and complete differentiation after sec-
tioning.
1. Fix the material and wash as required by the fixative (Chapters 4
and 5) .
2. Stain by one of the following methods. Stain in Grenacher's alum
or borax carmine as directed for whole mounts in Exercises 15 and 16.
206
MISCELLANEOUS TECHNIQUES 207
Stain in alum hematoxylin, using a dilute solution as described in Exer-

cise 17; or stain regressively in Ehrlich's or Harris' alum hematoxylin
(Exercises 32 and 33). Staining in the dilute solution will require ev-
eral days; blocks to be stained regressively should be left in the stain
for several hours or overnigh t depending upon size.
3. D ehydrate, infiltrate and embed in paraffin or celloidin.
4. Section and affix paraffin sections to slides. Celloidin-embedded
material may be processed without affixation.
5. Process the sections to 95 per cent alcohol and examine. If the stain
is satisfactory proceed to step 6. If the stain is too dark , differentiate in
0.5 to 1.0 per cent hydrochloric acid in 70 per cent alcohol followed by
a wash of clean 70 per cent alcohol to stop the dcstaining and remove
the acid. Hematoxylin-stained material should be blued in 70 per cent
alcohol to which a pinch of li thium carbonate has been added. Wash in
clean 70 per cent alcohol before counterstainin g. If the stain is too light
the sections may be processed to water and restained.
6. Counterstain carmine preparations in 0.2 per cent fast green in 95
per cent alcohol. Counterstain hematoxylin preparations in 0.2 per cent
eosin Y in 95 per cent alcohol; 10 to 15 seconds in either hould suffice.
7. Rinse in 95 per cent alcohol to remove the :iuperflcial . tain from the
slide.
8. Wash through two changes of 100 per cent alcohol, leaving for 5
minutes in each change. Substitute carbol-},:ylol for free celloidin sec-
tions.
9. D ealeoholize and clear in two changes of xylol, leaving for 5 min-
utes in each. Mount t he cover glass.
Exercise 42
Methylene Blue and Eosin
1. Fix, wash, dehydrate, embed, section and affix the material by the
usual methods. Mercuric chloride fixatives are recommended.
2. Process the sections to water.
3. Stain in 2 per cent eosin Y for 5 to 15 minutes or until the tissue
is heavily stained and bright pink.
4. Rinse well in distilled water to remove all superficial stain from
the slide. Avoid basic water which will remove the eosin from the tissue.
5. Stain in 0.2 per cent methylene blue (aged aqueous solution) for 2
to 15 minutes. The time required will vary with the tissue and fixation,
and must not be excessive or the eosin will be removed.
6. Rinse in distilled water to remove the superficial stain.
7. Transfer to 95 per cent alcohol. Leave only until the excess of
208 GENERAL ZOOLOGICAL MICROTEC~QUES
methylene blue has been removed and the slides begin to appear pink
rather than blue. If the material does not differentiate, add a small
quantity of colophonium to the alcohol.
8. Rinse in 100 per cent alcohol and transfer to carbol-xylol to com-
plete dehydration.
9. Clear in two changes of xylol and mount the cover glass.
The chromatin should be a bright blue; cytoplasm, pink; mucin, blue
to purple
Exercise 43
Feulgen's Nuclear Stain
Feulgen's nuclear stain represents one of the first histochemical tech-
niques. The material is hydrolyzed and the thymonucleic acid stained
with reduced basic fuchsin (p. 83) . The stain should be a bright red
or magenta color and limited to the chromatic areas. It is well to run a
control series of slides, processing the COIl troIs through all steps except
hydrolysis. Mercuric chloride fixation is generally recommended, al-
though any good chromatin fixer may provide good results. The period
required for proper hydrolysis will vary following different fixation pro-
cedures, and must be determined by trial. This technique may be ap-
plied equally well to smears or sections.
1. Prepare, fix and wash smeaTS; or fix, wash, dehydrate, embed, sec-
tion and affix tissue blocks.
2. Process the material to water.
3. Place in cold, 1 normal hydrochloric acid for 1 minute.
4. Hydrolyze in 1 normal hydrochloric acid at 60°C. for 4 to 15 min-
utes. The best time must be determined by trial.
5. Rinse in distilled water. _
6. Stain in reduced basic fuchsin for 1 to 3 hours.
7. Wash the slides through three changes of the following solution:
10 per cent sodium bisulfite ...............•....... ..... ......... 5 cc.
Distilled water .... . .............. .. . ... ............•.......... 100 cc.
Leave for 5 to 10 minutes in each change.
8. Examine the slides and process additional material if necessary.
Vary the time in the hydrolyzing solution and the stain.
9. Wash the slides in running water for 15 to 20 minutes.
10. Dehydrate through 95 per cent alcohol.
11. Optional. Counterstain with 0.2 per cent fast green in 95 per cent
alcohol; rinse well in 95 per cent alcohol.
12. Complete dehydration through two changes of 100 per cent al-
cohol.
13. Dealcoholize and clear through two changes of xylol and mount
the cover glass.
Exercise 44
Flemming's Triple Stain
The greatest difficulty encountered with this stain is in retaining the
crystal violet which is removed by water, lower alcohols, and the orange
G counterstain. This technique is recommended for onion root tips fixed
in Karpechenko's fluid , or other tissues showing numerous dividing
cells.
1. Fix the material in Flemming's fluid or a chromic acid fixative such
as Karpechenko's. Wash as required by the fixative.
2. Dehydrate, infiltrate, embed, section and affix by the usual meth-
ods.
4. Stain in 1 per cent aqueous safranin 0 for 30 minutes to overnight.
5. Rinse well in distilled water.
6. Differentiate in 0.5 per cent hydrochloric acid in water until the
stain has been removed from the cytoplasm; the chromatin should be a
bright pink or red.
7. Rinse in distilled water to arrest the destaining process.
8. Stain in 1 per cent crystal violet in distilled water until the spindles
are a deep violet; remove before the chromatin is stained (10 minutes or
more).
9. Rinse in distilled water, examine and return to the stain if neces-
sary.
10. Rinse in 70 per cent alcohol and then in 95 per cent alcohol.
11. Counterstain in a saturated solution of orange G in 95 per cent
alcohol for 10 to 15 seconds. Rinse in 95 per cent alcohol and then in 100
per cent alcohol.
12. Place the slide on a glass plate on the microscope stage and flood
the slide with clove oil. Examine the material and when the cytoplasm is
orange, spindles violet and chromatin red, transfer the slide to xylol.
13. Complete clearing through two changes of xylol and mount the
cover glass.
The orange G may also be introduced in an aqueous solution or as a
saturated solution in clove oil. In the latter case flood the slide with the
orange G-clove oil solution and then differentiate in clove oil.
Exercise 45
Masson's Triple Stain
There are many modifications of this technique, which involves basi-
cally the use of iron hematoxylin as a primary stain, secondary staining
with acid fuchsin or xylidene ponceau, differentiation of the connective
tissue with phosphomolybdic or phosphotungstic acid, and counter-
staining the connective tissue with aniline blue, light green or fast green.
1. Fix, wash, dehydrate, embed, section and affix by the usual meth-
ods.
3. Stain in Reidenhain's iron hematoxylin, using ferric ammonium
sulfate as the mordanting and destaining solutions (Exercise 31).
4. Wash well in running water for 30 to 60 minutes.
5. Stain in 1 per cent acid fuchsin for 5 minutes.
6. Differentiate in 1 per cent phosphomolybdic acid until the con-
nective tissue is free of the stain.
7. Rinse in distilled water.
8. Dehydrate to 95 per cent alcohol.
9. Counterstain in 0.2 per cent fast green in 95 per cent alcohol. A
short staining period of 10 to 15 seconds will usually suffice. Rinse in 95
per cent alcohol and examine. The connective tissue should be green.
Return to the stain if necessary. The stain may be diluted further for
greater control over the staining process.
10. Dehydrate through two changes of 100 per cent alcohol for 5 min-
utes each.
11. Dealcoholize and clear through two changes of xylol for 5 min-
utes each and mount the cover glass .
.
Exercise 46
Heidenhain's Mallory-Azan
This procedure is rather difficult to master, but is excellent for dem-
onstrating cell types in pituitary or pancreatic tissue. The material
must be fixed in ReIly's fluid (Zenker-formal) , saturated mercuric chlo-
ride, or some other fixative which is acid-free so that the cytoplasmic
granules will not be destroyed during fixation. The material may be
processed in paraffin or celloidin and should be sectioned at 3 to 5 mi-
crons.
1. Fix, wash, dehydrate, embed, section and affix the material to
slid
2. Process the slides to distilled water.
3. Stain in 1 per cent azocarmine in 1 per cent glacial acetic acid.

Leave overnight at 37° or for about 1 to 2 hours at 55°C.
4. Cool to room temperature.
S. Rinse well in distilled water to remove the superficial stain.
6. Differentiate in 0.1 to 0.07 per cent aniline oil in 95 per cent al-
cohol. The chromatin should be red, the cytoplasm pink. Differentiation
must be carefully watched, and usually requires a very short period
(about 10 to 15 seconds). If destaining is very rapid it may be controlled
by further dilution of the analine oil with 95 per cent alcohol.
7. Stop the destaining in 0.1 per cent glacial acetic acid in 95 per cent
alcohol; leave about 30 seconds.
8. Hydrate the slides to water.
9. Mordant in 7 per cent phosphotungstic acid for 1 hour or over-
night.
10. Dilute the aniline blue-orange G stain (p. 86) with three parts
of distilled water (25 cc. of stain to 75 cc. of distilled water). Stain for 1
hour or more.
11. Rinse in phosphotungstic (or phosphomolybdic) acid and ex-
amine. Return to the stain if necessary.
12. Rinse very briefly in 70 per cent alcohol, place in 95 per cent and
examine quickly. If the aniline blue is too dark, rinse again in 70 per
cent alcohol.
13. Complete dehydration in two changes of 100 per cent alcohol.
14. D ealcoholize, clear and mount from xylol.
It may be necessary to substitute two washes of dioxane for the ethyl
alcohol in order to retain the stain during dehydration. Use with ade-
quate ventilation. In the finished slide the granules of the acidophiles
should be red to orange-red. Granules of the basophiles should be dark
blue in a light blue cytoplasm.
Appendix
I. Reagents, Formulas and Resins
Most of the formulas for fixatives, stains, etc., have been presented in
the text (Chapters 4 and 7). A few reagents warrant a more general con-
sideration here; and a few formulas are presented which have not been
given earlier.
A. General Rules jor Preparing and Handling Solutions

1. All solutions should be kept in clearly and correctly labeled con-
tainers.
2. When stock solutions are provided nothing should be poured back
into the stock bottle. If you take too much solution it is better to discard
the excess rather than risk contamination of the entire supply by at-
tempting to conserve a small amount.
3. Distilled water should be used in preparing solutions unless other-
wise specified.
4. When diluting acids always. add the acid to the diluent, never the
diluent to the acid. The acid should be added slowly.
5. Use clean containers for preparing solutions.
6. Strong acids and bases must be handled with care.
7. Avoid inhalation of acid and other fumes, particularly osmic acid
or mercuric chloride powder. The technique laboratory should be well
ventilated and volatile fluids such as xylol, toluol, dioxane, chloroform,
ether, etc., should be kept covered as much of the time as possible.
8. Flammable solutions should be kept away from open flames and
sparks. Be particularly careful with ether and with ether-alcohol-cel-
loidin mixtures.
9. Always wash your hands before smoking or eating after working in
the laboratory. Many fixatives and other solutions are poisonous.
212
APPENDIX 213
B . Accuracy Required in Preparing Solutions

It will be noted that formulas have been presented with what may ap-
pear to be an inconsistent degree of accuracy. Thus, 0.5 cc. of acid may
be combined with 100 cc. (not 100.0 or 99.5) of alcohol. This has been
done intentionally, since it indicates the degree of accuracy required in
the measurements. The standard practice is to print formulas to the de-
gree of accuracy of the smallest component. Thus, 0.5 cc. are combined
with 100.0 cc. in one formula; 1 cc. combined with 100 cc. in a second
formula; and 100.00 cc. combined with 0.24 gram in a third. Although
this may appear more consistent in print, it is not realistic. If a volume
is given as 100.00 cc. it should indicate that the measurement is to be
made in a volumetric flask. For most technique purposes (such as prepar-
ing fixatives and staining solutions) ordinary graduates provide the
necessary degree of accuracy. Volumes of 1 cc. and less should be meas-
ured in graduated pipettes. It is well to use a small graduate for small
quantities (thus use a 1O-cc. rather than a 1000-cc. graduate for meas-
urements from 1 to 10 cc., and so on). Most weights have been given to
tenths of a gram and the balance used should be accurate to one one-
hundredths of a gram. A triple-beam balance reading to one one-hun-
dreths of a gram is ideal for the technique laboratory. Unless milligrams
are specified, an analytical balance is not required for routine prepara-
tions in microscopic technique.
C. Reagents
Acids. The acids most commonly employed in technique procedures
are acetic and nitric acids in fixatives, and hydrochloric acid in decalci-
fying and destaining solutions. When percentages are indicated they are
based upon dilutions of glacial acetic acid (99 per cent) ; concentrated
nitric acid (about 70 per cent), and concentrated hydrochloric acid
(about 39 per cent). Thus, 1 per cent hydrochloric acid for destaining
refers to a solution prepared by adding 1 cc. of concentrated hydro-
chloric acid to 99 cc. of alcohol or water as specified. The exact percent-
ages of the concentrated acids vary slightly with different suppliers and
grades and this must be taken into account when a "normal" solution is
specified.
Alcohol. References to "alcohol" in technique procedures mean ethyl
alcohol unless otherwise specified. Isopropyl alcohol may be substituted
for ethyl alcohol in the dehydration and hydration series. Methyl alco-
hol is preferred for the fixation of smears before staining in Giemsa's.
The substitution of tertiary butyl alcohol for the highel percentages of

ethyl alcohol before paraffin infiltration has been discussed in Exercise
25, Section B. Like dioxane, tertiary butyl alcohol is miscible with both
water and paraffin, but it is usually utilized after partial dehydration
with ethyl alcohol.
Dilutions of alcohols should always be prepared from 95 per cent (or
96 per cent) and never from the much more expensive absolute alcohol.
A simple method of calculating the dilution is to take the number of cubic
centimeters of 95 per cent alcohol as the percentage required (thus use 70
cc. for 70 per cent) and add enough distilled water to bring the final vol-
ume to 95 cc. For 50 per cent alcohol, use 50 cc. of 95 per cent alcohol and
dilute to prepare 95 cc. of 50 per cent alcohol. Larger or smaller volumes
are prepared on a proportional basis. For example: 700 cc. of 95 per cent
will provide 950 cc. of 70 per cent; 35 cc. of 95 per cent will provide 47.5
cc. of 70 per cent. For technique purposes the latter dilution may be 47
or 48 cc., since the exact percentage in the graded series of solutions for
hydration and dehydration is not critical.
Beechwood Creosote.Xylol. Beechwood creosote may be used as a
clearing agent in place of xylol or carbol-xylol. It has considerable
dehydration capacities and, therefore, may be utilized like carbol-xylol
to assure complete dehydration of thick material; or when 100 per cent
alcohol is not available or must be avoided. It is suitable for processing
free celloidin sections, being substituted for carbol-xylol in the dealco-
holization-clearing series (Exercise 37). Beechwood creol:iote if' fre-
quently combined with xylol in mixtures of 1: 1 or 2: 1 by volume.
Carbol.Xylol. Carbol-xylol, as the name implies, is a combination of
carbolic acid (phenol) and xylol. Various proportions are used for this
solution, but a combination of 1 part of melted phenol crystals to 4 parts
of xylol, by volume, is satisfactol'Y. Phenol is a strong dehydrating agent.
The solution is used between 100 per cent alcohol and xylol to insure
complete dehydration of large whole mount materials. It is also used in
place of 100 per cent alcohol for final dehydration of free celloidin sec-
tions. Carbol-xylol may be substituted for 100 per cent alcohol in the de-
hydration series when 100 per cent alcohol is not available.
Celloidin. Parlodion sheets are simply mixed with a 1 : 1 mixture of
anhydrous ether and 100 per cent alcohol. Shake the container occasion-
ally and allow plenty of time for the sheets to go into solution. As much
as a week may be required.
When nitrocellulose solutions are prepared from cotton wetted with
alcohol, an allowance must be made for the alcohol already present. It
is difficult to prepare solutions having a precise percentage of nitro-
APPENDIX 215
cellulose, since it is difficult to determine exactly how much alcohol i

present in the material. Fortunately, the exact percentage of the infiltra-
tion series of solutions is not critical and "10, 20 and 30 per cent" solu-
tions might well be designated "thin, medium and thick," respectively.
If the nitrocellulose stock is specified as containing 70 per cent nitro-
cellulose by weight, the following mixtures will give approximations of
the requi.·ed percentages. For 20 per cent nitrocellulose ("medium solu-
tion"): mix 144 grams of nitrocellulose-alcohol with 200 cc. of 100
per cent alcohol and 250 cc. of anhydrous ether. For 10 per cent nitro-
cellulose ("thin solution"): dilute the above mixture with an equal
volume of anhydrous ether and 100 per cent alcohol, 1: 1 by volume. For
30 per cent nitrocellulose ("thick solution"): mix 216 gram of nitro -
cellulose-alcohol with 162 cc. of 100 per cent alcohol and 250 cc. of anhy-
drous ether.
Ringer's Solutions. Ringer's solutions should be employed for mois-
tening tissues which must be held for any period of time before fixation ;
or for objects to be observed in the living state under the microscope.
For warm blooded vertebrates, prepare the solution as follows :
Sodium chloride ..... . ..... .. . .... . . ...... . . . ... . . . . . . 8.5 gm.
Potassium chloride .. . ..... . ............. . .... ..... .. . . 0.25 gm.
Calcium chloride . ... . ........ . . . ....... . . .. . . . ... ... . 0.25 gm.
Sodium bicarbonate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 gm.
Distilled water ... . . .. ............. . . . .. . . . . ........... ]000 cc.
Some workers prefer to increase the potassium chloride to 0.30 gram
and reduce the calcium chloride to 0.20 gram. For cold blooded verte-
brates and invertebrates, reduce the amount of sodium chloride to 6.5
grams.
Saline Solution. A simple physiological saline solution is adequate for
washing tissues before fixation. For invertebrates and cold blooded ver-
tebrates use 0.75 per cent sodium chloride in distilled water; for warm
blooded vertebrates increase the percentage of sodium chloride to 0.9
per cent .
. Toluol. Toluol is synonomous with toluene; the latter is the correct
chemical name. Toluol is recommended for routine dealcoholization be-
fore paraffin infiltration. It does not harden the tissue as much as does
xylol. It is also preferred over xylol as a solvent for resinous mountants.
Xylol. Xylol is synonomous with xylene; the latter is the correct
chemical name. Xylol is recommended for deparaffination of attached
sections and for dealcoholization and clearing of sections before mount-
ing.
216 GENERAL ZOOLOGICAL M1CROTECBNIQUES
D. Resins
Canada Bal8am. Natural Canada balsam is very slow drying, and
solutions of the dried gum redissolved in toluol or xylol are recommended.
A thin solution may be used for mounting cover glasses on sections, al-
though synthetic resins are now generally preferred for this type of
mount. Balsam is generally employed as a mountant for whole mount
materials. With small flukes, arthropods, or delicate objects such as
hydroids and certain embryos, etc., it is advisable to use a medium-thick
syrupy balsam. If the object is mounted in an open "cell" with the
cover glass resting on the material, or supported by a tripod of glass
chips (fig. 7, C), an excess of balsam should be used to allow for shrink-
age as the mountant hardens. If material is mounted within a solid wall
in rather thin balsam, air pockets are very apt to develop. If this should
occur, flame the cover glass and carefully remove it from the prepara-
tion; add more mount ant and mount a clean cover glass. It may be prac-
tical to leave thick-cell mounts of delicate objects uncovered for several
days on a slide warmer; they must be carefully protected from dust.
Additional balsam is added after the initial mountant has thickened and
the cover glass is then placed on the preparation. Delicate embryos and
hydroids, etc. cannot be mounted in thickened balsam since they are too
fragile to handle in a viscous fluid.
Thick mounts of relatively tough materials (such as flukes) or mounts
of minute objects which tend to drift in the mountant even when the
slides are kept flat, should be mounted in thickened balsam which is es-
sentially solid when cool. Material which is mounted in thickened bal-
sam will usually require infiltration with the mountant as described in
Exercise 17. The balsam may be thickened by permitting some of the
solvent to evaporate off (loosEln the lid, or uncover and protect from
dust). Thickening may be hastened in a convection oven, but excessive
heat must be avoided or the balsam will darken. When the material is to
be mounted, the balsam is warmed (by placing the container in a hot
water bath) and the slide used for mounting is also warmed (on a spread-
ing table such as those used for affixing paraffin sections). The balsam
containing the infiltrated material is also warmed. The desirable thick-
ness of the balsam is a matter of experience. The balsam should be es-
sentially solid when cool, but fluid enough to permit mounting when it
0
is warmed to about 40 C. When preparing large mounts in thickened
balsam it will be easier to mount the cover glass if it is first dipped in
toluol.
Canada balsam tends to be acid and marble chips or balls should be
APPENDIX 217
kept in containers of the solution. If an acid mountant is used the basic

stains will fade very rapidly.
Colophonium. Colophonium has been used as a mountant but is no
longer recommended for this purpose. This resin, in 95 per cent alcohol,
is specified for destaining methylene blue. It is present in small quanti-
ties in alcohol which has been stored in barrels. If the 95 per cent alcohol
used will not differentiate methylene blue, a small amount of colopho-
nium may be added to the solution; apparently, any other natural resin
may be substituted.
Euparal. Euparal is a combination of a number of resins. It has a
relatively low refractive index (about 1.48) and may be obtained in a
green phase (Euparal, vert). This latter phase is preferred by many tech-
nicians for covering hematoxylin preparations. The material is mounted
directly from alcohol, usually from 95 per cent. A special solvent, "Eu-
paral essence," should be obtained for thinning the mountant.
Hydrax. Hydrax has a relatively high refractive index (about 1.82)
and is popularly used for the mounting of diatoms and similar objects.
It is soluble in toluol, xylol or dioxane. The solid resin may be u ed in
the manner described for thickened balsam, the material being mounted
in warmed Hydrax which will be essentially solid when cooled. Materials
to be mounted in Hydrax should be dealcoholized and cleared in xylol
toluol or dioxane.
Synthetic Neutral Re8in8. A number of synthetic resins are now
available and almost every large biological supply house offers a differ-
ent product. These mountants meet the specifications outlined in the
text regarding refractive index and neutrality (p. 106) and it can only
be suggested that they be tried in the laboratory to test for general ea e
of handling, shrinkage, brittleness, fading of specifi c stains, coloring with
age, and so on. Most of them may be obtained dissolved in toluol or xylol
or as dried resins. They are usually employed in a 50 to 60 per cent solu-
tion. Since I have not tried many of these it seems inadvisable to rec-
ommend a specific product. The neutrality of these products will be
maintained only if the solvent employed is also neutral. It is well to keep
marble chips in the solution as directed for Canada balsam.
II. Di8po8a1 of Wa8tes
Many institutions have specific regulations regarding disposal of
wastes, and these should be followed . If DO such regulations exist the
following suggestions may be helpful. The disposal of wastes presents
a real problem in a large laboratory with many students. Wastes should
be disposed of promptly and not allowed to accumulate.
Acids and bases may usually be discarded in the sink with a quantity
of water. If a concentrated solution is involved pour this into a large
volume of water and then discard the diluted solution with a good flow
of water.
Alcohols of 70 per cent or more, whi<;h are not contaminated with
xylol, toluol, etc. should be saved in a separate container for use in al-
cohol lamps.
Alcohols below 70 per cent, and those which are contaminated with
other reagents may be discarded in the sink along with a good flow of
water.
Animal remains should always be placed in a separate container.
These are usually disposed of by incineration.
Celloidin should never be discarded in a sink. Containers should be
provided and emptied frequently. It is well to keep such waste wet with
water. The waste may be disposed of by burning in the open in small
quantities. Do not throw it into an incinerator nor attempt to burn a
large quantity at one time. It is best to make arrangements for its dis-
posal with the maintenance department.
Clearing agents, such as toluol, xylol, terpineol and chloroform, should
be placed in separate containers for disposal. These are often discarded
in the sink along with quantities of water, but in any volume they may
present a fire hazard or, at the least, complaints from other persons
served by the same sewer system. Arrangements should be made with the
maintenance department for their disposal. They may, for example, be
disposed of by pouring them out in an open area in a dump where they
will safely burn or evaporate off.
Paraffin should never be poured into sinks. This includes mixtures of
paraffin with toluol or xylol, etc. Paraffin chips from trimming blocks
should be saved, filtered, and reused. Waste paraffin should be disposed
of in open cans and may be discarded by the usual means for solid
wastes. Never throw paraffin into an incinerator.
Ether rarely presents a disposal problem, since it usually evaporates
off before it is necessary to discard it. Empty ether cans should be left
open when they are discarded, since a small quantity of ether in a closed
can is highly explosive if the can should be incinerated. If a quantity of
waste ether must be disposed of, special arrangements must be made
with the maintenance department.
m. Equipment Usually Needed by the Individual for
Routine Technique Work
Noncorrosive glass microscope slides, 25 by 75 mm. (1 by 3 inches)
Cover glasses
APPENDIX 21
Thickness No.1, 22-mm squares for routine preparations

Thickness·No. 1 or 2, 18- or 22-mm. rounds for whole mounts
Thickness No. 1, 24 by 60 mm. for serial preparations
Sharp, fine scissors for fine work
Sharp, heavy scissors for routine dissections
Two to four dissecting needles
Four to six, No.3 to 5, tapered, Camel's hair brushes
Fine tipped, curved forceps
Straight, heavy forceps
Four to six, rubber-bulbed pipettes
Diamond or carborundum pencil or glass-marking ink
Slide labels of good quality
India ink.
Crow quill or similar pen
Single-edged razor blades
Heavy weight, double-edged razor blades
Wooden or plastic slide boxes
Optional: Section lifter
Cover glass forceps
IV. Scheduling of Work
The scheduling of work for a semester course in technique will vary
considerably, depending upon the number and length of the periods
available. Other adjustments will be required, depending upon the num -
ber of students, availability of equipment and materials, and the par-
ticular interests and backgrounds of the students. In general , it is better
to schedule laboratory sessions for three or even four hours, rather than
for two. Thus, two two-hour periods each week are less desirabl than
a single four-hour session. The processing of tissues through paraffin ,
and the staining and completiDn of slides are more readily adjusted to
the longer periods. The best situation would be to have two, 3-hour pe-
riods each week.
The scheduling of work should be arranged so that tissue blocks are
stored overnight (or longer) in 70 per cent alcohol. Tissues being in-
filtrated with paraffin may be left in toluol-paraffin at room temperature
from one period until the next. Usually all of the tissues required for
the semester can be fixed during one period; they are processed during
the next period to 70 per cent alcohol, where they may be stored until
needed. From 70 per cent alcohol the tissues are processed to toluol-
paraffin; infiltrated and embedded; sectioned and affixed; stained and
completed, during subsequent periods. When periods are long enough it
may be possible to combine certain units of work. The sections, how-
220 GENERAL ZOOLOGlCAL NUCROTECHNIQUES
ever, must be affixed at least one period before they are processed. For
class work the use of Bouin's fluid and F.A.A. for the fixation of tissue
blocks is recommended, since these simplify the handling of material
through postfixation procedures and the tissues may be left in either
fixative for considerable periods of time. When tissues are fixed in fluids
requiring prompt removal and/ or washing in running water, special ar-
rangements wi ll be required.
When scheduling the work every effort must be made to avoid leaving
tissues in alcohol below 70 per cent alcohol, where they will tend to
macerate; or in 9.5 and 100 per cent alcohols, where they may over-
harden. Tis ues should not be left overnight in the infiltrating paraffin
in the oven , except under special circumstances discussed in the text.
The best result with taining procedures will usually be achieved if
the material is stained, differentiated and completed during one period
There are several exceptions to this (as in Grenacher's borax carmine
and iron hematoxylin techniques, where the tissues may be stained from
one period until the next).
Several procedures are usually carried out simultaneously. Thus, one
tissue is being infiltrated while another is being stained and so on. This
necessitates careful scheduling on the part of the individual. Students
who have difficulty accomplishing the work in the time allotted should
prepare a written schedule of work for each period.
The semesters' work may be divided into four portions as follows:
I. The microscope, Exercise 1.
Supravital staining and methyl green fixing-staining, Exercise 2.
Fixation of tissues, Exercises 3 through 7.
Preparation and mounting of unstained materials, Exercises 8
through 14.
II. Stained whole mounts, Exercises 15 through 20.
Smears, Exercises 21 through 23.
III. The paraffin method, Exercises 24 through 33.
IV. The celloidin method, Exercises 35 through 39.
The frozen method, Exercise 40.
Miscellaneous techniques, Exercises 41 through 46.
The separation between the four portions is not as sharp as is indicated.
Thus, fixation and po tfixation processing of tissues for the entire se-
mester occurs in the first portion of work. Blocks should be processed
into paraffin while smears are being prepared; and infiltration with
celloidin could be initiated while work with paraffin sections is in prog-
ress.
The numbers of unstained materials, whole mounts and tissues proc-
APPENDIX 221
essed through paraffin, celloidin and frozen techniques will vary with
the time available. If laboratory time is very limited, the proportion of
the course assigned to the paraffin method should probably be increased.
If celloidin and frozen methods are not included, the use of the paraffin
method may be extended by processing paraffin sections through a num-
ber of different staining techniques, including safranin 0 and fast green
(Exercise 37, Section B) and those procedures outlined under miscella-
neous techniques in Exercises 41 through 46. If frozen techniques are
not included, it is recommended that mesenteric spreads be fixed in
formalin (Exercise 5) and stained in sudan II (Exercise 40, Section B).
V. Selected References
It is strongly recommended that the beginning technician examine all
or some of the following volumes in order to obtain a broader concept
of the types of technique methods available. Most of these books pro-
vide extensive references to the original literature.
CoNN, H. J ., 1953. Biological stains, a handbook on the nature and uses
of the dyes employed in the biological laboratory , 6th ed., The
Williams & Wilkins Company, Baltimore.
COWDRY, E. V., 1948. Laboratory technique in biology and medicine, 2nd
ed., The Williams & Wilkins Company, Baltimore.
DAVENPORT, H . A., 1960. Histological and histochemical techniques, W.
B. Saunders Company, Philadelphia.
GRAY, P., 1954. The microtomist's formulary and guide, Blakiston Com-
pany, Inc., New York.
LEE, B., GATENBY, J. B., AND H . W. BEAMS, 1950. The microtomist's vade-
mecum (Bolles Lee), 11th ed., Blakiston Company, Philadelphia.
LILLIE, R. D., 1954. Histopathological technique and practical histo-
chemistry, Blakiston Company, Philadelphia.
JOHANSEN, D. A., 1940. Plant microtechnique, McGraw-Hill Book Com-
pany, Inc., New York.
SASS, J. E., 1958. Botanical micro technique, 3rd ed., Iowa State College
Press, Ames.
G . .v . ~ llbrsry
U. A.S. BANGAlORE
UNIVERSITY UBRARY.
5 OCT 1971
Ace. NO ,~ii zal
CL.NO. _ _ __
Index
Accesaion numbers, 24 Ammocoetes larvae, 46
.Acetic acid, 29, 213 Amphibian embryos and larvae, 46
Acetic-alcohol, 31 Amphibian blood, 110
Acetone, 118 Anemones, 43
jAcid fuchsin Annelids, 44
. formulas, 82, 86, 87, 88 Anesthetizing, 41
procedures, 99, 185-187, 197-198, 210 Aniline
Acid hematoxylin. &e Hematoxylin, Ehr- blue W S
lich's. formulas, 83, 86, 87
Acids, dilution of, 213 procedures, 100, lIS, 116, 185-187,
disposal of, 218 197- 198, 210-211
&e specific acids. dyes, 82-90
A.F A . fluid, 31 oil, 211
Affixatives, 163, 164 Anticoagulant, 111
Affixing . Aperture diaphragm, 3, 5
celloidin sections, 199-200 Arthrop'ods
paraffin sections, 166-176 double embedding, 200
common difficulties, 167-170 exoskeleton mounts, 72
serial sections, 171-176 fixation of, 45
Agar-agar embedding, II\inute objects, 140 staining whole mounts, 94-
Alcohol ~. Ascaris, 31, 44
dilution of, 214 :r Ascidians. See Tunicates.
disposal of, 218 ~." :. ""'" .-. ., Aves. See Birds.
dehydration with . Sec' J:?_eey'dration. Amoebae t ,,42 •
ethyl, 213 . Azan :·Se~ Heidenhain's Mallory-azan.
anesthetizing with, '41, 45 Azocarmine ' G-
-acetic fixative, 31 formula, 83, 86
fixatives, 29, 31, 32 procedure, 210-211
See also Dehydration. Azures, 85, 88
-formalin-acetic fluid, 31
iso-propyl, 118, 213 Bacula, 100
methyl, 112, 118, 213 Balsam, Canada, 216
n-butyl, 118 infiltrating with, 96, 98
tertiary butyl, 119, 135, 136, 214 mounting in, 93, 96, 98
Alizaran red S storage of mounts, 27
formula, 83 thickening, 96, 98, 216
procedure, 100-101 Basic fuchsin
Alum formula, 83--84
carmine. See Carmine, Grenacher's procedure, 82, 208
alum. Bdelloura, 43, 53
hematoxylin . See Hematoxylin, Harris' Beechwood-creo8ote-xylol, 73, 214
alum. Benzene, 118
223
224 INDEX
Berlese 's gum chloral, 71-72 Cartilage

Bird embryos fixation, 49
fixation of, 54, 56 in situ staining, 101- 102
staining and mounting, 92-94 Cason's Mallory stain
Bladder, 51 formula, 87
Bleaching procedure, 114-115, 116, 187
after osmic acid, 38 Cedar oil, 118
pigmented materials, 72 Celloidin method, 20, 1lS-2OO
Blpod smears, 110-114 affixing sections, 199
Blood stains, 85, 90 attaching the block, 190
Boats, embedding, 124-125 disposal of, 218
Bone double embedding, 200
decalcification, 49 hardening blocks, 189-190
fixation, 49 infiltration with, 189
ground sections, 75-76 preparation of solutions, 214-215
in s'itu stai ning, 100-101 processing sections, 196-199
mounting, 64, 73, 75-76 removal of, 198
sectioning. See Celloidin method. sectioning, 191- 196
Borax carmine. See Carmine, Grenacher's solvents for, 188
borax storage of blocks, 191
Botani cal F. A. A., 31 types of, 188
Botanical material Cestodes, 44
fixation, 57- 59 Chick embryos
embedd ing, 135 fixation, 54-56
softening hard blocks, 160 staining and mounting, 92-94
sectioning. See Celloidin method. Chitin, 79
Bouin 's fluid Chloral hydrate, anesthetic, 41
formula, 32 mountant, 7I
postfixation treatment, 33 Chloroform
Bryozoa, 44, 53, 92 dealcoholizing with, 118, 200
Bulk tissue staining, 206 hardening celloidin blocks, 190
Butterfly wing scales, 14, 63 killing wi th, 41
Chromic acid
Campo,n ularia, 53 fixatives, 36, 37
Canada balsam. See Balsam . postfixation treatment, 37
Carbolic acid, 214 Chromo-nitric fluid, 37
Carbol-xylol, 73, 197, 200, 214 Cleaning
Carbon dioxide gas, 201 cover glasses, 103
Carmine, 78, 91 finished slides, 26, 27
Grenacher's alum lenses, 9, 13
bulk staining, 206-207 osmic acid containers, 37-38
formula, 79 slides, 103-104
minute objects, 97 Clenran e distance, defined, 2
whole mounts, 92-93 Clearing, defined, 18, 119
Grenacher's borax Clinical microtome, 201, 202
bulk staining, 206-207 Clove oil, 199, 209
formula, 79 Cochineal, 78
whole mounts, 94-96 Coelenterates, 43
Carnoy's fluids, 31 Collagen fibers, 35
INDEX 225
Colophonium, 208, 217 Ehrlich's acid hematoxylin. See Hema-

Color image, defined, 6 toxylin, Ehrlich's acid .
Columbia jars, 106 Embryos
Concave mirror, 3 amphibian, 46
Concentration of bird and repti le, 54- 56, 92-94
protozoa at fixation, 42 mammalian, 46
whole mount material, 96 Embedding
minute objects in paraffin, 136-140 agar-agar, 140
Condenser, substage, 3 boats, 124-125
Connective tissue stains, 83, 88 celloidin, 190
Coplin jars, 105 containers, 124-125
Corrosion techniques, 74-75 gelatin, 203
Corrosive sublimate. See Mercuric chlo- paraffin, 126-131
ride. Eosin
Critical illumination, 4 formula, 84
Crystal violet procedures, 98, 182-183, 184
formula, 84, 85 Epsom salts, 41
procedure, 209 Erythrosin, 84
Counterstains, defined, 78 Ether
Cover glasses disposal of, 218
cleaning, 104 killing with, 41
mounting, 68, 99, 106, 178 with celloidin, 189
storing clean, 104 Ether-alcohol, 189
supports, 65 Ethyl alcohol. See Alcohol.
thickness, 12 Euparal, 61, 72, 73, 177, 217
Cutting facet, knife, 142, 143 Exoskeleton mounts, 72
Eyes, 36, SO
Dealcoholizing, 18, 19, 118, 177
Decalcification, 35, 43, 49 F.A.A . fluid, 31
Dehydration Fasciola, 43, 53
before celloidin, 189 Fast green
before paraffin, 117, 118, 134, 135 formu las, 84, 88
minute material, 97, 137 procedures, 82, 80, 94, 95, 98, 198, 210
plant tissues, 58, 135 Fat, 29, 90, 204-205
sections, 177 Feathers, 73
smears, 110 Fecal samples, 66
whole mounts, 92-93 Fecal smears, 42
Deparaffination, 176 Ferric ammonium sulfate, 81, 109
Desilification, 43 Feulgen's nuclear stain
Destaining. See various staining proce- formula, 85
dures. procedure, 208
Digestive system, 49-SO Field diaphragm, 5, 6
Dioxane, 118, 134-135, 211 Field of view, 2
Double embedding, 200 Fixation, 39-59
Dry mounts, 17, 63-66 animal tissue blocks, 47- 51
invertebrates, 42-46, 51-54
Earthworms. See LumbriCU8. larvae and embryos, 46, 54- 56
Echinoderms, 46, 96, 98 mesenteric spread. 56
226 INDEX
plants, 57-59 Glycerine, 50, 66-68

smears, 112, 115 Glycerine jelly, 50, 68-71
Fixatives, 28-38 Golgi apparatus, 29
acetic acid, 29 Grenacher's alum carmine. See Carmine.
acetic-alcohol, 31 Grenacher's borax carmine. See Carmine.
A.F A., 31 Grinding bone, 75
alcohol, 29, 31 Grinding knives, 144-145
alcoholic Bouin's, 32 Gum arabic, 71
Bouin's fluid, 32 Gum chloral, 71
chromic acid, 29, 36
chromo-nitric, 37 Hair, 73
Flemming's osmic fluids, 38 Hard materials, softening, 160
formaldehyde, 29, 30 Harris' alum hematoxylin. See Hema-
formalin, 29, 30 toxylin, Harris' alum.
Hollande's fluid, 32 Haversian canals, 76
Karpechenko's fluid, 37 Heart, 48, 50
Lee's fluid, 37 Helminth eggs, 66
mercuric chloride, 29, 33-36 Hematoxylin, 80
osmic acid, 37-38 Ehrlich's acid
Perenyi's fluid, 37 formula, 80
picric acid, 32 procedure, 181-183, 207
potassium bichromate, 29, 36 Harris' alum
Schaudinn's fluid, 35 formula, 80
Smith's fluid, 36 procedure, 97, 183-185, 207
sublimate-acetic, 34 Heidenhain's iron ,
Susa's fluid, 34 formula, 81
Flatworms, 43, 94 in Masson's, 210
Flemming's fluids, 38 sections, 179-181
Flemming's triple stain smears, 10S-110
formula, 85 Heparin, 111
procedure, 209 Helly's fluid, 34
Flukes, 43, 94 High-dry objective, 12
Foraminifera, 64-66 High viscosity celloidin, 188
Formalin Hirudinea, 45
affixing fluid, 164 - HoJlande's fluid, 32-33
fixative, 29, 30 Holothuroidea, 5( 74
neutral, 30 Hydra, 43
Formalin-acetic-alcohol, 31 Hydroids, 43, 53, 92, 94, 96
Formaldehyde, 29, 30 Hydromedusae, 52, 96
Formic acid, 30 Hydrochloric acid
Freezing method, 19, 201-205 cleaning solution, 104
Freezing microtome, 201, 202 destaining solutions, 95, 102, 184
dilution of, 213
Gelatin embedding, 203 Hydrofluoric acid, 43
Gentian violet, 101 Hydrax, 61, 72, 217
Giemsa's blood stain
solution, 85-86 Immersion lenses, 8, 13
procedure, 112 Immorsion oils, 8
Glare, 5 Infiltration. See various substrates.
INDEX 227
Insects. See Arthropods. Magnesium sulfate, 41

Intensity, light, 6 Magnification, defined, 6
Intestinal protozoa, 42, 138 Mammalian embryos, 46
~n vertebrates. See specific references. Mammalian tissue fixation, 47
fixation, 42-46, 51-54 Mallory's triple stain
Iodine-alcohol, 35 formula, ~7
Iron alum . See Ferric ammonium sulfate. for celloidin sections, 197-198
Isolation techniques, 19, 74 for paraffin sections, 185-187
Iris diaphragm for spreads, 99
lamp, 4, 5 Masson's triple stain
substage, 3, 4 formul a, 88
procedure, 210
Janus green Mayer's albumen affixative, 164, 170, 171,
formula, 86 199
procedure, 21 Mercuric chloride
fix atives, 29, 33-35
Kaiser's glycerine jelly, 68 postfixation treatment, 35
Karpechenko's fluid, 37 Mesentery
Killing, 41 fixation, 56
Knives, microtome, 141-145 staining, general, 99
Kohler illumination, 5, 11 staining, fat, 204
Methyl green
Labels formula, 88
embedding containers, 26, 126 fixing-staining, 22
microtome pegs, 26, 133 Methylene blue chloride
nitrocellulose blocks, 190 formula, 88
section trays, 26, 172 for cartilage, 101
slides, 26, 27, 65 with eosin, 207
working-labels, 25 See also Giemsa's and Wright's stains
Lamp Microscope, 1- 15
microscope, 3 focusing, 9
diaphragm, 4, 5 Microtome
Larvae, 46 knives, 141- 145
Lavdowsky's fluid, 31-32, 55 pegs, 132
Leeches. See Hirudinea. Microtomes
Lee's fluid, 37 clinical, 202
Lepidoptera wing scales, 14, 63 freezing, 201, 202
Leptosynapta, 54 rotary, 20, 147-151
Light intensity, 6 sliding, 191, 194, 201
Lithium carbonate, 33, 207 Mitochondria, 29, 82
Liver, 118, 160 Mollusca, 45, 96
Living material, 17 Mordant, defined, 78
Low power objective, 10 Mossman's fluid, 31, 32
Low viscosity celloidin, 188 Mounting media, 8, 16,60,61,66,68, 71 ,
Lumbricus, 44, 45 106, 216-217
Lung, 50 Multiple embedding, 121, 129
Maceration, 19, 31 N.A., 8

Magnesillm chloride, 41 Navashin's fluid, 37
228 INDEX
Nematodes, '44, 53, 66 Parasagittal sections, 40

Neutral red Parfocal, defined, 10
formula,88 Parlodion, 188, 214
procedure, 21 Perenyi's fluid, 37
Nile blue, 101 Perisarc, 92, 94
Nuclear reaction . See Feulgen's nuclear Phenol, 68, 214
stain Phosphomolybdic acid, 82, 87, 100, 185.
Numerical aperture, 8 197
Nitric acid Phosphotungstic acid, 82, 86, 87, 185, 197,
cleaning with, 104 211
corrosion with, 74 Physiological saline, 39, 215
decalcification with, 49 Picric acid
fixatives, 36-37 counterstain, 89
Nitrocellulose destaining solution, 82
characteristics of, 188 fixatives, 32, 33
disposal, 218 postfixation treatment, 33
preparing solutions, 214-215 Pituitary, SO, 210-211
See a180 Celloidin method. Placoid scales, 74
Planaria, 53
Obelia, 52, 53 Plant tissnes
Objectives, 2 embedding, 135
Oculars, 2 fixation, 57-59
Onion root tips, 59, 179, 209 softening, paraffin blocks, 160
Opaque mounts, 64 See also Celloidin technique.
Orange G Platyhelminthes, 43, 53
formulas, 85, 86, 87, 89 Poriferans, 43, 52, 74
procedures, 98, 99-100, 114, 115, 116, Postfixation treatment, defined, 29
1~186, 197-198, 209, 210-211 acetic acid, 30
Orientation of tissues alcohol, 32
in paraffin, 126, 127 chromic acid, 37
in microtome, 152-153, 158 mercuric chloride, 35
Osmic acid osmic acid, 38
fixatives, 37-38 potassium bichromate, 36
preparing solutions, 37 Potassium bichromate
postfixation treatment, 38 fixatives, 36
Ovens, paraffin, 121 Potassium hydroxide
clearing, 101
Pancreas, SO, 210-211 corrosion, 72, 74
Paper boats, embedding, 124-125 Preparing solutions, 213
Paraffin Primary stain, defined, 78
affixing sections, 166-176 Progressive staining, defined, 78
disposal of, 218 Protozoa
embedding procedure, 123-131 blood, 113
filtering, 121 fixation, 42, 52
handling, 120 fecal preparations, 42, 43, lOB
infiltrating with, 123 fixation-staining, 22
grades of, 119 sectioning, 136
• melting, 120 staining, lOB, 116
ovens, 121 supravital staining, 21 \.
&eetionilllt, 151- 161 whole monnts, 96 ".w ~..!.
INDEX 229
Radula,45 Sliding microtome, 191, 194. 201
Razor blades, 146 Smears, 18, 108-116
Razor blade holders, 145-146 blood, 110
Reagents, 213-215. See also specific re- fecal, 42, 108
agents. protozoa, 42, 52, 108
Records, 24-27 sperm, 87, 108, 112, 114
References, 221 tissue, 108, 114
Refractive image, defined, 6 vaginal, 87, 114
Refractive index, defined, 7 Smith's fluid, 36
Refractive indices, 7, 50, 61, 217 Softening hard blocks, 160
Regressive staining, defined, 78 Spermatozoa, 87, 108, 112, 114
Reptiles, embryos, 54 Spicules, 43, 52, 74
Resins, 216-217 . See a180 specific resins. Spleen, 118, 160
Resolution, de.fined, 6 Sponges, 43, 52, 74
factors affecting, 7, 8 Spreads
Rheostat, 6 fixation, 56
Ringers solutions, 39, 215 staining, 99, 204
Ripening hematoxylin, 81 Squashes, 18
Root tips, 58, 59, 179, 209 Stains, 77-90
Rotary microtome, 20, 147-151 See also specific stains.
Starfish. Sec Echinoderms.
Safranin 0 Stomach. See Digestive system.
formula, 89 Storage
procedure, 198, 209 celloidin blocks, 191
Sagittal sections, 40 fixed tissues, 31, 32, 35, 118, 129
Saline solution, 39, 215 paraffin blocks, 129, 162
Schaudinn's fluid, 35 sections, 161
Sea cucumbers, 54, 74 slides, 27
Sealing mounts, 62 Subdivision of tissues, 40, 47, 48
Sections, cutting of Sublimate-acetic, 34
celloidin, 193-196 Substage condenser, 3, 4
frozen, 201-204 ubstage iris diaphragm, 3, 4
paraffin, 151-162 Sudan II, III
Sections, thickness of, 152 formula, IJO
Serial sections procedure, 204-205
defined, 174 Supports, cover glass, 65, 96
preparation, 171- 176 Supravital dyes, 86, 88
Sharpening knives, 141-145 Supravital staining, 21
Shell vials, 105 Susa's fluid, 34-35
Skin, 50 Synthetic neutral resins, 106, 217
Slant of the knife, 191- 192
Slide boxes, 27 Tapeworms, 94
Slide racks, 105 Terpineol, 118
Slide ringer, 61 Testis
Slides fixation, 51, 114
cleaning, 26, 27, 103 smears, 114
handling during processing, 104-105 staining, 179
labeling, 26, 27 Tilt of knife, 191
storing, '}~ Tissue blocks, fixation, 47-51
S .•rmers, 164-165 TililSue smears, 108, 114
230 INDEX
Toluidine blue 0 Undulating membrane, 113

formula, 90 Uterus, 51
procedure, 101-102
Toluol, toluene, 118, 177-178, 215 Vaginal smears, 87, 114
Tongue, 48 Van Wijhe's stain
Transverse plane, 40 formula, 90
T rematodes, 43 procedure, 101-102
Trichloroacetic acid, 35 Vital dyes, 86, 8S
Triple stains. See Cason's, Flemming's,
Heidenhain's Mallory-azan, Mal- Warming table, 165
lory's, and Masson's. Washing tissues, 57
Types of preparations, 17-21 Wastes, disposal of, 217-218
Water bath, 165, 170
Tuni cates, 46, 94
Whole mounts, 17, 91-102
Turbellarians, 43 Wrights stain
Turn table, 61 formula, 90
procedure, 112
Unstained preparations, 60-76
Urinary bladder, 51 Yolky material, 46, 54, 118
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UNIVERSITY LIBRARY. BANGALORE-S600~
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GENERAL

(.::::I:=LJf1!:::::n~~:2 Labo"'.'Y Tuhnician

Reprinted March, 1964

Indian Edition Published August 1968

Library of Congress Catalog Card Number 60-128J1

Indian edition published by special arrangement with the

This book has been published with the assistance of the

Sales area of Indian Edition: Japan, Taiwan, Okinawa,

Price Rs. 6'50

This book is a product of ten year' of teaching a general course in

good technician, having mastered and understood the basic procedures

PREFACE . . . . . ..... . . . . . . . . . . . . .. .... . . . . . . . . . . . . . . • • . . . v

Fixation of Larvae and Embryos ... . ....... .. .... . . 46

The Coal Tar Dyes and Stains . . . . . . . . . . . . . . . . . . . .. 82

CHAPTER 13, The Paraffin Method, III. Affixin g a nd

III. Equipment Usually Needed by the Individual for

The theoretically ideal illumination source is a point of light, but

A second compromise involves the use of a pair of planoconvex lenses

Refractive Index. The relative velocity of light passing through a

It should be kept in mind that although mo t images are a combina-

mitted through the material. The inherent capacity of an objective to

2. Do not tip it excessively or the oculars may slip out!

Setting Up the intermediate Objective (16 mm.)

cover glass or preparation is so thick that there is not sufficient clear-

THE COMPOUND MICROSCOPE 15

In view of the characteristics of the light microscope and the princi-

tion," which stabilizes the material in as near a lifelike condition as

stead, the material is dehydrated by replacing the water with other

solution. It may be examined in the initial solution or prepared as a

infiltrated tissue block is then hardened by cooling, the block is trimmed,

necessary for paraffin infiltration. The fixed material is dehydrated

a considerable period and notice if within 30 minutes or more the color

6. Mount of sponge spicules or sea cucumber skeletal plates.

Before beginning a technique project, a system of records should be

fixing a letter in alphabetical sequence after the accession number, JJ-

Cleaning, Labeli,.ng and Storing the Finished Slide

The initial step in the processing of an organism or tissue for perma-

water for 12 hours or overnight. If the material is to be stored in alcohol

Formula 1 is in more general use than Formula 2. Chloroform in-

Lavdowllky'll mixture is excellent. for the fixat.ion of small embryos.

Matl'rilll an hr stored in thi olution for a considerable time with-

This fixative is particularly good for flatworms to be prepared as whole

turated aqueoUll mercuric chloride . ce.

Fixation of protozoa smears is completed in 15 to 20 minutes.

Iodine, saturated in 70 per c nt alcohol ........... . 5 cc.

With large blocks of tiB8Ue, removal of the mercury may require a

In the case of alcoholic mercuric chloride fixath·e , the ti ue i

chromic cleaning solution, after which they are thoroughly washed

Fix the material for 24 hours to several days.

In order to obtain as near a lifelike picture of thc material a po-

chloride fixatives flood with saline. If the tissue is subdivided or trimmed

are cut perpendicular to a flattened surface are generally referr d to

specimen. The weight should be sufficient to flatten without crushing

or magnesium chloride. Anesthetize LumbriCtl8 by adding alcohol very

F. Nematodes for Unstained, Glycerine Jelly Mounts

G. Sea Cucumber for Body Wall Mounts or for Corrosion Preparations

Fixation of Bird and Reptile Embryos

An alternate method of removing the embryo is dependent upon its

6. Formalin-fixed material should be stored in 5 p r cent formalin