Plant Pathological Methods Fungi & Bacteria

T h i s i s an a u t h o r i z e d f a c s i m i l e o f t h e o r T g i n a l
book, and was produced i n 1974 by m i c r o f i l m -

xerography by Xerox U n i v e r s i t y M l c r o f i lms,
Ann Arbor, M i c h i g a n , U . S . A .
PLANT PATHOLOGICAL
METHODS
Fungi and Bacteria
JOHN TUITE
Department of Botany and Plant Pathoiogy
Purdue University
Lafayette, Indiana
Copyright O 1969 by Bilrgess Publishing Con~pany
All rights reserved
Printed in the United States of America
Library of Congress Catalog Card N ~ t n ~ b c69-1
r 7858
AiTectionately Dedicated
to
My Devoted Wife Camile

and to
George Cummins and A. J. Ullstrup
Preface
A part of the literature concerning methods of plant pathology are brought together here for the
investigator but particularly for the student and those heking adequate tibrary facilities. The intent is
not to solidify particular ways of doing things but rather t o "open up" the literature and t o describe
currently used methods and to a small degree their rationale. In so far as possible I have included
methods from the literature which have been endorsed by a second party or successfully tried by me.
There are, however, some methods which are unverified and possibly unsuitable. 1 hope they are not
too numerous. I would like t o thank the many persons who have helped me. Those that I recall are: S.
A. Anderson, K. L. Athow, J. B. Bancroft, G. B. Bergeso~!,P, Best, K. R. Bromfield, J. A. Browning.
E. E. Butler, R W. Caldwell, C. M. Christenscn, J. E. DeVay, J. J. Favinger, F. E. F r o s h e i ~ r ,K.
J. Green, G. A. Cries, F. W. Holmes, D. Hornby, J. Hunter, W. G. Keyworth, C. H. Kingsolver, C. H.
Livingston, P. hlislivec, M. B. hioore, G . C. Papavizas, Betty Rice, J. Rice, R. 1. Samson, J. Schafer,Nf.
C. Schnathorst, J. R. Shay, M. C. Shurtleff, R. D. Wilcoxson, E. B. Williams, and J. Williams. 1 wish to
especially thank C. B. C u m i n s , R. W. Curtis, and A. J. Ullstrup for their considerable help and
encouragement.
Table of Contents
Preface..................................................................... i
w e
................................................................. iv
List of Tables
................................................................ v
List of Figures
Chapter
1 Medla and Nutrient Solutions Used by Plant
................................................. 1
Pathologists and hlycolopsts
2 Disinfection and Ster~ltzation:Stedlzation of Laboratory
....................................................... 81
Equipment and hled~d
3 Isolation of Bacterrophage and Plant Pathogcn~c
............................................ 92
Actinomycetes. Bacteria and Fungi
.........................................112
4 Diagnosing the Causes of Plant Diseases
......................................................141
5 Increase of Inoculum
6 Establishment of Disease: Inoculation. Infection
andDiseasedSymptorns ..................................................... 176
...............................................193
7 Preservation of Aitcroorgan~srns
8 Microscopic Techniques.....................................................204
................................... 224
9 Writing for Publication: by George B . Curnmins
Appendu .....................................................................227
Ustof0rg;lnisms ................................................................ p3
Index ............... ....................................................... 237
List of Tables
.
Table I Listing of Media by Use .....................................:..........page3
.
Table 2 Literature on Culture hledia ............................................... 8
.
Table 3 Recommended Time in an Autoclave for
Sterilization of Various Volumes........................................... 82
.
Table 4 Recommended Time and Temperature for
Sterilization with Dry Air.................................................. 84
. .................................. 86
Table 5 Antiseptics and Disinfectants and Their Uses
................................ 88
Table 6 . Some Firms that Sell Bacteriological Air Filters
.
Table 7 Compounds Commonly Added t o Media to
...........................................9 4
Inhibit Fungi or Bacteria or Both
.
Table 8 Diagnostic Characters and Associations of Some
..................................................113
Causes of Plant Diseases
. ............................ 116
Table 9 Hosts and References on Culture. Pests and Diseases
Table 10.References Which Consider D l j z a ~ of ............................. 118
s Nany Hosts
Table 1l.Culture Collections ...................................................... 121
.................. I 2 6
Table 12.Some Diagnostic Features of Genera of Plant Pathogenic Bacteria
Table 13.Some Fungi that Require or are Stimulated
by "Light" to Spomlate ..................................................144
.................................. 147
Table 14.Check List for Inducing Sporuhtion of Fungi
Table l5.Media and Procedures to I n d u e Scxud Reproduction
of Pythium and Phytophtlrom ............................................. 155
raole 16.Ways of Producing Asexual Stages of Various Species
of Pythium and Phytophtlrom ............................................. 157
....................................186
Table 17.Saurces of Equipment for Moist Chambers
Table 18.Recommended Methods of Preserving Various Groups
ofMinooqanisms ....................................................... 194
.
Table 1 The Relationship of Density of Sulphuric Acid
and Relative Humidity ................................................... 228
.
Table 2 The Relationship of Specific Gravity o r Refractive Index of
............................229
Aqueous Clyccrinc Solutions and Relative Humidity
.
Tablc 3 The Relative Humidities of Various Saturated Salt
Solutions at Various Temperatures .......................................... 230
List of Figures
Page
Figure I. Arnold Sterlllzer ....................................................... 83
Figure 2. Sintered Class Filter ...................................................85
Figure 3. Swrnny FJter Adapter ................................................ 87
Figure 4. Microspatula ......................................................... 105
Figure 5. Biscuit Cutter ........................................................105
Figure 6. Micro-spear ...........................................................105
Figure 7 .Darley Isolator ...................................................... 105
Figure 8. Keyworth lsoldtor ......................................................105
Figure 9. Rukngs of the Spenser Hemacytometer wrth the
..................................... 184
Improved Form of the Neubauer Ruling
Figure 10. Plastic Bag Supported by W~reFrame to
Give a Moist Chamber...................................................185
Figure 11.Metal Moist Chamber ................................................... 185
. ....................................... 185
Figure 12 Moist Chamber Used for Large Areas
.
Figure 1 3 Homemade Dew Chamber ................................................185
. ...........................................137
Figure 14 Class Lyophllrzation Apparatus
........................................... 199
Figure 15.Metal Lyophilization Apparatus
. .......................................... 217
Flgure 16 Van Tieghem Cells in a Petri Dish
.
Figure 17 Thm Culture Cell ......................................................217
Media and Nutrient Solutions
Used by Plant Pathologists and .Mycologists
Introduction
Suggestions on Preparation of Media and Related Subjects
Table 1. Luting of media by u s
A. I o h t e , culture, andlor identify Actinornycetes
B. Isolate andlor culture bacteria
C. l o k t e and culturr: of dermatophydc or other &d pathogenic fungi
D. Imlate and culture from wil andlor water
E. I r ~ l a t efungi from plant m a t e d
F. Induce sporulation in fungi
C. Culture and/or for the study of the nutrition of fungi and some bacteria
H. Media to aid in the identification of fungi
1. MedL t o aid in the identifiation of bacteria
J. Culture plant tissue or intact plants
K. Pknt nutrknt solutions
Table 2. Ltterature on culture media
GLtnvare Cleaning
P d c a t i o n of Agar
Literature Cited
Media and Their Ingredients
INTRODUCTION
Since Micheli, many substrates have been used for the culture of microorganisms. As Thom points
out (24), investigators often develop a medium optimum for a particular organism but with no real
attempt to devix one that would support growth of a number of organisms, or gne that could be easily
duplicated. Some of the media listed here are not of this kind but they are extremely useful. The media
ue grouped in table I according t o their use in:
A. The isolation, culture and/or identification of Actinomycetes
B. The isolation and/or culture of bacteria
C. The isolation and culture of dermatophytic fungi and other animal pathogens
D. The isolation and culture of fungi from soil and/or water
E. The isolation of fungi from plant material
F. The sporulation of fungi
G. The culture and/or the study of the nutrition of fungi and some bacteria
H. The identfication of fungi
I. The identification of bacteria
J. The culture of plant tissue
K. The nutrient culture of piants
For sources of commercial media, references on media, and the nutrition of particular groups of
microorganisms see table 2,
SUGGESTIONS ON PREPARATION OF h1EDlA AND RELATED SUBJECTS

In media containing mineral salts, dissolve the salts one at a time. Some prefer to dissolve
chemicals separately and then add them as solutions. If higS04 is included, it should be dissolved first.
A pH below 6 aids solutions of the chemicals. Solubilities of many antibiotics and amino acids can be
found m Kavanagh (10). Vitamins are usually soluble in 2Wo ethyl alcohol and many are soluble in
water.
Because chelates such as EDTA can maintain a constant supply of an ion, they are being used
increasingly in place of the inorganic salts which precipitate more readily with a change in pH of the
medium or by autoclaving (1 1). Buffers (7) are often recommended to maintain the pH of a mehum
(2), but they inhibit some fungi, particularly the water molds (2, 3). Choice of the source of the
carbon-nitrogen and their amounts may lessen pH changes (4), or suitable amounts of acid or a h l i m n
be added at intervals (3). Inclusion of calcium carbonate or magnesium carbonate in the medium may
be helpful for acid producing microorpisms. Some prefer t o add these sokd bufiers after autoclaving.
Common constituents of media such as malt extract, casein hydrolysate, yeast extract, peptone
and tomato paste are complex rnaterhls, and depending on the amount used and sometimes the
particular batch, they may fulfill partly or wholly the organism's requirement for carbon, nitrogen,
growth factors (20) and trace elements. Attempts at purification or elucidation of grotvth factors
involved have not been entirely successful ( I 5, 16).
Media including decotions of natural products, such as potatoes, seeds and plant foliage are
usually prepared by boiling the materials for 20 t o 40 minutes followed by filtering through
cheesecloth and/or cotton if a clearer medium is desired. Other ingredients and melted agar are added
prior t o sterilization. Some natural medu if exposed to strong light may be inhibitory (27).
In critical studies it is recommended that heat labile substances, such as certain sugars, vitamins
and many antibiotics (for properties of individual antibiotics see (10, 17) be sterilized for a minimum
period of time when incorporated into the medium. It is better to sterilize them separately, either by
filtration (sintered glass or membrane fdters such as millipore filters are superior to seitz filters) or in
some cases by heat, and then add them aseptically t o the medium prior to its use. Glucose when
autoclaved with certain amino acids inhibits the growth of some fungi (14) but increases the growth of
an organism when autoclaved with u2Po4(26). It has been suggested (21), aside from the breakdown
of sugars into more available compounds, that autoclaving may be stirnulatory because of the
production of chelating agents.
Cereal grains should be autoclaved for longer periods than ordinary media (40-60 minutes at 121 C
or on 2 successive days) to insure sterilization. See chapter on steriiization for other details.
Agar is the solidifying agent of choice. It is usually melted in an Arnold sterilizer in half the fmal
volume of the medium. Be sure that there are no lumps when the other ingredients are added. The usual
commercial agars are comparatively clean and have a high gel strength, thus permitting use at a
conantration of 1.2% rather than the 2% often stipulated in older recipes. The lower the pH of the
medium the more agar, however, is required. Details on agar may be found in Syke (22).
In nutritional studies agar is usually omitted from the media because of contamination with
vitamins and minerals, but agar may be partially purified by pyridine and/or by washing with various
dvents. Details of cleaning of % a s s w e and purification of agar are described separately.
Nobk agar is an agar washed with wdter, available commercially from Difco, who also produce
mother purified agar (washed in various solvents and mare expensive) for use in nutritional studies.
Consolidated Laboratories, Inc., Chicago Heights, Hlinois, makes an agar (Oxoid No. 2) purified by an
h=cxchange process that is useful for mrrobiological assays.
Media and Nutrient Solutions 3
Purification of inorganic chemicals is difficult and somewhat controversial, thus the following
references should be consulted prior to research in mineral nutrition (2, 5, 13, 18, 19, 23). Growth
rubstances can be removed from glucose (8) and casein hydrolysate (1 2) by treating acid solutions (pH
3) t w i a with Norit A charcoal and filtering with the aid of supercel (1 part of glucose to 20 parts
Norit and 1 part of casein hydrolysate to 10 parts Norit).
If pure water is required, it should be freshly prepared and be either twice glass distilled water or
glaw distilleddeionized water. Deionized water, although extremely low in ions, may contain organic
eontaminants from resins or bacteria. Addition of charcoal (0.5%) to water containing toxic heavy
metals is often valuable.
As cotton plugs may add minute amounts of contaminating substances to media, it may be
desirable to substitute one of the following, meanwhile recognizing changes in aeration rates.
Apparently preautoclaving reduces amounts of contaminants given off by cotton plugs.
1. Squares of cellophane (Dupont type 450 PT) held in place with a rubber band (9).
2. Squares of washed aluminum foil. Air may be limiting in some cases.
3. Stainless steel caps (morton enclosures), available from Bellco Glass Co., Vineland, N J .
4. Polypropylene enclosures. (hlfg, by Biological Research Inc., St. Louis 21, Mo.)
5. Polyurethane foam plugs (apparently gives off a volatile substance which is inhibitory to some
organisms).
6. For static cultures and short experiments, cap flasks with inverted glass jars or beakers.
7. In limited instances an organism may be grown in petri dishes without agar.
8. Milk fiiter discs are used primarily to increase air exchange.
Most of the recipes are given in g r m s per liter. The following are some frequently found units:
milligram (mg) @ .OOl g
microgram (pg) = .000001 g
gamma (7)= .000001 g
One milligram of a substance in a 1 = 1 ppm. A Molar (hi) contains one gram-molecular weight in
1 I of solution. A normal solution contains one equivalent of the active reagent in grams in 1 1 of
solution. Percentage solutions: Percent means parts in 100 parts. Solutions may be made up according
to weight, volume or any combination. Small percentages - less than 3% are usually made up on a wt/wt
basis, for example 3 dl00 g of solution. If the concentration is critical the method used shouid be
reported.
TABLE 1. LISTING OF MEDIA BY USE

Media commonly used to:
A. Isolate, culture and/or identify actinomycetes Peptone yeast extract iron agar
Basal mineral salts agar Porter pirnarcin medium
Bennett agar (also a good sporulation medium Pridham yeast malt extract medium
for Streptomyces) Ridham and Cottlieb trace salts
Carbon utilization medium Silica go1 medium
Casein glycerol medium Tomato paste oatmeal agar
Giycerol asparaginate agar Tnce salts solution
Glycerol asparagine agar Tyrosine agar
Inorganic mlts starch medium Tyrosine caseinate nitrate medium (Strepto-
Jensen agar mycs scabies)
Krainsky agar Tryptone yeast extract broth
Lingappa and Lockwood chitin medium Water agar
Oatmeal agar
Modh and Nutrient Solutions
B. h h t e andlor culture bactaia Glucose nitrate agar (modified)

Agrobacterium isolation medium Hay infusion agar
Beef peptone agar (nutrient agar).- Hemp seed
Burkholdcr medium (Cot)nebacterium sepedo- Malachite greencaptan medium (Fuszriurn and
nicum) ~ m c h a e t terrestis)
a
Carrot discs (Agrobacterium turnefacie~s) Martin medium (as is and modified)
Desoxycholate agar Milk agar (nematode trapping fungi)
Diet food medium Nash and Snyder PCNB medium (Fusarium) see
Elliott agar also PCNB (modified)
Emerson medium modified (Erwin& amyb- Noopeptone rose bengal aureomycin agar
ma) Ohio medium (OAES)
Fluorescin medium (King agar) PCNB (modified) (Fusrium)
Hugh and Leifson carbohydrate medium PDASV
Ivanoff isolation medium (Xrmthonwrtas stew* Reischer agar (Modified) (Pythium)
atti) Reynolds medium (Chitin-decomposing
Peptone water microbes)
Potato dextrose agar Russd Basidiomycete medium
Psetdomonas nwrsprunomm isolation medium Sea water agar
Pyocyanin mediurn Sea water glucose medium
Ring rot medium (Cbrynebacterium sepedoni- Sea water glucose peptone agar
dm) Snake Skin (Aphanomyces)
Soil extract agar Soil extract isolation agar ( Verticilliurn dahlkre,
Reynold medium (Chitin decomposing mi- K albostrum)
crobes) v-8 juice medium (modified) no. 252 ( T h i e t
Tetrvolium medium (Paeudomms solanu- vbpsis basimkr)
ccmurn) Warcup medium (as is and modified)
Thornton medium E. I d a t e fungi from plant material (See also
Tryptone glucose yeast media used to isolate fungi from soil and/or
Van Gundy and H'alker medium water.)
Wieringa pectate gel Alcohol agar (Vetticillurn)
Yeast extract medium (Eminirr arnylovora) Botran-isolation medium (Asperg'llus~us)
Xanthornows vesica.tori0 isolation medium ST Corn meal agar
Xanthomonas vesicaroria isolation medium X-1 Czapek solution agar
C. lmlate and cultute dcmratophytic or other ~ h p c k d o agar
x
animal pathogenic fungi Dutch elm medium (Ceratocystis ulrni)
Littman oxgall agar Fomes annosus-isolation medium
Mymphil agar Malt agar
Mycological agar Mdt mh agar (storage fungi)
Mycosel agar (Commycs immftis) Oak wilt identification agar (Csatocystis fags.
Sabouraud destrox agar ==m)
D. I d a t e and culture fungi from soil andlor w a t a PMA
Alcohol agar ( VerticiRium) Potato dextrose agar
Botran-Isolation medium (A ~ p e ~ l t u s ~ s ) Ru~senbasidiomycete medium
Cehlose asparagine mdhrm Tomato juice sdlt agar (storage fungi)
Carrot discs ( T h i c l r r v i o ~Ceratocflis fimbri- Water agar
ata) F. Induee sporulatlon m fungi (With a representa-
Corn meal agar tivc fungus)
Corn meal PPM @hYto@rhx. and Pythium) Alfalfa decotion agar (S~lemtrlchumgramink)
Dextrose peptone y e a agar (modified)
Medim and Nutrient Solutions
d
Alfalfa seed decotion agar (Alternark brasicae)Hemp seed agar (Phytophthora)
Alphacd medium (a variety of fungi) Kennedy & Erwin EDTA medium (Phytoph-
Aphanomyces washing solution (A. ~uteiches) thora sp.) L:
see alro Glucose-peptone medium Kldn y bean agar ~ h y w p h t h o r a f r r p p ~ e )

Apple infusion agar (Vertturkr inrrequalis) Lactose wein hydrolysate medium (Hebnh-
Armstrong Fusarium medium ( F u ~ r i u m thospltium Nrcicum and H. &ma)
oxyspo~m) b n i a n agar (Mapy Ascomycetes)
Adhana and Hawker medium A (Melanospara Lima bean agar ~hytophthoraspp.)
desmenr = Sordak destmem) Lima bean agar (Ritynchosprium w cnb)
Barnett maltose casamino medium (Cerato- Lukens and Sisler synthetic medium (Ahemad
cyst&fa aceorurn) sohni and H,vagans.)
4
Bean agar Phytophtlwra cactorum)
Bean juice agar (Colletotricltum Wemuthi-
Malt agar
Maltose peptone broth (Aplzanomyces eutei-
""*' r(
Bean meal aga Phytophthora cinnamoni)
Bean pod agar
ches)
Maltose tartrate medium (hfeektnconim fulig-
ineum)
BPSM A (Diplocmpon earlia~u) Neapeptone glucose medium (Cblletornmchum
Bilai medium (Fusaria) Indemt flianum)
Bonner Addicott medium (P/~matotriclutm Neurospora sex synthetic agar
ornnivorum) Nutramigen agai (Ceratorysrisfagaceurn)
Carrot agar (Pythium spp.)
Carrot decotion gar (Cerwspora spp.)
S
Oat meal agar Phytophtfwra spp.)
Oat grain agar (Phytop/rthoracinnamornt)
d'
Charcoal wa t e Pltytophthora pmasitica) '
Cerelose ammonium nitrate medium (F.
Oak wilt agar (See Barnett maltosecaamino-
biotin medium)
oxysparum'$ Orchard grass agar (Scolecotrichm g m h i s )
Chickpea sucro (Phytophthora infestans) Paddy rice medium 4'
CMC medium (Gibberella zeae) Pea brat h (Pythium and Phytophthora)
Corn meal agar
Corn steep dextrose agar
,
Pea r e d (Pythiwn and ~ h ~ t o ~ l t t h o r ~
Petri medium (Pythium and Phytophrhora)
Diet food medium Peat percolate (Phytophthora erytivoseptica
Dung agar vaf. pi$
Dung oatmeal agar (Pilobolus) Phytone dextrose agar (StemphyEim bobli'cki)
Elm extract agar (Ceratocystis ulmi) Plant parts
Emerson YpSs agar (AIlomyces) Pon tecorvo minimal medium (Aspegilhs
Filter paper yeast extract (SorcSarirr) niduhs)
Gnin Potato carrot agar
Glucose alanine agar (Copinus lagopus) Potato dextrose agar
Glucose casamino acid medium (Fusarium Potato ma1t agar (Chaetomiwn)
0xysPQwm) 'd Potato marmite agar ( R h i w c t o n ~
spp.)
Glucose nitrate rterol {~hyto~hthoraand Prune agar
Pythhrm) Radish agar (A phomyces raphano
Glucose yeast agar (Copniuts Ibgopus) Rape seed agar (Phytophthora)J
G l w s e peptone agar (Gbeospltum mztsrmrm) Rice polish agar (Pirim&& orpae. H e m -
Glucose pept one medium (Apftorwmyces thosporium oryzae)
euteiches) Rye seed medium ( ~ h ~ t o ~ h t~~~~~)
dra
Green bean agar (Col&totrichumbgenarfum) Sach's agar (Hetmhthos~riumspp.)
Harrold medium (Eremsars) Schizophyllum fruiting medtum
Hcmp seed J Soil extract agar ,
Hemp seed decotion (Phyrophrhoro spp.) Soil extract (Phytophthora spp,)
6 Mcdia and Nutrient Solutions
J
Soybean agar (Phytophthora infestans) Vegetable oil nitrate agar (Phytophthora and
Sucrose poline (Drechskra spp.) Pythium )
Timothy agar (see orchard grass agar) Vogel medium N (.l'eumspra)
Tochinai solution (F~tsariurnoxysplnrrn) Water agar J
Trione mked cereal agar (Tilletia controversp) Wills mineral nlts medium (Phytophtliora sp)
Trione Dwarf bunt synthetic (Tilletia contra- Yeast extract agar (Cluetomiurn)
vem) Yeast glucose (Aphnomyces sp)
V-8 juice agar
In addition, a variety of natural products are used
-
Seeds other plant parts -other
bean, corn carrot slices filter paper
oat ,pea grass leaves snake &in
rice
sorghum potato slices
wheat sweet clover stems
soybean stems
C. Culture and/or for the study of the nutrition of Machilis medium A
fungi and some bacteria Maltose-tartrate medium
Amino acid substitute for casein hydrolysate Microelement solution
Amino acid substitute for yeast extract Neurospora complete medium
Aphanomyces synthetic Ncurospora minimal medium
Armstrong Fusarium medium Neurospora sex medium
Asthana and Hawker medium A Park medium
A-Z solutions (3 versions) Pectin medium
Barne tt maltose casamino medium Pectin polypectate medium
Carbon utilization medium (For actinomycetes) Petri medium
Cellulose asparagine medium Pfeffer medium
Corn steep dextrose Pontecorvo minimal med~um
Cutter host rust medium Rautella and Cowling cellulose medium
Czapekdox agar Richard solution
Czapek sblut ion agar Sach's agar
Elliott agar Sea water agar
Fothergill sucrose ammonium sulphate medium Starch agar
Fries medium Steinberg unit optima dibasl medium
Fungus resistance testing medium Stan pectate medium
Clllcose asparagine medium (2 versions) Starr spirit blue agar
Glucose casamino acid medium Starr Xanthomonas synthetic
Clucase casein hydrolysate medium Sucrose ammonium tartrate nitrate medium
Glucose nitrate agar Sucrose proline agar
Glucose nitrate sterol medium Tochinai solution
CIyccrol asparaginate medium Trace element salution
Hugh and Leifson carbohydrate medium (For Trione dwarf bunt synthetic
bacteria) Van Cundy and Walker medlum (modified)
Kennedy and Erwin EDTA medium , Vogel medium N
K ~ O Pagar Wieringa pectate gel
Lukem and Sisler synthetic medium Wills mineral salts medium
H d h a d Nutriurt Solutions 7
H. Medm t o aid in the identification of fungi

Includes more useful or commonly used media in diagnosis. Any medium that allows normal
sporulation of fungi and actinomycetes could be included.
Medium
__L
For we with
Botrytis separation agar B. cinerea and B. alli
Cantino PY agar Bhstochdielh and other aquatic fungi
Corn mea9
Czapekdox
Phytophthora, Pythium and others
2 spergillus and Penicillium particu la$y
Dung agar Pllobohs and other coprophilic fungi
Emerson YpSs medium Allomyces and other aquatics
Gallic acid agar Wood rotting fungi
N t z medium (modified) Agarics and boletes
Malt agar A variety of fungi
Oat meal Same as corn meal
Petri medium J t h y t opht fmra
Potato dextrose agar A variety of fungi
Sabouraud dextrose agar Dermatophytic fungi
Snake skin A phnnomyces
Sucrose prohne agar Drecllslera, Helmirlt hosporiunt
Synthetic Mucor Muooralcs
Sod water Aphanomyces spp.
TaIboys medium Verticitlium aibo-atrum and V, dahliae
Tannic acid agar Wood rotting fungi
Wheat straw agar q*erroclraeta
I. Media to aid in the identification of bacteria J. Culture plant tissue or intact plvlts
Andrade indicator Burkholder and Nickell tumor mehum
Carbohydrate fermentation medium Cutter host-rust medium
Desoxycholate agar White nutrient solution (1963)
Dimmick medium White plant tissue medium modified by Hilde-
Eosin methylene blue agar brandt, Riker and Duggar
EMB medium White medium modified (1962)
Fluorescin medium K. Plant nutrient solutions
Hayward 0-F medium Crone solution
tiugh and Leifson carbohydrate medium Hoagland solution
K o a r citrate medium Hoagland and Knop solution (Modified) (Used
Motility medium in sterile culture of plants)
Pcptone water Liong Ashton nutrient solution
Pyocyanin medium Shivc and Robbin nutrient solution
Starr pectate medium Tris EDTA Algae medium
Stan spirit blue agar Withrow nutrient solutions
Tetrazolium medium
Tyrosine casein hydrolysate medium
8 Medh and Nutrient Solutions
TABLE 2. LITERATURE ON CULTURE 3IEDI.4

Actinomycdw
Cochrane, V. W. 1961. Physiology of Actinomycetes. Ann. Rev. Miaobiol, 15: 1-26.
*ling, E. B. and D. Cottlieb. 1966. Methods for characterization of Srrepromyces species. Int. J.
Syst. Bacterial. 16:313-340.
Waksman, S. A. 1961. The Actinomycetes Vol. 11. Classification, identification, and descriptions of
genera and species. Williams and Wilkins, Baltimore.
Agaric Fungi
Singer, R. 1962. The Agaricales in Modern Taxonomy. J. Cramer, H'einheim.
Algae
Starr, R. C. 1960. The culture collection of algae at Indiana University. Amer. J. Bot .47:67-86.
Animal and Human Fungal Pathogens
Ajello, L., Lucille K.Georg, W. Kaplan, L. Kaufman. 1963. Laboratory manual for medical mycology.
Public Health Serv. Pub. 994. Wash., D.C.
Beneke, E. S. 1957. Medical mycology laboratory manual. Burgess Pub. Co., 3linneapolis.
Emmons, C. W., C. H. B~nford,J . P. Utz. 1963. Medical mycology. Lea and Febipr. Philadelphia.
Moss, E. S. & A. L. McQuown. 1960. Atlasof Medical !4ycology. Williams and \\'ilkins, Ba!timore.
Aquatic Fungi
Cooke, Wm. B. 1963. A laboratory guide to fungi in poIluted waters, sewage, and semge treatment
systems. Pub. Health Serv. Pub. 999-\i'P-I.
Emerson, R. 1958. Mycological organization. hiycologia 50:589421.
Johnson, T. W. Jr. and F. K. Sparrow, Jr. 1961, Fungi in oceans and estuaries. J. Cramer, Weinheirn.
Sparrow, F. R.Jr. 1960. Aquatic Phycomycetes. Univ, blich. Press, Ann Arbor.
Cochrane, V. W. 1958. Physiology of fungi. John Wiley, Sew York.
Bacteria
Committee on Bacteriological Technic of the Society of Amsican Bacteriologists. 1957. Manual of
miaobiological methods. hlcCraw-&U, New York.
Cowan, S. T. and K. J. Steel. 1965. hianual for the identification of medical bacteria. Cambridge
University Press, London.
Dowson, W. J. 1957. Plant dlseases due to bacteria. Cambridge Univ. Press, b n d o n .
Edwards, P. R. and W. H. Ewing. 1962. Identification .of Enterobacteriaceae. Burgess Pub. Co.,
Minneapolis.
Levine, M. and H. W. Schoelain. 1930. A compilation of culture media for cultivation of micro-
organisms. Wiltiarns and Wilkens, Baltimore.
Skerman, V. B. D. 1959. A guide to the identification of the genera of bacteria. Williams and Wilkens,
Baltimore.
Stapp, C. 1961. Bacterial plant pathogens. Oxford Univ. Press, London.
Sykes, G. 1956. Constituents of bacteriological culture media. Cambridge Univ. Press, London.
Commscial Sources of Media
Anom. 1959. BBL products, culture media, materials and apparatus for the microbiological laboratory.
Baltimore Biological Laboratory, Inc., Baltimore 18.5hyland.
Anom. 1961. C O M B products for the chemical laboratory preparations. Consoli&tcd Laboratories,
Inc., Chicago Heights, Illinois.
Anom. 1959. Difco manual of dehydrated culture media and reagents. Difco laboratories, Detroit 1,
Michigan.
Aaom. 1963. Fisher bacteriolopcal culture media. Chi* 5 1, l ~ o i s .
-6 ( G d )
Aimworth, G. C. 1961. Dictiotl~ryof the Fungi. Cornmnwalth Mycobgial Institute, Kew,Surrey.
Fries, N. 196 1. Growth factors: metabolic factors limiting growth, bacteria and fungi. In Encyclopedia
of Plant Phys. 14:332400.
Lilly, V. G. and H. L. Barnett. 1951. Physiology of the fungi. McGraw-Hill, N.Y.
Riker, A. J. and Regina S. Riker. 1936. Introduction to research on plant diseases. John Swift @.,St.
fnuis.
Higher Plant Nutrient Culture
Hewitt, E. J. 1952. Sand and water culture methods used in the study of plant nutrition.
Commonwealth. Bur. Hort. Tech. Com. 22, East Malling.
Hoagland, D.R. and D. I. Arnon. 1950. (Rev.) The water culture method for growing plants without
soil. California Agr. Exp. Cir. 347.
Withrow, R. D.and Alice P. Withrow. 1948. Nutriculture. Purdue Agr. Exp. Sta. Cir. 328.
Insect Mimobial Paraates
Steinhaus, E. A. '1963. Insect pathology. Academic Press, New York.
Microbiological Assays for Amino Acids, Antibiotics and Vitamins
Kavanaugh, F. 1963. Analytical microbiology. Academic Press, New York.
Plant Tissue Culture
Butcher, D.N. and H. E. Street. 1964. Excised root culture. Bot, Rev. 3 0 5 13-586.
' Gautheret, R. J. 1955. The nutrition of plant tissue cultures. Ann. Rev. Plant Physiol. 6:433484.
Cautheret, R. J. 1959. La culture des tissus vegetaux. Masson and Cie,Paris. ,
Hildebrandt, A. C. W. Tulecke, G.Cuinn, E. Ball ,nd A. H. Haber, and J. C. O'Kelley (ed). 1963. Plant
tissue culture and morphogensis. Scholar's Library. New York.
Narayanaswami, S. and K.Norstog. 1964. Plant embryo culture. Bot. Rev. 30:587-628.
Paul, J. 1960. Cell and tissue culture. \\'illiams and Wilkens, Baltimore.
White, P. R. 1963. The cultivation of animal and plant cells. Ronald Press, New York.
Soil Nematodes
Sasser, J. W. and W, R. Jenkins. 1960. Nematology. Univ. of N. Carolina Press, Chapel Hill.
Ymsts
Cook,A. H. 1958. Chemistry and biology of yeasts. Academic Press, New York.
Wickerham, L. J. and K. A, Burton. 1948. Carbon arjimilation tests for the classification of yeasts. J.
Bacteriol. 56:363-371.
GLASSWARE CLEANING
Of the solutions commonly used in the cleatling of glassware, each has its supporters and
detractors. The argument is that although the traditional dichrornate-acid solution does the p b ,
extremely small amounts of residue are toxic and are difficult to remove.'~lso,handling s ~ c ha &on&
mrrosive solution is undesirable, But on the other hand it is claimed that the other materials do not
give biological purity (some vigorous dissenters). Whatever the cleaning agent, all glassware should be
-
rinsed thoroughly and should be visibly clean no droplets of water a?pearing a t sides upon draining.
1. Sulfuric adddichromate
Despite the stated disadvantages of dichromate-acid it is widely used, but it should n& be used
on sintered glass ftlters. The following method by White (28) overcomes the toxic residue problem.
1. Soak glassware in 100 rnl. of saturateu sodium dichromate and 1 gallon of concentrated
sulfuric acid for at least 4 hr.
2. Rinse in hot ~ ~ i tapn waterg at least 10 times.
3. Rinse twice in single distilled water.
4. Rinse once in double glass distilled water.
Medh and Nutrient Solutions
Aaotha formula for acid dichromate:

Sodium dichromate m g
Distilled water 800 rnl
Cow. sulfuric acid 1200 ml
Grind dichrornate crystals in a mortar and dissolve in hot water.
Cool t o room temperature and add acid slowly.
1. Soak glassware in a mixture of 95 ml. of concentrated H2S04 plus 3-5 ml of concentrated

HN03.
2. Rinse with distilled water after a contact time of 1-48 hr, particularly recommended for
pipettes (I 0).
1. Place glassuare in equal parts of concentrated H2S04 and HNOj in a pyrex dish.
2. Heat for 30 to 60 rnin. in a hood. Cool.
3. Wash several times in tap w t e r and rinse twice with distilled water (5).
4. H N O ~
1. One part of concentrated H N 0 3 to 9 parts of water will oxidne organic matter without
having a residue (1).
5. Detergents
1. Imrnerot in a solution of Kelvar 1 tablespoon per gallon of water. (bryandotte Chem. Corp.,
Wyandot te, hi ichigan)
2. Boil gently for 15 min.
3. Rinse several times with tap water and twice with distilled water. Thir procedure is supposed
not to etch glass (25). Other detergents recommended are Alconox. Haem-sol and sodium
bury1 sulfate. The latter may be followed by a 1-2 min. rinse of 75% sulfuric acid. Other
detergents, particularly alkaline ones, are alx, probably suitable.
PURIFICATION OF AGAR
If agar is nsessiuy to a growth study and the purity or price of commerchlly purified agar is not
suitable, the following methods are suggested: Elimination of all impurities is not claimed.
Robbim, W. 1. & Annette Hervey (1 5)
1. Difco agar is percolated with several volumes of 5% aqueous pyridine. Use pyridine under a
hood
2. FoUow by 1% HCI.
3. The agar is then neutralized with calcium hydrdxide.
4. Wash free of excess calnum.
5. T n a t with 95%ethanol and dry at a low temperature.
1. 1 Ib of agar b placed in a 6 1 flask.

2. Add 5 1 of distilled water and 500 rnl of pyridinc and allow to stand 24 hr.
3. Imert 1 piece of 6 mm glass tubing of sufficient length to admit air, tie a piece of cheesecloth
over the neck of the flask,invert flask and drain.
4. Wash the agar 3X with distiUcd water,
5. Wash the agar 2X with 95% ethanol and anow alcohol to stand overnight.
6. Dry agar in thin layers between washed cheesecloth.
Rocedure takes about 10 days.
1. Phcc agar in a muslin bag.

2. Immerse in distilled water for 2 or 3 days.
3. Transfer agar to a vessel containing about half the volume of liquid for preparation of medium.
4. Dissolve agar by heating and follow usual methods of media preparation.
LITERGTURE CITED
1. Cavanaugh, Gail hl. 1956. Formula and methods IV.of the marine biological laboratory chemical
room. hfiirine Biol. Lab., Woods Hole.
2. Cochrane, V. W. 1958. Physiology of fungi. J. Wiley, Sew York.
3. Crascman, Jean M. 1954. The nutrition of Chynidiwn and ,~lacrochybium.Amer. J. Bot.
4 1 :302-3 10.
4. Dimond, k E. and G. L. Peltier. 1945. Controlling the pH of cultures of PenicilBum notatum
through its carbon and nitrogen nutrition. Amer. J. Bot. 32:46-50.
5. Donald, Catherine, Beverly I. Passey and R. J. Swaby. 1952. A comparison of methods for
r e m o w trace metals from microbiological media. J. Gen. hlicrobiol. 7:211-220.
6. Dowson, Mr.J. 1957. Plant diseases due to bacteria. Cambridge Univ. Press, London.
7. Gomori, G. 1955. Preparation of buffers for use in enzyme studies in S. P. Colowick and N. 0.
Kaplan (ed). Xiethods in enzymology. Vol. 1. Academic Press, Sew York.
8. Hutner, S. 11. 1944. Growth of the photosynthetic bacterium, Rhodospirillum rubnun. Arch.
Biochcm. 3:439444.
9. Jemnison, bI. W., Mciva D. Newcomb and R. Henderson. 1955. Physiology of the wood rotting
Basidiornyrcies 1. hlycologia 47 :275-304.
10. Kavulaugh. F. (ed.). 1963. Analytical microbiology. Academic Press, N. Y.
11. Klein, R 51. and Georgia E. Manos. 1960. Use of metal chelates for plant tisue cultures. Ann.
New York Acad. Sci. 88:41-25.
12. Landy, M. and D. M. Dicken. 1942. A microbial assay method for 6 B vitamins using kctobcilIus
cosci and a medium of essentially known composition. J. Lab. Clin. Med. 27:10861092.
13. Lilly, V. G. and H. L. Barnett. 1951. Physiology of the fungi. SlcGraw-Hill, N. Y.
14. McKeen, W. E. 1956. Interaction product of glycine and dextrose toxic to Phyrophtbra fraarirre.
Science 123:509.
15. Robbins, W. J. and Annette Hervey. 1%0. Growth substances for Polypoms schweinitzii.
Myoologia 52:94&957.
16. Robbins, W. J. and Annette Hmey. 1963. Unidentif~dfiftrate growth SUbst,anCZS for several
fungi. Myalogia 55:59,-64.
17, Spector, W. S. 1957. Handbook of toxicology. Vol. 11 Antibiotics. W. B. Sanders.
18. Steinberg, R. A. 1950. Trace element impurities in nutrient solutions for fungi. Arch. Biochem.
28: 111-1 16.
19, Stiks, W. 196 1. Trace elements in plants. Cambridge Univ.Press, b n d o n .
20. Stokes, J. L., Marion Gunncss and J. W. Foster. 1944. Vitamin content of microbblogial culture
mcdis, J. bctniol. 47:293-299.
12 Media and Nutrient Solutions
21. Street, H. E. 1957. Nutrition and metabolism of plant t i m e cultures. J. Nat. Canc. Inst.
19:467-494.
22. Sykes, G. 1956. Constituents of bacteriological culture media. Cambridge Univ. Press, London.
23, Taber, W. A. 1957. The influence of inoculum on variability in comparative nutritional
experiments with fungi, Can. J. 5licrobiol. 3:803-812.
24. Thorn,C. 1954. The colony. blycologia 46: 1-8.
25. Umbreit, W. W., R. H. Burris, J. F. Stauffer. 1964. hfanometric techniques. Burgess Pub. Co.,
Minneapolis.
26. Ward, C. W. B. 1964. Stimulation of growth of a low temperature basidiomycete due to heat
sterilization of a culture medium. Can.J. Bot. 42:283-285.
27. Weinhold, A. R. and F. F, Hendrix, Jr, 1963. Inhibition of fungi by culture media exposed t o
light. Phytopathology 53: 1280.1284.
28, White, P. R. 1963. The cultivation of animal and plant cells. Ronald Press, N.Y.
MEDIA AND THEIR INCREDLESTS

(Media formuhe)
The media included here are called by their apparent common names and are listed alphabeti-
cally. If a medium lacked a name it was called by its major and/or other constituents if they were
different enough from previously named media, otherwise by its function or by the name of the person
who devised it.
Ingredients of the media are given on a 1 basis unless so noted. Units and description of
ingredients are that of the author cited. Some investigators report composition of media solely on the
basis of molarity, a minor inconvenience or unfortunately do not give full names of compounds or the
amount of hydration.
1. Agrobacterium I.wlation Medium

Mannitol 10.0 g Highly specific for the isolation of
Sodium nitrate 4.0 g Agrobactmium turnefaciens A. -
Magnesium chloride 2.0 g radiobacter group from soil. pH
Calcium propinate 1.2 g adjusted to 7.1 with 1N HCI. See
Magnesium phosphate 0.2 g also no. 43.
Magnesium sulfate 0.1 g
Sodium bicarbonate 0.075 g
Magnesium carbonate 0.075 g
Am 20.0 g
Distilled water 11
After autoclaving and at 50-55 C add the following:
Berberine 275 ppm
Sodium selinite 100 PPm
Penicillin C (1,625 units/mg) 60 PPm
Streptomycin sulfate 30 PPm
Cycbhexamide (85-100% active) 250 PPm
Tyrothricin (pure) 1 PPm
Bacitracin (65 unitslmg) 100 PPm
Schroth, M. N., J. P. Thompson and D. C. Hildebnnd. 1965. Phytopathology 55:645-647.
2. Alcohol A p t
Agar 75 1
Streptomycin nitrate 100 ppm = 0.1 g
Water 11
90 ml are dispensed in 250 ml flasks and autoclaved, Suggest 11 Ib for I S min if Streptomycin
sulfate is used. After cooling to 44 C, add 0.5 ml absolute ethanol to each flask. The diluted soil
samples are added and the plates poured and incubated 10-15 Qys in the dark. Used t o detect
Verticillium in soils and according to R. J , Green ('personal communication) works well for isolation
from roots. See also soil extract isolation agar.
Nadakavukazen, M, J . and C. E. Horner. 1959. Phytopathology 49527-528.
3. Alfalfa Decotion Agar

Alfalfa leaves
Dextrose
Agar
Water to make a 1
Blendorize leaves in water, filter through cheesecloth and add to melted agar containing dextrose.
Used to induce sporulation of Scolccwtrichurn grantinis.
Cobb, G. W. 1961. MS Thesis. Purduc University.
Graham, J , H., R,W. Zoiders and S. W, Braveman. 1963. P b t Dis. Reptr, 47:255-256.
4. Alfalfa Seed Decotion

Alfalfa Seed 100 !I Used for the sporulation of
Water made up to a 1 Altsnaria brassicne.
McDonald, W. C. 1959. Can.J. Phnt Sci. 39:409-416.
5. Alphacel Medium
Alphacel (obtained from Nutritional 20 8
Biochemicals Corpora tion, Cleveland,
Ohio, a non-nutritive cellulose)
MgS04 Ig
KH2m4 1.5 g
NaN03 1g
Coconut Milk 50 ml
Agar 128
Water 11
Adjust pH t o 5.6 and sterilize 20 lb/20 min. Coconut milk filtered through several layers of
cheesecloth and autoclaved at 15 lb/lS min. Keep at 6 C until needed. A valuable medium for the
sporulation of fungi.
Sban, B. J., J. B. Routien and Virginia P. M i k , 1960. Mycob* 52:4763.
6. Alphaal Medium (modified)

Same as medium 5 with the addition of:
Hunt's tomato paste 10 0 A good sporulation inducing
BeechNut baby oat meal 10 g medium for many fungi.
Sbm.B.1, J. B. Routien and Vuginu P.M i l k . 1960. Mycobgia 52:47-63.
7. Amino Acid Substitute for Casein Hydrolysate

MgllOO ml
5.O For the "amino acid block"
DL-alpha-alanine 19.0 approach to replacement of casein
DLvaline 75 0 hydrolysate see G.R. Seaman.
LLeucine 48.5 1962. Experiments in microbial,
D-lmleucine 48.5 physiology, and biochemistry.
DLphenylanine 39.0 Burgess Pub. Co., Minneapolis.
Lt yrosine 66.O
ttryptophane 22 .o
DLthreonine 35.0
D-glutamic acid 21 8.0
Laspartic 41.O
Lproline 90.o
Lhyboxyproline 2 .o
DLserine 50-0
kystine 3.O
Lmethionine 34.0
Dsrginine HCI 30.0
D-histidine HCI 25 .O
D-lysine (HC13)2 60.0
Treat with excess CaC03 and filter. Did not substitute completely for natural casein hydrolysate,
Robbins, W. J . md Roberta Ma. 1945. Arner. J. Bot. 32.509-523.
8. Amino Add Substitute for 1% Yeast Extract (Difco)
h r g i n i n e HCI For purines and pyrimidines

D h s p a r t i c acid supplied by yeast extract see G. R.
DLglutamic Seaman, 1962. Experiments in
Gvcine (anhydride) microbiology, physiology and
DLbistidine HCI biochemistry. Burgess Pub. Co .,
DLrifoleuckrc Minneapolis. For minor element
DLkucine aonantration see Grant, C. Laand
DL4ysme HCI D. Rama. 1962. J. Bacterhl.
Dhdhionine 84:869-870.
Dphmylalanim
D M hre oninc
DLtryptophane
Dttyrosine
DLvaline
h@m, G. C, urd C. B. D m y . 1960. A m . I. Sot. 473758-765.
9, Andrade Indicator
Acid fuchsin 05 g Dismhrc!fuchsin in dist. water and
NaOH 1N 16 rnl add NaOH. If after several hours
Distilled water 100 mf not decolorized, add 1 or 2 ml of
alkali, pH 5-8 pink-yellow.
Edwards, P. R. and W. H. Ewing. 1962. Identificatbn of Entcroblcteriaccae. Burgess Pub. Co, Minnerpohs.
10. Aphanomyces Synthetic (Haglund & King)

Dglucose 5.0 g
line 0.75 g
Monobasic potassium phosphate 2.0 g
Magnesium chloride 0.05 g
Manganese chloride 0.605 g
Zinc chloride 0.005 g
Ferric chloride 0.00s g
Lmethionine or Lcystine 5 t o 20 ppm
Adjust pH to 5.5, Aphartomyces euteiclzes requires a reduced form of sulfur for growth, like a
number of genera in the Saprolegeniaceae.
Haglund, W. A, and T, H. King. 1962. Phytopatholopy 52:315-317.
A man ctaborate medium (SSI-2) for the production of oospons is given by Papavizas, G. C. and C. B, Davy.
1960. Amor. 1. b t . 47:75&765.
11, Aphanomyces Washing Solution

CaClz After A. euteiches grown on a
KC1 broth containing 2% peptone and
MgS04 05%glucose for 48 hr at 24 C
Adjust to pH 6.5 replace broth with washing solution
twice, immediately, and again after
-
2 and 4 hr Xiotile spores produced
1620 hr later.
Mitchen, J. E. and C. Y. Yang. 1966. Phytopatholog 56:917922.
12. Apple Infusion Agar

Apple leaves (dead) airdry Steam appk kavcs in 500 ml water
Malt extract for 30 min. Add mah extract and
Ae~lr melted agar. For the production of
perithecia of Vent& ~ w l i s .
Medium stems to be replaad by thl
fobwing one.
Kcitt, G. W. and M. H.Langford. 1941. Am!. J. Bot. 28:SOS820.
13. Apple Infufioa Agar

Dextror 5g Usd for the production of perithecia
Potatoes 4og of Ventwia irt~equalix Crow at 16
Apple leaf decotion as prepared above 20 C for 10-14 days, then at 8 C
Agar 17g until perithecia ripen, usually about
Water to make a 1 34 months.
Boone. D. M. and G. W. Keitt. 1956. Amer.l. Bot. 43:226-233.
Sucrose or glucose 2% Uwd to increase wilt Fusmia. 72 hr

M8SO4 0.003M at 28 C, fksk shaken occasionally,
KC1 0.02 2M
w 2 m 4 0.008M
Ca(NO3)2 0.0356M
-
0.2 ppm of each cation See Barnett microelement
solution no. 143 for a similar mixtue.
Armstrong, G. hI. and Joanne K. Armstrong 1960. U. S. Dcp. Agr. Tech. Bull. 1219.
15. Asthana and Hawker Medium A

Clumse
KNOB
Mas04
Agar
Distilled water
A mediwn intensively used to study perithecia formation of Afehnospora destruens, now Sordmia
dcsmtens (Shear) Hawker,requires thiamine and biotin depending on strain.
Arthurr. R. P.and LiKYr E. Hawker. 1936. Ann. k t . 50:325-t43.
16. A-ZSolution (Hiss and Reed)

Media and Nutrient Solutbm
17. A-ZSolution
CuS04 S H 2 0
MnC12 4H20
ZK12
Ca(N0& 6ti20
BaC12 2 H 2 0
(NH4)6 h f 0 7 0 / c ,
Water
Use 1 ml per 1 of culture medium, commonly used for fungi.
For I8 l redistilled m t a .
Robbins, W. J. and F. K3ranaugh- 1938. Phnt PhysioL 13:611-619.
19. Barnett Maltose Casamim, Medium

hf altose 5g
Difco Caslrnino Acid? (tech, grade) 1.0 g
Kt12P04 1.Og
M@04 7H20 0.5 g
"
Mn 1
Fe (as sulphates)
Biotin (0.5 g yeast extnct may be
substituted for sont isolates)
0.2 mg
0.2 mg
0.1 mg
5 4%
I see miaonutrient solution No. 143
Distilkd water 11
Agar 20%
Adjust pll to 6.0, autoclave at 15 Ib 15 rnin. Used for the production of perithecia of Ceratoc~rstis
fqacecuum.
20. Bas11 b l i n d Sllltr Agar

(NH4)2S04 2.64 g Adjust pH to 6.8-7.0with IN NaOHl
KHIP04 anhydrous 238 g or IN HCI. Used for culture of
K2Imj' 3 f i 2 0 5.65 g Srreptomyces with addition of
MgS04 71i20 1.00 g sterite carbon source at a concen-
Pndham and Cot tlieb trace salts (No. 192) 1 rnl tration of about 1%.
Distilled water 11
b r 1s g
SbL& E. B. and D.GrrLLb. 1966. la.J. Syst. Blctcrid. 16:313-340.
Media and Nutrient Solut~ons
21. Bean A p r
Great Northern white beans Autoclave beans in 500 ml water
Ghrcose and add melted agar and glucose.
AB~= Used for zoospore production of
Distilled water Phytophrlara cactoruttt.
22. Bean Juice Agar

Bean Juice from c a ~ e green
d beans 215 rnl
Agdr 10.0 g
Water 285 ml
For mnidial increase of Colletotricltum lindemzcthiatturn.
23. Bean Meal Agar

Ground dwarf bean seed (Variety Canadian Wonder)
30 g
Agar 20 g
Boil ground seed for 5 min and filter, add liquified agar. Used t o induce asexual reproduction of
Phyropirthwa cirrmn~oni.After 8 days growth, transfer to \Vill non-sterile soil extract. Also used for
zoospore production of P.fragarhe w ~ t htransfer t o pond water,
Hickmm, C. J. and Pamela M. Gcwde. 1953. Nature 172:211-212.
Royie.D.J.and C. J . Hickman. 1 9 6 4 . C ~ J.. Uot. 4?:311.318,
Stamps. D. Jean. 1952. Bnt. hlyc. Soc., Trans. 35:248-253.
24. Bean Pod Agar

Green string beans 200.250 g Edgerton suggests drying beans with
(34 hr a t 50.60 C in water) b w heat and grinding to a fine
Agdr 15 g powder, 20 g is about equal to 250 g
Water 11 of fresh beans. It should be stored
in a dark place. This medium is
awilable from Difco.
v'
25. BeefPeptone Agar (nutrient agar, meat infusion)
Beef extract
Peptone
Agar
Medk and Nutrient Solutions
26. Beef Peptone Agar

Minced lean meat 250 g
Peptone 10 g
NaCl 5E
Yead extract 0.05 percent
Water 11
Combine ingredients and place in refrigerator for 24 hr, steam for 3 hr and when cool, strain through
muslin and filter through cotton-wool. Make up to a 1 and adjust pH to 7,2.
Dowson. W. J. 1957. Plant diseases due to bacteria, Cambridge University Pnss. London.
27. Beef Peptone Agar

Same ingredients as No. 25 plus the following:
Dextrose 100
Yeast extract 58
28. Bennett Agar

Yeast extract IB
Beef extract 1g
N-Z amine A * 28
Glucose 10 g
Agar 15 #
Distilled water 11
For sporulation of Streptomyces spp. Adjust pH to 7,3 with NaOH.
*Acid hydrolyzate of casein.
29. Bilai Medium (modification by Joffe)

KH2P04 Strips of cellulose lens paper are
KNO3 added. According to Joffe, medium
Mss04 always induced conidia in Fusaria.
KC1
Starch powder
Glucose
Sucrose
Water
Joffe, A. 2.1963. MycologiD 55:271-282.
30. Bonner-Addicott Medium

KNo3 0.008 M Cultures of Pnymatoltichum
bigso, m 0 0.00015 M omnivorum s c reported t o sportdate
fi@Oo)z 4H20 0.001 M on this medium when grown under a
KH2po4 0.00008 M 16 hr photopriod under white or
KC1 0.0087 M blue light (Woods et al),
Glucose 20.0 g
Ferric tartrate
Agar
Bomer. J. and F. Addicott. 1937. Bot. Gaz. 99:144-170.
Woods,R., H. E. Bloss and C . A. Cries. 1967. Phytopathology 57:228.
31. Botrytis Separation Agar

KC1 0.1% Used to distinquish between
KH2P04 0.15% Botrytis citierea and B. alli, the
&NOj 03% 1atter.spccies is sharkly restricted
MgS04 0.05% by krbose,
Casein hydrolysate 0.5%
Yeast extract 0.3%
Glycerol 0.5%
Laorbose 0.25%
Agar 2.0%
Netza, D. and Irene Dishon. 1967. Phytopathology S7:795-796.
32. Botran Isolation Medium

K2 HP04 0.5 g Medium used to isolate Aspergillus
MgSO, 7H20 0.5 g mvus from soil and peanuts,
Pcptone 0.5 g Mycelium of R hizopus stoloriifer
Yead extract 0.5 g restricted in the Botran amended
sucrose 20 medium. Adjust pH to 5.5 with IN
Rose bengal 25 mg HCI or NaOH before autoclaving.
Streptomycin sulfate -
50 mg Added after autoclaving
Ebtran (Upjohn Co.) -
10 ppm (of active ingedient) if technical grade,
(2,6 dichloro-4 nitroaniline) dissolve in acetone
DistiUed water 1 1
B8ll.D. K. and J. L,Cmwford. 1967. Phytopathology 57:939941.
Ekan pod ='g Make a distilled water extract of

Sucrose 5g bean pods, sterilize sucrose
Mdt extract 51 separately. Use to obtain conidia
*LSar 17 g of D i p b c a r p n earliana.
Distilled water to make a 1
Dhmrntui B. N. 1967. Can. J. Bot. 45:1525-1543.
34. h a d Crumb Agar

Commercial bread crumbs Used to permit luminescence of
(without preservatives) miow fleshy fungi.
Bacto-agar
Tap water
BerIiaer, Martha D. 196 1. Mycobpi 53:8440.
35. Burkholda Medium

Potatoes
Peptone
Na2HP04
NaCl
Sodium citrate
Asparagine
Dextrose
A@f
Water to make a I
Add ingredients to the p t a t o broth water. Medium supported good growth of Grynebacreriwn
sepedonicum Useful in isolation from potato, See also ring rot medium,
Burkholder. W'. H. 1938. Am=. Potato J. 15:243-245.
36. Burkholder and Nickell Tumor Medium

-
Final
0.002 M
Concentrations
B
-
PPM
0.1
0.003 M Mn 0.1
0.002 M Zn 0.3
0.001 M Cu 0.1
0.003 M Mo 0.1
0.002 M Fe 05
MgClz 0.001 M
Sucrose CP mo Used for growth of virus wound tumors.
Thiamin 100 411
Pyr idoxin 800 %I1
Niacinarnide 800 ug/l
Difco Noble Agar 1%
Stock Solutions Vitamin Sol.
O.IM K!03 Thiamin HCI
0.1M Ca(S03)2 Pyndoxin HCI
0.1M KH2P04 Nicotinic amide
0.1M hfgso, Water
0.1M CuC12
0.1M Kfl Trace elements mgll
0.1M MgC12 H3BO3
Suaose MnClz H20
V~taminsolution ZnClt
Trace ekmmts Na2hfo04 =2H20
Wata F~2(C4H406)3
(glass double distilled)
Adjust pH to 5.2. Make up 500 ml of 2% agar. Sterilize agar and nutrient solutions ~parntely.Mix
equal parts of each.
M d h and Nutrient Solutions
37. Cantino PYC Agdr

Peptone
Yeast extract
Glucose
Agar
For culture of Blartocbdiella. If broth culture u x d , necesrary to haye brorncresol purple indicator
and intermittent neutralization with NaOll. pH 6.8.
Horenstein, E. and E. C. Cantino. 1961. Brit. MycoL Soc.,Tnns. 44:185-198.
38. Carbohydrate Fermentation Medium (Fdwards and Ewing)

Peptone 10 s Adjust to pH 7.1-7.2. Tube with
Meat extract 3g inverted tube at 121 C 15 min,
Sodium &lor ide 5g Glucose. lactose, sucrose & mvnnito!
Andrade's indicator 10 rnl concentration is 1% while dulcitol.
Distilled water 1 1 -
salicin are 0.5%. Those unhrlined
m y be added t o medium prior to
sterilization. Others sterilized by
filtration or in a I CGsolution at
121 C.
Edwards, P. R. and W. H. Ewing. 1962. Identification of Enterobacteriaceae. Burgess Pub. Co.,BIinneapolis.
39. Carbon Utilization Medium

(NH4)2S04
u12P04
K21m4
MgS04 7 H 2 0
C S 0 4 5k120
FeS04 7 H 2 0
MnC12 4H20
ZnS04 7 H 2 0
Difco Agar
Distilled water
Medium adjusted to pH 6.8-7.0, tubed in 9% ml amounts and autoclaved. At 45 C, sterile aqueous
mlutions of carbon added to give a final concentration of I percent. Water soluble a r b o n
compounds were sterilized by Seitz filtration, those insoluble were added to medium before
autoclaving. Used to determine carbon utilization of actinornycetes.
Pridhrm,T. C. and D. GottHeb. 1948. J. BactAPl. 56:107-114.
40. m o t Agar
Carrot (grated)
Aw
Water
Soak tissue in water for 1 hr then boil for 5 min. Filter through cotton-wool.
Grated carrot
Agar 17 1
Add grated carrot to a depth of 2.5 crn in a test tube and add 10 rnl of water agar. Encourages sexual
reproduction of species of Pytiu'um. String beans was a good substitute for carrot.
Jolunn, Hekn. 1929. Phy lopathology. 18:710.
42. Carrot Decotion Agar

Finely chopped carrot leaves mg
Asar 12 g
Water 11
Leaves finely ground in food chopper. Steam without pressure in 500 ml water for 1 hr, strain and
add 500 ml of melted agar. Induces sporulation of a number of species of Cezmspra.
Upatrick. R. A. and H. W. Johnson. 1956. Phytopathology 46: 180-181.
43. Carrot Discs

To isolate Thiehviopsis basicola from soil. (To detect crown gall bacteria see Plant Dis. Reptr.
42: 1279-1281 and to detect Ceratocj*stisfin~briatasee Phytopathology 58: 123-124.) 5mrn carrot
discs placed on moist filter paper, soil spread over surface of discs and water atomized on it. Left for
2 t o 4 days at room temperature. Soil washed off and the discs placed on fresh moist filter paper,
continue incubation. Yarwood, C. E. (1946. hlycologia 38:346-348,) Lloyd, A. B. and J . L.
Lockwood (1962. Phytopathology 52: 13 14-1315) cautioned against using carrots contaminated
with C/nlmopsis tilielavwLdes, a fungus remarkably similar to T,basiwb. hlalby, 0. C. and M.
Alexander (1958, Phytopathology 48: 126-128) have quantitated this method.
44. Casein Glycerol Medium

Casein (Difco) "vitamin free"
Glycerol
KN03
NaCl
K2 m 4
MgS04 7H20
c&03
FeSO4 7H20
A8Pr
Adjust pH to 7.2. In a comparative study of a number of media, this medium found to be the best
for the isolation ofStreptonzyces. Soil diluted to lo5.
Kurlar, E. a d S. T. Williamr 1964. Nature 2023928-929.
45. Casein Starch Medium (modified no. 44)

Starch substituted for glycerol, used for the isolation of Sneptomyces.
Kuda,E. *ad S. T. Wilfinmr 1964. Nature 202:928-929.
Ammonium sulphate Cellulose is prepared as a 4%
Lasparaghe suspension. Whatman standard grade
Potassium dihydrogen phosphate cellulose powder for chromatography
Potassium chloride is ball-milled for 72 hr. Add this to
Crystalline magnesium sulphate other dissolved substances and
Calcium chloride autoclave for 20 min at 10 Ib. The
Difco yeast extraft final pH is 6.2, used for isolation of
Cellulose allulolytic fungi from soil. They
Agar are detected by a clear area.
Water t o a 1
EQgins, H. 0. and G. 1. F. Pugh. 1962. Kature 193:94-95.
47. C a e b s e Ammonium Nitrate Medium

Ccrelose Used to increase Fusarium
NH4N03 oxysporurn f. sp. lycopersici.
KH2P04
MgS04 7kI20
FeClj 6H20
Water
Sheffer, R. P. and J. C. H'alker. 1953. Phytopathology 43'1 16-125.
48. Charcoal Water

Charcoal For inducing sporulation of
Tap water Phytophtiioru parasrtrca, apparently
after growth on potato dextrose
broth. Valuable in eliminating toxic
ions as well as adding unknown
stimulatory substances. Couch, J.
N. (1939. J. Elisha Mitchell soc.
55:208-214) used 1 level teaspoon
of charcoal/l. Filter after 1 hr and
then autoclave.
49. Chick-pea Sucrose Agar

Chick-pea (Cicer mierfnwn)
Sumse
4w
Dist ilk d water
Wash seed in tap water for an hr then soak in distilled water overnight. Drain off water and mash
see& with pestle and mortar. Add 1 1of water and steam for 1 hr, and screen through gauze. Sucrose
and agar are dissolved separately and combined, making up t o volume. Medium is tubed and
rtctilized for 15 min at 10 Ib. For culture of Phyrophthora infestans. Garden peas may be
lubffituted but not quite as good for sporulation according t o the author.
Kay, Y. A. 19S3. Phnt Path. 2: 103.
50. CMC Medium

Carboyxmethylcellulo~ 15.0 g Used t o produce abundant
(CMC7MP-Ilercules Powder Co.) macrocanidia of Gibberelk zeae
(Dissolve by mixing in a blender in warm water) when grown on a shaker for 4 days,
N1i4NO3 1.Og
KH2P04 1.0 g
MgS04 7 H 2 0 05 g
Yeast extract 1.0 g
Distilled water 1I
Capcllini, R. A, and J . L. Peterson. 1965. hlycalbgia 57:962-966.
51. Corn Meal Agar (Shear)

"To 40 g of corn meal add 1 I of water. Keep at a temperature of 58 C, never over 60 for 1 hr. Filter
through paper, add 1.5% agar, stcam for 1 % hr., filter and tube. Autoclave for 15 n~inat 115 C."
Don't over-fdl containers. Scc oatmcsl agar no, 159 for lilterrlate advice on sterilizing.
Shear, C. L, and N. E. Stcvcns, U. S. Dept. A g . Hur. Plant Ind. Ck. 131.
52. Another Version of Corn hIeal Agar

Corn meal 6c'g Place corn meal in a fine cloth bag
Agar 12& and steam for !/L hr, use extract.
Water t o make a 1
53. Corn Meal PPP hledium

Difm corn meal agar II Keep antibiotics in a sterile wat K
Pimaricin NaSalt
*Penicillin G Na-Salt
Polyrnyxin B sulpl~ate
*Heat labile
100 PPm
50 PPm
50 PPm I solution in the dark, add after
sterilization, although only Penicihn
is heat labile, For isolation o f
Phytophtlwra and Pytiu'um from
plant roots.
Eckert, 1, W. and P. H. Tsaa. 1962. Phytopathology 52:771-777.
54. Corn Steep Dextrose

Dextrose (Cerelose) 4og A generally used medium for
Corn steep liquor (Stales's) 4og growing microorganisms to detect
NaN03 3g production of biologically a c t i n
warn4 0.5 g compounds.
MgS04 7 H 2 0 0.25 g
CaCOJ 35 g
Deionized water 11
Curtis,R. W. 1957, Plant Physiol. 32:56.59.
Medla and Nutrient Solutions
55. Crone Solution (modified by Bond)

Dipotassium phosphate
KC1
CaS04 2H20
MgS04 7H20
Ca3(P04)2
Fe2(S04)3(soluble)
Boric Acid (H3P03)
Manganese sulphate
Copper sulphate
Mix all salts and grind to ;I fine powder. Add 1.0g to 1 1 of water. To solidify add 7.5g o f agar. For
[yowing seedlings, for two other versions see Allen.
Allen, 0. N. 1959. Evperimcnts in sod bacterroloyy. Burgess Pub Co., M~nneapoLs.
56. Cutter Host-Rust Medium

Green-coconut milk 150 ml
Trace element (Burkholder and Nlchell) s o l u t ~ v n 1 ml
Complete vitamin solution 10 ml
Sucrose 30 g
Agar ( h f c o Noble) log
Indoleacetic a c ~ d0.01%solution (filter sterilized) I0 ml
Half-strength Knop solut~onto 1 1
This medium is mixed arid autoclaved at 15 lb for 20 min.The approximate pH after autoclaving is
53.The composition of the various solutions used are:
Burkholder and Nickell trace element solutiop
(Bot. Caz. 110:426-437)
Vitamin solution in ug/ml

Thiamine 100
268 Riboflavin
252 Pyridoxine
1825 Calcium panto thenate
Half-strength Knop Sol. mg/l Para-am ino-be nzo~cacid
Ca(NO& 4H20 720 Choline
125 Inositol
125 Folic acid
125 Biotin
I
Cutler, V, M, $1. 1959. hlycologia 51:248-295,
57. Czapek Solution Agar (Difco and BBL Czapek-Dox)

NaN03 This medium is so named and
K2 HP04 described in the manuals of the
MgS04 7!j20 Aspergilli, The authors attribute the
KC1 formula t o Dox, but his medium as
FeS04 7H20 it appears in their cited reference
Sucrose (U.S.Dept. Agr. Bur. Anim. Bull,
120) is different as it includes
KH2P04 and only 2 g of NaN03.
Czapek's original aslution (1 902,
&it. zur Chem. Physiol. Path. Band
1. Heft 10-1 3, pp. 538-560) includes
1 g KH2P04,0.5 g hlagnesium
sulphate, 0.5 g KCI, 0.1 g Ferrous
sulphate, no nitrogen or carbon is
specified.
This medium may be modified by adding 1% wnc. corn steep Liquor. Adjust pH to 7.0with N NaOH
as the corn steep is acid; and if medium is unadjusted, medium may not solidify. Xerophytic
Aspergilli such as members of the A. gloucus group require 200 g of sucrose.
Raper, K. B. and Dorothy I. Fernen. 1965. The penusArn..rgillur. Williams and H'ikin, Baltimore.
Thom,C. 1910. Cultural studies of Petricilh'um. U.S. Drp Agr. Bur Anim. Bull. 1 1 8.
58. Czapek-L)ox A p r (Difco Czapck Solution)

Contains the same ingredients as Czapek solutior~agar (57) except that there are 2.0 g of NaN03.
According t o Thorn and Church (1926, The Aspergilli) this has little effect on the Asptrgilli. Strictly
adhering to the original version of A. W. Dox, !UI2PO4should be instead of K2tfP04. Thorn and
Church used the latter because the final reaction is neutral or only very slightly acid. 50th Difco and
BBL use K2l P 0 4 as do most versions.
Dox,A. W. 1910. U.S. Dcpt. Agr. Bur. Anim. Ind. BuU. 120.
59. Desaxychohte Agar

Peptone pH should be 73 or 7.5. Store
Lactose -
medium in dark inhibits Gram (+)
NaCl but not Gram bacteria.
(0)
Ferric ammonium citrate

Dipotassium phosphate
Sodium dcsoxycholate
Neutral red
Agar
Lesson, E. 1935. J. Path. Baa.403581.599. A h Mutun, Rorc et aL, 1959. Aud. J, BioL S d 12:223-232.
Medh and Nutrient Solutians
60. Dextrose Pcptone Yrast (modified)

Dextrose 5.0 g Yeast extract
Peptone 1.Og QwU
NH4N03 1.0g Sodium propinate
K2IP04 1.0g A w
MgS04 7 H 2 0 0.5 g *Chtortetraqcline
FeCII 61i20 traa *Streptomycin
Used for isolation of fungi from soil. Sterilized at I I Ib., 15 min.
*Fresh solutions added after the media ha been sterilized and cmAcd to approximately 48 C.
hplvizas.G, C. and C. B. Davey. 1959. Soil Sci. 88: 112-1 17.
61. Diet Food hledium

Metrecal Edward Dalton Co. Used for the general cultiviltion of
j
Evansville, lndhna many plant pathogenic fungi and
or bacteria.
Sego Pet,Inc.
1
St. Louis, hlksouri
Agar
Water
Stoner, M. F. 1967. Phytopnthology 57:447.
62. Dimmick Medium

Glucose A superior medium for determining
K2 lIP04 nitrate positive organisms. Dimmick
NaCl believes lack or use of small amount
MgSO, 7H20 of caldum prevents phosphate
NaNOj deficiency. Huisingh and Durbin
Agar consider it a good medium for
Distilled water studying growth responses of
(may use CaC12 Agrobacrerium and useful for
identifying 3-keto-glycoside-negative
isolates.
Dknmkk, Isabel. 1947. Can. J. Res. (C)25:271-273.
Huiringh, D.and R. D. Durbin. 1967. Phytopathobgy 57:922-923.
63. Dung Agdr

Fresh horse dung Boil mixture for 15 min, then filter
Water through cheese cloth. Twelve g of
agar is added and boiled until melted,
Bring t o volume. To clear the
medium add the whites of 2 eggs
and their shells to SO ml of water
and stir. Add to dung decntion that
is at 60 C, stir and steam for 1 hr
and fitter through cottonwool.
B u k , A. K 193 1. R c ~ ~ d on
u rfungi. VoL 1V. Longmans, Green & Co, Loadon, N.Y. & Toronto.
Mcdla and Nutrient Solutions 2Y
64. Dung Agar

Bovung (commercial dried cow manure)
Na21LP04 1 21i20
M2m4 M/2W I Used t o buffer medium to pH 65.
According t o Hesseltine et a1 (see No, 65) pH rrwy be low for RbboLs.

McVickar, D. L. 1942. Amer. J. Bot. 29: 372-380.
65. Dunpatmeal Agar

..Bovung (commercial dried cow manure) 125 g Stand for 2-3 hr and then filter
Tap water II through 2 thicknesses of cotton
g a w and filter paper.
B. Oatmeal Boil for 5 min and strain the hot
Tap water suspension through 2 thicknesses
of ootton gauze.
Add 15 g or agar to a mixture of 500 rnl of A and 500 ml of B. Autoclave for 15 min at 15 Ib, Good
stock medium for Pilobolus. Be sure pl l is above 7.
66. Dutch Elm Medium

Potato dextrose agar Both antibiotics are added prior to
Cyclohexin~idc(actidione) 200 ppm autoclaving. Used for the isohtion
Streptomycin sulphate 10 PPm of Ceratocytis ulmi. F. W. Holmes
(personal communication 1966)
considers PDA adequate under
most circumstances.
Schncider, I. R. 1956. Plant Dis. Reptr. 40:816-821.
67. Elliott Agar

Potassium monobasic phosphate 1.36 g Dextrose
Sodium carbonate 1.06 g Asparagine
Magnesium sulphate 0.5 g Agar
Distilled water 11
Elliott, J. A. 1917. Arner. J. kt.4:439476.
68. Elmextract Agar

Elm sawdust or shavings from living trees, 120 g
includes bark, s p w o o d and heart wood
Agar 4og
Distilled water 2400 ml
Gently boil elm shavings in 1500 ml water for 10 min. Srrain through screen wire. Remaining
material washed with distilled water and strained into f i s t fdtrate ro bring to final volume of 2400
d.Add 240 ml to each of ten 500 ml flasks, add 4 g of agar, steam until melted and then autoclave
at 15 lb for 15 min. If no elm sawdust is available, water agar may prove a satisfactory substitute.
Used t o detect Cerotocystis ultni. Wood chips 0.5 cm thick from branches 0.5 to I.? cm used,
Coremia were produced at 22-25 C in 5 to 7 days,
Hat,J. H. 1960. P h t Dis. Reptr. 44.806-807.

69, Emerson YpSs Agar
Yeast extract (Difco) Used in the study of life cycles and
Soluble starch taxonomy of Altomyces and other
KzIP04 acquatics,
Mg334
Agar
70. Emersons hieditl~n(modified)

Bacto peptonc For long-time storage of Erwinb
Beef extract omylowm grow in screw capped
Dextrose vials, then store at 3 C. Buffer
Yeast extract medium to pH 6.8 with stock buffer,
Trypticase then add trypticase.
Water
Stock Buffer Solution
Distilled wa tcr
K21fPo4
KH2P04
Rchhardl, J. F. and D,Powell. 1960. Phytopathology 50:685-686. hlodified in a personal communication to E, B.
Wllliam~
71. Eosin Met hylene Blue Agar (EMS)

Peptone (Gelysate or Bacto) loll Medium should be mixed often
Lactose 5g while pouring plates, Colonies of
Sunose 5g Erwin& nigrifidens and other
Dipotassium phosphate 28 members of the Enterobacteriaceae
Eosin Y 0.4 g develop a greenish metallic sheen.
Methylene blue 0.065 g
Agar 13.5 g
Distilled water 11
Edwards, P. R. and W. H,Ewing, 1962. Identification of Entcrobacteriaceae, Burgess Pub, Co., Minneapolis.
Zdtoun, .'k M. and E. E. Wilson. 1966. Phytopathology 56: 1381.13115.
72. Filter Paper Yust Agar

Filter paper Blend filter paper in water, for
Yeast extract perithecia formation of Sordaria.
Agar
Tap water
Ingold, C. T. and V. G. During. 1957. Ann. Bot. 21:465-477.
73. Fluorcscin 5ledium (Medium 8)

Proteose peptone no. 3 20g Pigment of Psrtrdonzonus observed
Glycerol C. P. 10 g most frequently is fluorescent and
K2!POs (anhydrous) 1.5g of a greenish yellow hue. According
MgS04 7Ii20 1sg to Caribaldi, I. A. (1967. J. Bacterd
Agar 15 g 94: 1296-1 299) 10% sterile egg white
pH 7.2 added t o the medium a t 45 C
enhances pigment production,
King, Elizabeth 0..Sfartha K. Wud and L). E. Raney. 1954. J. Lab.and C l n . hled. 44:301-313,
74. Fomes Annosus Isolation Medium

Bacto-peptonc
MgS04
KHP04
Pentachloronitrobenzcne
Streptomycin
Agar
Lactic Acid (505)
Ethyl alcohol (95%)
*] ~
20 ml
d after
d sterilization.
Kuhlman, E. C. and F. F. Hcndriu, Jr. 1962. Phytopafholo~52:131~1312.
75. Fothergill Sucrose Ammonium Sulphate Sledium

Sucrose 4% w/v Used in a study of the mineral
K2HPO4 OA8M nutrition of Rhizopus srobnifer.
MgS04 7)j20 0.01 2hi
( M i d 2 so4 0.025M
::]
Mn
2 ppm
Add sterile phosphate solution
a f t a autoclaving to avoid possible
precipitation.
Fothergill, P. G. and M. hf. Yeoman. 1957. I. Gen hlicmbiol. 17:631639.
76. Freezing Agar

Diced potatoes ='g Add potatoes to 500 ml tap water
Dextrose 8.0 g and autoclave at 15 Ib for 15 min,
Yeast extract 0.5-1 .0 g mash and filter through 4 layers of
Activated charcoal 0.5 g cheesecloth, bring the volume to 1 I
Agar a.0g with distilled water and add other
medients.
77. Fries Medium

N11, tartrate K2 HP04
NH4N0 Glucose
MgS04 7H20 Yeast extract
KII~PO~ Water
Use at the rate of 0.1 rnl/100 ml of medium.
Frirs,N. 1938. Symb. Bot. Vpsal. 3: 1-188.
78. Fungus Resistance Testing Medium

Glucose Adjust pH to 6.8 prior t o autoclaving.
NH4N03 Medium designed to give maximum
KC1 growth of a number of fungi used in
K, 10'0, testing materials that resist fungi.
WSO4
FeS04
ZnS04
MnS04
NaCl
Klausmeier, R. E., Janice L. K y l z i g , U'. A. Jones and V. L. Hnrnrnersley. 1963. Dev. Ind. hlicrobiol. 4:306-313.
79. Gallic or Tmnic Acid Medium

Difco powdered malt
Agar
Gallic or tannic acid
Water t o make I I
Agar and malt added t o 850 rnl of water, the remaining 150 ml placed in a separate flask. b t h are
sterilized for 20 min at I S Ib. N'hile the sterile water is stiU hot, the gaUic or tnnnic acid is d~ssolved
in it; then this solution is added to the stightly cooled malt agar and thoroughly mixed with it before
being poured directly into sterile phtes. See also Noble, Mildred Can. J. Bot 36:91-99 for rapid test.
Davidson, R. W., W. A. Campbelland D. J. Blaisdell. 1938. J. A g . Rrs. 57:683695.
Nobk, Mildred J. 1965. Can. J. Bot. 43: 1097-1139.
Glucose 1% Used for the production of fruiting

dl alanine 0.1% bodies of Coprinus bgopus. See
Thiamine HCI 500 P8P also medium no. 90. Suggest
K2 HP04 0.2% separate sterilization of glucose and
M@O4 7H20 0.02% K2 HPO4
wr 296
81. Glucose Asparagine Agar

Reveloped from a chemical analysis
Gluoox
Asparagine of potato tubers. Designed t o
replace PDA, at least for certain
K3PO4 species o f Fusarium.
MgS04 71120
A P ~
Water
Brown. W. 1925. Ann. Bot. 39:373-408.
82. Glucose Asparagine Medium

Glucose Medium used for thiamine assay.
Asparagine 1g Schopfer, W. H. 1945. Experientia
M@04 7t120 0.5 g 1:148, p. 210.
KH2Po4 15 g
Distilled water 11
L U y , V. G. and 11.1. Barnett. 1951. Physiology o f the fun$. h1cC;raw-Hill. Ncw York.
83. Glucose Casammo Acid YIedium

Glucose 15 g Used to increase Fusariurn oxysparu~
Casamino acids 1.5 g f. sp. lywpersici.
Yast extract 1.0 g
KI12P04 1.5 g
MgSO4 1g
Trace elements A-Z from Iioagland solution 10 ml
Dimond, A. E., C. H. Plumb,E. hi. Stoddard and I. C. tiorsfall. 1949. C o ~ c c t i c u Agr.
t Exp. Sta. Bun. 531.
84. Glucose Casein Hydrolynte Medium

Glucose
Casein hydrolyate equivalent to 2 g casein
M f i 0 4 ' 71.i20
IUj2P o 4
Fumaric acid
Na2C03
Mn++as sulfate
FcHas sulfate
&++as sulfate
Distilled water to make 1 1
Lilly. V.G. and H...I Bamatt. 1951.Physiology o f the fungi. hlffirew-Hill, Hew York, p. 211.
85. Clucw Nitrate Agar
1g Most bacteria and fungi grow
1 ml each
of a 10%
solution
150 1OW1001.) pH adjusted to 7.
86. G h r c o Nitrate
~ Agar (modified)
Addition of 40 ~g Actidone, previously sterilized by Ntration through an ultrafine sintered glass
filter, eliminates growth of a number of fungi while Actinomycetes grow well. See Corke, C. T, and
F. E. Chast. 1956. Can.J. Microbiol. 2:12-16. For most purposes Actidione can be added directly to
the medium and sterilized with it.
87. Ghcose Nitrate Sterol hledium

Glucose 5.4 g Adjust pH to 6.0. Autoclave for 10
NaNOj 1.5 g min without sterol, Add 20 mg of a
KHzP04 1.og sterol in 2 ml of ether to surface of
?d@O4 7H20 0.5 g 25 ml of agar. Anyone of these
ABar 174 sterols: Ergosterol, Phytosterol,
Sterol 0.8 g stigmasterol, B-si tosterol, cholesterol.
Thiamine hydrochloride (2 ml of a 1000 ppm) After several hr inoculate, Keep in
(dock solution) dark. For oospores of Phytopittlww
Distilled water 1I and Pythium.
Hendrix, J. W. 1964. Sdencc 144: 1028-1029. See abo 1965 Phytopathology 55:790-797
88. Glucose Peptone A g r

Glucose 10.0 g For high conidial production of
bcto-peptone 2.0 g Gbevsporittnt ntltsarunt. Grow on
KHzm4 0.5 8 slants at 27 - 30 C .
hlH4 71i20 0 5g
Agar 15 8,
Water 11
Coos, R. D. and Marylou Tschcrsch. 1962. Mycolagia 54:353-367.
89, G l u c o Peptone
~ Medium
Glucose For zoospores of Aplutanornyces
Peptonc euteiches. Grow for 2 days at 24 C,
replace with Apllanoniyces wasting
solution no. 1 1.
Mitchell, J. E. and C. Y. Yang. 1966. Phytopathology 56:917-922.
Glucose Used for the production of fruiting

Yead extract bodies of Coprinus hgopus. Suggest
K2 HP04 rparate sterilization of glucose and
MgS04 7H20 K2HPO4. See also medium no. 80.
AIPr
MdeL. IW. F. 1956, Ann. Bot. 20:307-330.
M d i r and Nutrient Solutions
91. Glycerol Asparaginate Medium

Glycerol Used in the study o f Actinomycftd
K~t1m4
Sodium asparaginate
CaCOj
Agar
92. Giycerol Arparagine A p r (Medium 5, Sl~irlingand Gottlieb)

L-asparagine (anhydrous b~sis) 1 .O g pH should be 7.0-7.4
Glycerol 10.0 g Used in the study of Streptonlyce~,
K3!11'04 1.0g
Trace salt solutio~~
no. 139 1.0 rnl
Agar 20 g
PrLlhsm,T.G. and A. J. Lyons. Jr. 1961. J . Bactcriol. 81;431441.
Shirling, E. B. and I). Gotttieb. l n t . J . Syst. Bacterial. 16:313-340.
93. Grain
Corn, lima bean, oat, pea, sorghum, wheat, etc.
Seed usually soaked in water and in case of pea (Thurston, 11. D. 1957. Phytopathology 47: 186) and
white seeded sorghutn (\!111tehead, 51. D, 1957. i'hytopathology 17:450) soaked overnight, rinsed
with water, and autoclaved intact in flasks. hlixture of cereal grains are often used. It is
recommended that these grains be autockved in small amounts at 121 C for 40 m i l or at 121 C for
20 min on 2 successive days.
94. Green Bean Agar

Finely ground and cooked green bean seed 400 g Used to produce conidia of
Agar mg Collerotricizum hgerlarium, See
Water to make a I also Littrell, R. H,and W. M. Epps.
1965. Plant Dis. Reptr. 49:649-653,
Goode M. J, 1958. Phytopathology 4k79.83.
95. Hartold Medium

Malt extract 2% (20B) For sporulatian and growth of
Sucrose (400 g) Eremascus.
Difco Yeast extract 0.5% ( 5 g)
Agar 2og
Water I1
Hurold, C. E. 1950. Ann. Bot. 14: 127-147.
96. Hay Infusion Agar

Decomposing hay
Distilled water
50,
11
] Autoclave for 30 min at 15 Ib.,
filter.
Infusion filtrate
K2HP04
Agar 15 g
i: ] Adjust to about pI1 6.2.
Useful in the isolation of Aspergilli and dark spored hyphomycutes.

Thom,C. and K. B. Raper. 1945. Ximud of the Aspcrgilb. \Viharns S Wilkins, Baltimore.
97. Hayward 0 - F hledium

Designed to determine oxidative - fermentative rnctabolisrii of carbohydrates
NH4H2P04 I .O g Adjust pH t o 7.1 with 40% W/V
KC1 0.2 g NaOH before add~ngagar, sterilize by
MgS04 7H20 0.2 g autochving, Sterilize a 10% W/V
Peptone 1 .O g solution of carbol~ydrateby auto-
Bromthynlol blue 1% W/V 0.3 ml claving but filter sterilize, cellobiose,
Agar 1.5 g fructose, maltose, arabinose and
Water, distilled 11 galactose. Add 5 ml of carbohydrate
solution to 45 ml molten coolcd baul
medium in test tubes. For oxidative-
fermentative test of Zlugh and Leifson
(1953 J. Bact. 66:24-26) stab inoculate
2 tubes and cover one Yo to 5" deep
with sterile melted petrolatum. Acid
production gives a yellow color in
sealed tube indicating fermentative
metabolism of carbohydrate, not
characteristic of Xanthonmnlas.
Hayward, A.C. 1964. J. Appl. Baa. 27:26S.
98. Hemp Seed

Place root hairs or soil in water, add boiled henip seeds (may be split). Isolate fungi growing out
from hemp seed. Emerson, R. (1958, Mycologia 50:589421) recommends that the seed be viable
and they be boiled for 10-20 min.
Matthews, V t h a D. 193 1. Studies on the genus Pyrhium. Univ. o f N. Carolina Press, Chapel Hill.
99. Hemp Seed Agar

Hemp seed 1008 To induce sexual reproduction of
Agar 208 Phytophtlara mpsici and P.
Distilled water to make I I prasifia, inoculate with small discs
of pairs o f isolates about 15 mm
apart and grow at 21 C for 5-7 days,
Satour, M. and E. E. Butler. 1967. Phytopathobgy S7:SlO-515.
Medk and Nutrient Solutions 37
100. Hemp Seed Dccotion

Haasis, F. A. and R. R. Nelson (1963. Plant Dis. Reptr. 47:705-709) used an aqueous hemp seed
decotion 10 pt hemp seed to 1 pt water added to water agar in a proportion of 1 :I 0 to induce sexual
reproduction in Pl~}~tophthora.
101. Hoagland Solution (1 950)

Solution 1 & I ml in a 1of solution
IM Kl12P04 136 1
Ihl KK03 101.1 5
1M Ca(N03)2 164.1 5 Solution B g/l ml in 1 1of solution
IM MgS04 1 20.3 2 Iron tartrate 5 .O 1 ml
Solution 2 (necessary to add at regular intervals)
- 1 h!1i4H2P04
~ 132.07 1 Alternate Solution B - Disxrlve 26.1 g ethylene-
Ihl KNOl 101.1 6 diamine tetra-acetic acid in 268 ml of I N KOII..
1M Ca(N03)2 164.1 4 Then add 24.9 g FeS04 *71120and dilute to
1M M@04 120.3 2 one 1. After aerating overnight to produce the
-
Solution A
H3BO3
MnCl2 4U2O
ZnS04 7 i i 2 0
;:if 1
0.22 1 ml
PPM
0.5
0.5
0.05
stable ferric complex, the pH should be about
5.5. One ml provides 5 ppm to one liter of sol.
-one addition enough. After Jacobsen, L.
(195 1. Plant Physiol. 26:411413.)
Add 1 ml of solution A to either solution 1 or 2 and bring to a 1. Add solution B or its alternate.
Adjust pH t o 6 with 0.1N Il2SO4.
Hoaghnd. D. R. and D. 1. A+ &1950. California A p . Exp. Cir. 347.
f
102. Hoaghnd & Knop Solution (modified)
Macroelement s Ferric citrate sol.
Ca(N03)2 4H20 0.95 g FeC60sH7 5 H 2 0 10.0 g
KN03 0.61 g m1 (in I 00 ml)
M W 4 7H20 0.49 g Agar 5.0 8
NH42l2PO4 0.12g Distilled water to make I I
Microelements (in I000 ml water) Used in sterile culture of plants.
MnS04 4 H 2 0 3.0g *
7H20 0.5 g
H3PO3 0.5 g
CuS04 5 H 2 0 0.025 1
Na2Mo04 2H20 0.025 g
H1S04(sp.gr. 1.83) 0.5 ml J
Qn, T, R. A. Kilpaftick and A. E. Rich. 1961. Phylopathology 51:799-800.
103. Honey Peptone hledium

Honey A 6og
Difco Bacto-pept one 10 8
&ar 2'Jg
The pH is favorable t o fungal g r o w h and idubitory t o most bacteria.
Backus, I!. P. and J. F. Stauflir. 1955. Ifpwbgia 4 7 : 4 ? 9 4 3 .
Cnsklap, J. H. and Sfargoric E. Swill. 1957. htycologia 49:305-317.
104. Hugh and Leifson Carbohydrate 3ladium

Peptone (pancreatic digest of 0.3~ Used to detect fermentative and
casein preferred) oxidative n~etabolismof arbohydrates
NaCl 05% by gram negative bacteria fermentation
K2HP04 0.032 medium and Hayward's 0 - F medium.
Agar 03%
Brornthymol blue 0.003% (0.3 ml of 1% water solution added
to each 100 ml.of medium.)
Carbohydrate 1.Wo May require separate and special
sterilitation.
Hugh. R., and E. Leifron. 1953. J. Bscteriol. 66:24-16.
105. Inorpnic Salts Starch A g r (Shirling & Gottlieb no. 4)

Solution I.
Difco soluble starch 10.0 g Make a paste with a small amount of
distilled water (cold) to make 500 ml cold water and bring to 500 ml.
Solution 11. pH should be between 7.0 and 7.4.
K 2 W 4 (anhydrous basis) 1Ag Mix Sol. I and 11. Add agar and
MgS04 7H j 0 1.0 g steam for 20 min. Used for
NaCl 1.0g diagnostic tests with Strepromyces
(NH412S04 2.0 g
CaC03 2.0 g
Trace salts solution no. 239 I ml
Kuster, E. 1959. Int. Bull. Bact. h'omcn. Taxon. 9:98-104.
Shirting, E. 6. and D. Gottleb. 1966. Int. J. Syst. Bacterbl. 16:313-340.
106. Ivanoff Isolation Slcdium

Glycerol Dissolve salts with exception of
Ferric ammonium citrate sodium taurocholate by gentle heating.
Sodium taurocholatc Steam latter in 200 rnl water. ,\fix
Sodium chloride all ingredients and adjust pH to 7.0
Sodium sulfate by addition of 17 ml of N/I SaOH.
Dibasic potassium phosphate A skctive medium for Xanrirowwnru
Calcium chloride ttoyetti which allows isolation from
Magnesium sulphatc badly contaminated plant p a t s and
Aw t o m artificially infested soil.
M d i and Nutrient Solutions
107. lensen Agar

Dextrose 2.0 g An older medium used to isolate
Casein (dissolved in 10 rnl0.1 N NaOfi) 0.2 g Aainomyater. pH 6 5 4 . 6 .
K2m4 o5 g
M@04 7H20 0.2 g
FeCIS 6f120 Trace
Agar 15 g
Distilled water to make a I
Jenstn. 11. L. 1930. Soil Sci. 30:59-77.
108. Kennedy & Erwin EDTA Sledium
hhcrosalts Chclatcd iron stock I mln

KfI2FQ4 0.00 1 hl Ethylenediaminc tetra-acetic 26.1 g
KNOj 0.005 M acid (EDTA)
Ca(N03)2 4t1,0 0.005 M KOH 15il
h1gS04 71j20 0.002 M FeS04 71120 24.9 g
Distilled water to make a 1 1
hlicrosalts Stock I ml/l
H3B0, 2.86 g Used to induce sporangiai formation
hlnCll 41120 1.81 g of Phytophrltora when V-8 juice
ZnS0, 71120 0.22 g agar-mycelial disn are transferred
CuS04 S h 2 0 0.08 g to this medium for 24 hr.
flHi)6h107024 4ti10 0.2 g
Distilled water to make a I.
Kcnnedy, B. W.and D.C. Enbin. 1961. Brit. M y a I . Soc,,Tnns. 44:191-297.
109. Kidney Bean Agar

Ground dry red kidney bean 3og Used for oospore production of
Agar 17 g Phytophtirrro fragaripe.
Distilled water 11
Converse, R, H, and K. K. Sheroishi. 1962. Phytopathobpy 52:807-809.
110. Knop Agar

Ce(h'03)2 4H20 0.8 g Favorable for the germination and
Kh'03 0.2 g growth of some Slyxomycetes.
KH2po4 0.2 g
hlgSOI 7H20 0.2 g
FeS04 Trace
Am 2Qg
Water 11
Akxopoubs, C. J. 1960. Amer. J. Bot. 4 7 9 7 4 3 .
40 Mtdia and Nutricnl Solutions
111. Koser Citrate Medium

NaCl 51 Dissolve the salts In water and adjust
MgW4 7H20 0.2 g to pH 6.8 with N NaOH. Filter
NH4H2P04 1.08 through a sintered-glass funnel. The
K2m4 1.0 g medium should be colorless.
Distilled water II Sterilize at 1IS C for 20 min. All
Citric acid 2g ghss must be acid wash and alkali
free.
Cbann. S. T. and K. J. Steel. 1965. hlrmud for the identification of medical bacteria. Canbridge University Press.
landon.
112, Krainsky Agar

Glucose 10.0g Used in the study of Actinornycetes.
Asparagine 0.5 g
Ka fm4 0.5 g
Agar 158
Water II
113. Lactose Casein Hydrolyate Medium
Lactose 37.5 g Microelements (Used by Lilly and Barnett. 195 1,
Casein hydrolynte 3.0 g Physiology of the Fungi)
KH2P04 I ,O g Fe(N03)3 9H20 7235 mg
MgS04 0.5 g ZnS04 7 H 2 0 439.8 mg
Microelements 2 ml MnS04 4H20 203.0 mg
AfW 1% Dissolve one at a time in a 1of water. Add
sulfuric acid to yield a clear solution.
Adjust pH to 6.0. A good sporulation medium for Helrni~zthosporiumturcicum and f!, cmbotiurn
and probably for other tlelminthosporia.
M&a, I, and A. J. Ullstxup. 1962. Bull. Torrey Club. 89:240-249.
114. h n i a n Agar
Peptane 0.625 g According to W. Cooke, useful for
Maltose 6.25 g developing fruiting structures of
Malt extract 6.25 g some fungi and is a standard medium
KH2 P04 1.25 g for the study of cultures of
MgS04 7H20 0.625 g Ascomycetes and Splueropsidales.
A w 20.0 g Neopeptone or polypeptone may be
Distilled water 11 substituted for peptone.
Cook, Wm. 8. 1%3. A laboratory guide to fungi in polluted waters, sewage and sewage treatment systcmr Public
Herlth Ser. Pub. 999-Wpl,Cincinnati.
115. Lima k n Agar

Very fine ground dry lima beans 1% For growth and maintenance of
Dextrose 1 0 &! Phytuphtlwra infestans,
&r 10 E
Difco yeast extract 2g
Bean powder suspended in 800 ml water 4 5 min at 121 C then mixed together and autoclaved, in
mall amounts, for 30 min.
H. D. Thurston. 1957. Phylopathology 47: 186.
1 16. Lima B a n Agar

Dried lima bean illfusion and agar (difco) 18g Sporulation of Rliynclloslwrium
Agar 5g secalis and R, ortkosporum
Water 11
Schcin, R. D.and J. W. Kcrcb. 1956. Plant Dis. Reptr. 40:814-815.
117. Lima B u n Agar

Fordhock 242 dried lima bean seed 5g Autoclave with agar for 20 min.
Agar mg For use with test tubes, reautoclave,
Distilled water 11
n~egasperntaVU. sope.
Used for growing stock cultures of P1~)~lopl1tltora
Hilly, J. W. and A, I:, Schnitbc'nncr. 1962. Phytopatholopy 52:859-862.
118. Lima Bean Agar (frozen)

1 pkg of frozen Fordl~ooklinla beans 283 g
AP~ 408
Distilled water 21
Steam lima beans in 1 1 of water for 20-30 min, filter through a cotton towel, adjust the volume to
2 1, add 20 E; of agar and autoclave at I91 C for 15 min. Used for the production of sporangia and
oospores of various species ofPilytopltrltora.
119. Lima Bean (frozen)

Fmzen l i i a beans
Agar
Water
Blend lima beans in water, add melted agar, medium should bc kept out of the light as it becomes
fungistatic. Medium used for the production of oospores of Plt)aophthora ittfestans (Smoo t, J . J., F+
J. Cough, H. A. h m e y , J. J. Eichenmuller and 51. E. Gallegly. 1958. Phytopathology 48:165-171)
and increase and storage of Pl~)~topllthorasobe (Calvert, 0.H., L. F. Williams and M. D. Whitehead.
1960. Phytopathology 50: 1 31- 137).
120. Lingappa and Lockwood Chitin Medium

Colloidal chitin 2.0 g For isolation of Actinornycetes,
Agar 20 g although p k i n water agar is almost
Water 11 as good.
Colloidal chitin prepared as follows:
Crude unbleached chitin (Nut. Biochem. Co.) wadled alternatively for 24 hr each with 1 N NaOH
and IN IiCl (usually 54x1 then with 95% ethanol (34X).A clean white preparation is available
from Pfanstiehe Chemical Co, and does not require this pretreatment. Fifteen grams of cleaned
chitin is moistened with acetone and dissolved in 100 rnl of cold concentrated HCI by stirring for 20
min in an ice bath. The thick syrupy solution is filtered with suction tluough a thin glass wool pad in
a Buchner funnel into 2 1 of stirred ice cold dist. water precipitating the material as a fine colloidal
suspension. The residue is redissolved and refiltered usually 3 or 4X until little or no more chitin is
precipitated. The colluidal chitin is alternately sedirnented by allowing it t o stand and washed in 5 1
of distilled water until 4-5 vol of water is used. The material is stored in the refrigerator, usually
there is about 2-3 rng of colloidal chitin per ml.
Lhgappa, Y and J. L. Lockwood. 1962. Phytopatholoyy 52;317-323.
121. Littman Oxgall Agar

Peptone Frequently used for the isolation of
Dextrose fungi from animals. Dissolve 1.25 g
Oxgall of crystal violet in 25 ml of 95%
Agar ethanol and keep in a tightly stoppered
Crystal violet bottle. Add 0.2 ml/l of medium.
Littman, M. L. 1947. Science 106' 109-1 1 1 ,
122. b n g Ashton Nutrient Solution

Wt, in g
(for 100 1 of diluted nutrient)
KN03 20.2 or 33.6 or 50.5
Ca(N03)2 (anhydrous) 65.6 or 54.7 o r 82.0
NaH2P04 * 2 H 2 0 20.8
MgS04 7 H 2 0 36.9 Produces vigorous growth in a wide
Ferric citrate 2.45 range of plants. pfl 5-6.
MnS04 41 1 2 0 0.223
CuS04 S H 2 0
ZnS04 7 H 2 0
H3BO3
mH4)6M0702 4 4H20
I
(Al),S03 a 121.120 0.0186 For use with highly purified
G ~ ( N O ~ )1%~ solution
' of Ga. 0.05 ml salutiolu.
C0S04 7 t I 2 0 0.0028
NiS04 7 H 2 0 0.0028
'The gallium nltiate solution is obtained from hleous. hlatrhy Ltd. and is o r r of the EtMldard L L ~ p e ~ pgrade
wmpunds.
~n"
Hswitt, E. J. 1962. S a d and water culture methods uwd m the study of plwt nutition. Commonwealth Bur.
Hort, Tech. Com. no. 22. East Mdling.
Mcdh and Nutrient Solutions 43
123. Lukensand Sisler Synthetic Medium

Glucose 20 g Adjust to pH 6.0 with KOH. Used
Ammonium sulfate 3.0 g in conjunction with a filter paper
Magnesium sulfate 0.25 g technique to induce sporuktion of
Monobasic potassium phosphate 3.0 g Helntintiwsporium vagans and
Clycine 1.0 g Altermaria sahni (Lukens, R. J.
Niacin 20443 1960. Phytopathology 50:867-868
Biotin 1 Cg
1-inositol 200
Pyridoxine 10 Ccg
Folic acid 10 Ccg
Boron .O1 pprn
Mn 0.01 pprn
Zn 0.07 pprn
Cu 0.001 pprn
Mo 0.001 pprn
Fe 0.05 pprn
Distilled water to make a I
Lukens, R, J. and 11. D. Sislcr. 1958. Phytopathology 48:235-244.
124. Lutz Medium (modified)

Vitrwns malt extract1 10.0 g Magnesium sulpha te 0.1 g
Ammonium nitrate 1.Og Ferric sulphat e 0.1 g
Ammonium phosphate 1 .O g Manganese sulphate 0.025 g
Agar 25.O g
Medium suitable for almost all nonmycorrhizal species and many mycorrhizal species of the agarics
and boletes. Useful for diagnostic purposes.
' Obtained from Apoteksvuuncentrd, Stockhotm, Sweden.
Singer, R. 1962. The Agaricales, llafncr, N. Y.
125. Machlis Medium A (modification of lngraham Medium)

Thiamine IICI 0.15 mg KOH used to adjust pH to 7.0.
DL Methionine 0.1 g Glucose is autoclaved separately.
MgCl2 0.001 M A synthetic medium used for
KH2P04 0.0 1 hi nutritional studios ofAlbnryces.
w114)2m4 0.005 M
CaCll OD002 h1
MnC12 0 5 pprn hln
&SO4 0.1 ppnr Zn
0.5 pprn B
CdO4 0.1 pprn Cu
FeCIJ 0 5 pprn Fe
(N1i4)6M0702 4 0.2 pprn hlo
C0Cl3 0.2 pprn Co
Clucase 5g
Machlis, L. 1953. Amer. J . Bot. 40: 189-195,
126. Machfis Dilute Salt Solution

KHzP04 0.0001 hl A medium used to encourage spore
MgCl2 0.000l M discharge of ~ r i o u aquatic
s fungi.
CaCll OX)0002hl
KOH t o pH 7.0
Machiis, L. 1953. Amer. J . Bot. 40: 189-195.
-
127. Malachite Green Captain Medium
Sodium nitrate 2.0 g After autoclaving add fresh solutions
Dipotassium monohydrogen phosphate 1 .O g of malachjte green, captan and
htagnesium sulfate 0.5 g dicysticin (Singh & Nene). Used to
Potassium chloride 05 4I selectively isolate F~rsaritrmfrom
Ferrous sulfate 0.01 g soil. IIorton and Keen used it to
Sucrose 3og selectively isolate P}lrenoclzueta
Distilled water t o make a I terrest is.
hfakchite SO mg
Captan 100 mg
Dicrysticin (mixture of Streptomycin sulfate, put %g in 3 ml sterile water, and add
Pmcain penicillin C; and Sodium penicillin G) 1 ml to 200 ml of medium before
pouring.
Singh, R. S.and Y. L.Stne. 1965. Pknr Ois. Reptr. 49: 114-1 18.
Horton, J. C. and S . T. Keen. 1966. Phytopathology 56:908.916.
128. Malt A p r
Malt extract Do not "over" sterilize as agar will
Asar be hydrolized.
Water
129. blalt Agar (Bhkrke formula)

hfalt extract
Peptone
Dextrose
Agar
Distilled water
130. Malt Agar ( d i f r e d by Kauffman)

Malt extract For growth of most nonmycbrrhizal
Yast extract fungi of the agarics and boletes.
Peptone
hfaltose
Magnesium s u l p h t e
Cdcium nitrate
hlonopotassium phosphate
Agu
Wdh a d Nutrient Solutions
Difco Bacto malt a p r Standard medium for identification

Wco Bacto agar of Hymenomycetes.
Distilled water
Nobk. Mildred J. 1965. Can. J. Bot. 43: 1097-1139.
hfalt extract 3g
Yeast extract 2g
Dihydrogen potassium phosphate 05 g
hlagnesium sulplutc 0 5 !k
Agar mg
Distilled water 11
b n i a n , L. H. 1934. West l'irginia. A g . Exp. 5La. BuIL 262.
133. Malt Broth (liehrlich formula)

Difco malt extract U x d for growth and sporangial
Dextrose production of Pythiaccous fungi.
Peptonc
KH2P04
hfW4
Water
Mehrlich, F. P. 1934. Phytopatkalqy 24: 11 27-1 128.
Mchrhch, I-'. P. 1934. Phytopattalogy 25:432435.
134. Malt Salt Agar

NaCl A medium of high osmotic concen-
Agar tration used to isolatc storage mold!,
Malt extract i ~ . fungi
, able to grow on products
Distilled water stored at low relative humidities.
Various concentrations from 7.5 to
20% sodium chloride have been used'
Do not autoclave at 121 C for more
than IS min.
Chrktcnxn, C. hi. 1946. Cereal Chem. 23:322-329.
Kothcimer. J. 0. and C. $1. Christensen. 1961. Vallerstcin hb.Cornmue 24:21-27.
135. Xfaltose Peptone Broth

Maltose 3.0 g For zoospore production of
Peptone Aphanomyces euteiches.
Water II
Lockwwd, J. L. and J. C. Bahrd. 1959. Phj?opbhow 49:406-410 and Llanos, C. M. and J. t. tockwmd.
1960. Pbytopathobgy 50.826830.
136, Maltox Tartrate Medium

Maltose 208 Used for sporulation of.~~e&nconium
Ammonium tartrate 2.8 g fuligineum, at 25 C alternating light
KHzP04 1g and dark, inoculate by flooding.
MgS04 7H20 05 g
Thiamine 50 CB
Zinc
Iron
Manganese
Agar
0.2 mg
0.2 fq
0.1 mg
20 g
I See microelement solution no. 143
Timnick, Margaret B,, ti. 1.U ~ n e t and

t V, G.Lilly. 1952. Zlpcologia 44: 141.149.
139, Martin Media

137
(Martin's Original)
Ingredient -
RB-O
Water II
AW 20 g
Kt l2PO4 Ig
K2HP04
MgS04 7H20 0.5 g
Pcptone 5g
Dextr ose log
Yeast extract
Rose benpl 0.033 g
Streptomycin 0.03 g
Add streptomycin sulphate after autoclaving.
Used for the isohtion of fungi from soil and ~ i r W
. Yll and RB-hi?. are preferred to RB-0 for the
isolation of Thiebsiopsis busicola; KBhl:! is chosen by Tsao, P. H. (1964. Phytopathology
54:549-555). Do not expose to strong light as media containing rose benpl becomes very fungistatic
(Pady, et al. 1960.31ycologia 52:347-3SU).
140. hfartin Jledium (modified)

Modified by Papavizas and Davey (1959. Soil Sci. 88:11!-117) by addition of 5 g oxgall and 1 g
sodium propinate, to increase the isobtion of fun$ from soil.
141. Martin bldium (modified)
Modif~d by Singh, R. S. and J , E. hiitchell (1961. Phytopathology 51:440441). To isolate
PytiJum, add .01 or .05% Terraclor (penrachlaronitrobenzene) or pimaricin ,001 or .002X.The
latter is short-lived in media.
142. Mchrlich Agar (see Malt A p )

Dextrose Formula of C. M. Christensen.
Malt extract
Pcptone
MgS04 7H20
KH2P04
NaCl
Agar
Water
Roperson, C. T. 1958. Trans. Kansas Acad. Sci. 61: 155.162.
143. Minoelement Solution

723.5 mg Dissolve in 600 ml of distilled water,
439.8 mg add sufficient sulfuric acid to give a
203.0mg clear solution and bring up to volume.
Water 11 Two ml per 1 of medium is suitable
for most fungi, gives 0.2 mg of Fe
and Zn and 0.1 mg of Mn,
Lilly, V.G.and H. L. Barnett. 1951. Physiology of the fungi. hlcGnw-llill, N , Y.
144. Milk Agdr

Powdcrcd milk Used to isolate nematode trapping
br fungi by sprinkling 1 g or less of
Water soil on its surfice.
Tornurff, H'. J. 1959. Phylopathology 49: 11 3 (Abstr,)
145. Motility Medium

Gelatin Soak the gelatin in water for 30 min,
Peptone add the other ingredients, heat to
Beef extract dissolve. Put a piece of 3" glass
NaCl tubing with bottom end obliquely cut
Agdr in a 6 x !A" test tube, Add about 8 ml
Distilled water of medium and autoclave at 1 15 C
for 20 min, inoculate agar in upright
glass tubing. A motile bacterium will
grow down the tube up to the surface
in the test. Recommend using
~ b o p t i m i u mtemperatures.
Edwards,P. R, m d D. W. B ~ n e r 1942.
. Circ. Ky. Agr. Exp. Sta. No. 54.
Cowm. S. T. and K. J. Steel. 1965. Manual for the identification of mcdical bacteria. Cambridge Univ. Press,
Landon.
Dowron, 8'.J. 1957. Plant diunrs due t4 baderh. Cambridge Univ, Press. London.
48 Media a d Nutrient Solutions
146. Mycological Agar

Bacto soy tone 10.0 g Recommended by Difco for the
Dextrose 10.0 g cultivation and maintenance of
Agar 15.Og fungi,
Difco Laboratories. 19G2. Difco suppkrncnt;lry literature. Detroit.
147. hfycophil Agar

Phytone 10.0 g
Dextrose 10.0 g
Agar 16.0 g
Often used in cultivation of animal fungal pathogens, assay for antifungal activity; and if acidified, it
is claimed suitable for determinins yeast and mold counts in beverages, vitamins and other
preparations. Samo medium as hiycological agar.
Baltimore Biological Laboratory. 1959. BBL Products Catalog. Baltimore.
148. Xlycoxl Agar (BBL)

Phytone 10 s If using BBL product autoclave at
Gluwsc log 1 18 C for I5 min. Cool and use at
Actidione (L'pjohn) 0.4 g once or remelt only once. Used to
Chloromycetin (Park & Ilavis) 0.05 g isolate Coccodioides immitis and
Agar 15.5 g some other animal pathogens.
Distilled water to nuke a l
Geory. Lucille K., L. Agello and hi. A . Cordon. Science 115:387-389.
149. Nash & Snyder PCNB Medium

Difco peptone 1s g For the isolation of Fusarium from
K112P04 1.Og field soils. If used fresh, need not
MgS04 7 H 2 0 0.5 g be autoclaved. Also, exposure to
Streptomycin 300 mg'ppm diffuse daylight for the first few days
Agar mg necessary for profuse sporuhtion of
Pentachloronitro benzene Ig the fusria and inhibition of phycomycetel
(75% wettable powder, Terraclor) obtained from Olin Mathieson Chem. Carp., Baltimore 3,
Maryland.
N u h , Shirky M.and \V. C. Snyder. 1962. Phgtopathology 52567-572.
150. Neopeptone Rose Bengal Aureornycin Agar

Neopeptone (Difco) or Polypeptone (BBL) 5.0 g Aweomycin should be filter-sterilized
Dextrose 10.0 g and added before pouring. If pur-
Rose bengal 0.035 g chased in capsule, simply add
Aureomycin HCI 35.0 lrglml aseptically to sterile water. Add 5
AW 20.0 g mljl or 0 5 m1/100 of medium.
Recommended for the isolation of
fungi from sewage and swage
polluted water.
Coolu, Wm. B. 1963. A laboratory guide to fun@ in polluted wrters. rwagc and ! cwage Veatmcnt systems. Public
&&h Sa.Pub. NO. 999-WP-I.
151. Neopeptone Glucose Medium

Neopeptone 2.0 g For spdrulation of Culletotrichum
Glucoso 2.8 g lindemutltbnum.
MgS04 * 7H20 1.23 g
KHiP04 2.72 g
Agar 20.0 g
Water 11
Mathur, R. S., H, L. Barnet1 and V. C. Lily. 1960. Phytopathology 40:104-114.
152. Neurospora "Complete" Medium

Glucose 5.0 g Vitamin sol
Sucrose 5.0 g Thiamin
Hydrolized casein 5 .O ml Riboflavin
Difco yeast extract 2.5 g Pyridoxin
Spraydried malt syrup 5.0 g Pantothenic acid
Vitamin sol 10 ml Paminobenzoic acid
Agar 15g Nicotinarnide
Choline
Inositol
Alkali hydrolized
yeast nucleic acid 500
Folic acid 4 Cc pure
substance
Beadle,G. W. and E. L.Tatum. 1945. Amcr. J. Bot. 32:678686.
153. Ncurospora "Minimal" Medium

Ammonium tartrate Extensive aerial growth of
NH4N03 Neurospora can be prevented by
KHaP04 changing concentration of sucrose
MgS04 7 H a 0 to 1 g and adding 0,8g of Isarbose.
NaCl E. L. Tatum, R, W. Barratt and V.
CaC12 M.Cutter, Jr. (1943. Science
Sucrose 109:509-5 17).
Biotin
Bo
Cu
Fc
Mn
Mo
Zn
Badk,G. W.and E. L.Tatum. 1945. Amer. J. Bot. 32:67&686.
50 Mcdh a d Sutricnt Solutions
154. Neurospora Sex Synthetic

KN03 Favors sexual reproduction of
KH2P04 N e m s p r a , For use with aosses
MgS04 between mutants may be supplemented
CaCll with 0.25% yeast extract, 052 malt
NaCl syrup, 05% acid hydrolyzed casein
Biotin and 0.1%vitamin mixtures.
Trace elements Trace elements -, after (Eksdlc. G.
Sucrose W.ondE.L.Tatum.Amer.J.Eot.
Agar 32:678-686) in ms'l - Bo 0.01, Cu
Adjust to ptI 6.5 before autoclaving. O.l,Fe0.2,MnOB2,Zn2.0.
Westerpard, hl. and ti. K. 5Iitchell. 1947. Anrer. J. Bot. 34i573.577.
155. Nutramigen Agar

Nutramigen (baby food produced by
Mead Johnson & Co., Evansville, Indiana) 16 g For conidial production of
Asar 17g Ceratoc)rris f a g ~ c w u m for
,
Distilled water 11 perithecia, see media no. 19 and 156.
Fcrguz, C. 1954. htycolopi~46:43544 1.
Nutrient Agar - See Beef Pcptone.
156. Oak Wilt Agdr

Maltose 5.0 g Adjust the pH to about 6 before
Difco Casamino acids (tech, grade) 1.O g autoclaving 15 Ib 15 min. For the
KtilP04 lag production of perirhtcia
MgS04 7H20 05% Ceratocystis fagacearum
Zinc sulfate
Ferrous sulfate
Manganese sulfate
Biotin (0.5 g yeast extract may be
substituted for some isolates)
0.2 mg
I
0.2 mg For amount of cation see no. 119
0.1 mg
Agar
Distilled water
157. Oak Wilt Identification Agar

Glucose 3.0 g pH should be near 6. For the
Phenylalanine 05 8 isolation, sporulation and identifica-
1fig tion of Ceratocptis fagacemum
0s g Takes 3-5 days at temperatures
Ferrous (sulfate) 0.2 mgpmount between 20-25 C. See Campbell, R.
Manganese (sulfate:) 0.2 mg of and D. French. 1953. Plant Dis.
Zinc (sulfate) 0.2 mg, cation Reptr. 37:407. for another isolation
BDtin s Pg medium.
Agar 200
Distilkd water 11
Media and Nuvknt Solutions 51
158. Oat Grain hledium (Tuckn)

Soak non-hulled grains in distilled water 2-3 hr, pour off free water and autoclave 30 min 15 Ib.
Recommended for sexual reproduction of Phyropl~thorucinnamomi Inoculate with corn meal agar
discs, top flasks with aluminum foil and keep at 20 C for 2-3months.
Roylc, D. J. and C. J. Hickman. 1964. Can. J. Bot. 42:311-318.
159. Oatmeal Apr

Rolled oats Blend oats in 600 ml in IVaring
Agar Blendor for 5 min, add 400 ml of
Water melted agar. Put in prescription
bottles with caps and tightened. Do
not attempt to cool these bottles
rapidly as they will crack. Auto-
claving of 300 ml amounts for 90
rnin recommended by Goody and
Lucas seems unnecessary, 30 min
for 200 rnl amounts is adequate.
Modified fiom Coodhg,G. V. and G. B. Lucas. 1959. Phytopathology 49:277-281.
160. Oatmeal Agar (Shirling and Gottlieb no. 3)

Oatmeal 20.0 g Steam oatmeal in a 1 of water for
18.0 g 20 rnin,, filter through cheesecloth,
Distilled w t e r to make a 1 add trace salts, and bring to volume.
FeS04 7H20 0.1 g Adjust to pH 7.2 with NaOH. Add
MnCI2 4H20 0.1 g 1 ml agar and steam for 15-20 min. For
&SO4 7H20 0.1 g culture of Streptomyces.
Kuster, E. 1959. lnt. Bull. B a d . Somen. and Taxon. 9:98-104.
Shkling, E. B.and D. Gottlieb. 1966. Int. J. Syst. Bacterial. 16:313-340.
161. Ohio Medium (OAES)

Gbse 5g Used to isolate fungi from soil.
Yeast extract 28
NsNOl Ig
hlgS04 7H20 0.5 g
KH,pO4 1 kl
Streptomycin sulfate 50 mg
Chlorornyce tin 50 mg
oxga11 Ig
Sodium propinat e 10
Agu a g
Distilled water 11
Autoclave 1 1 Ib 15 rnin.
Wihm, L, E. and A.C. Schmitthenner. 1956.Ohio AH. Exp. Stn. Clr. 39.
167. PDA
Peeled, sliced potatoes A widely used and valuable mediun
Agar
Can be acidified with 25% lactic
Dextrose -
acid 3-5 drops1 100rnl melted aga
(Cerelose a crude form of when used for isolation of fungi
dextrose is sometimes used) from plant parts. Do not remelt
after acidifying. Agar will be too
soft.
168. PDA (another version)

Dehydrated potatoes Add dehydrated potatoes to 200 ml
Dextrose hot water. Add it and dextrose to
Agar melted agar.
Water
Lacy, M. L. and G. H. Bridgman. 1962. Phytopathology 52: 173.
169. PDASV
PDA Useful isolation medium which is
Streptomycin sulfate 100 ~ g l m l less inhibitory to fungi than hiartin
Vancomycin 50 ~(gtml media.
Latham, A.J. and hl, tl. Linlr. 1951. Plant [)is. Rcptr. 45:866-867.
170. PDTA
Potato dextrose agar I have found this medium to be
(with 5 g of dextrosell) valuable in the isolation of nonstora
Tergito1 NPX (Union Cwbide) 200 ppm fungi from seed. It does not, hower
Aureomycin 30 PPm effectively inhiiit all species of
Rhizopus. The addition of Botran,
about 8 ppm, seems to do the latter
Add Tergitol and Aureomycin just
prior to pouring.
171. PDTAS (PDA-NPX)

Difco Potato dextrose agar Found superior to OAES medium
Tergitol NPX (Union Carbide)
Streptomycin sulfate
Aureomycin
100 ppm
35 ppm
35 pprn
Steiner, G.W. and R. D. Watson. 1965. Phytopathology 55:728.730.
Hornby, D. and A. J. UUstrup. 1967. Phytopathology 57:76-82.
I for isolation of fungi from soil.
Added after
autoclaving
172. Pea Broth

Contents of one can (1 Ib 4 oz) of peas, wash thorougtily and add enough distilled water t o yield 4 1
of the liquid, bring to boil and allow to settie, siphon off and filer through absorbent cotton.
tconion,L. H. 1934. West Virginia Agr. Exp, Sta. Bull. 262.
173. Pea Broth

Dried whole or split peas 150 g Boil peas for 30 min in 1 1water,
Distilled water 11 strain through cheesecloth. Used for
the production of sporangia of
Phytopl~thomilicis.
Buddcnhagen, I. W. and R, A . Young. 1957. Phytopathology 47:95-101.
174. Pea Seed - For the production of zoospores of P ~ ~ ~ ~ r o p l t t linfesta~ls

iora
(1) Soak dried yellow peas overniglit
(2) Place I" layer of soaked pea in 250 ml flasks
(3) Add enough water to cover the pcas
(4) Incubate at 20 C for 7 to 10 days.
See also Chick pea and Rye seed medium.
Thurston, H. D. 1957. Phytopathology 47: 186.
175. Peat Percolate

Leach roughly estimated volume of peat with an equal volume of distilled water, dilute percolate
twenty times with distilled water. Use to induce sporangia of Phytophthora eryrluoseptica var. pisi
after having grown 2-3days at 18 C on bean agar.
Bywatcr, Jom,and C. J. Hickman. 1959, Brit. Llyc. Soc., Trans.42513-524.
176. Pectate Gel

Calcium agar base
Peptone (Evans) Sterilize separately at I21 C. Pour
Lenco in plates I cm deep and cover with
Calclum lactate a thin layer of pectin solution. The
Agar (Davis) gel sets fumly within 12-24 hr at
27 C.
Pectin solution
Pectate powder Make a paste of powder and alcohol,
(Sodium polypectate) bring to 100 ml and add EDTA and
Absolute alcohol sterilize at 121 C.
Disodiurn salt of EDTA
Distilled water t o make
Paton, A. 1959. Nature 183:1812-1813.
177. Pectin Medium

Pcctin 1% W/V (glucose free if assiying pectin
NaNOl 0.24 attacking enzymes) In hot autoclave
KC1 0.05% for 2 rnin at 10 Ib to prevent brcak-
K3HPO 0.10% down of pectin. Adjust pH to 6
MgS04 7H20 0.05% prior to sterilization.
FeS04 0.001%
&= 2%
YcDonnell. K. 1962. Brit. Mycot Soc.,Trsnr. 45:5562.
Media md Nutrient Solutions
178. Pectin-Polypectate hledium

NH4M03 1.0g Medium used for the production of
MgS04 7H20 2.5 g pcctinolytic and cellulolytic enzymes.
K)12P04 25 g
Citrus pectin Dissolve separately, add to mineral
Sodium polypectate
Double distilled water to make 1 I
"O g
5.0 ] salt solution, adjust pH to 5.0 and
autoclave.
Spolciing, D. H. 1963. Phytopathology 53:929-931.
179. Peptone Water

Peptone 10 g Dissplve by heating. Adjust the pH
NaCl 5 to 7.2 and filter if necessary.
Tap water 11
Skcrmm, V. B. D. 1959. A guide to the identification of the genera of bacteria. B'ilkins & Wilkins, Baltimore.
180. Peptone Yesst Extract Iron A p r (Shirling and Cottlieb no. 6)

Bacto-peptone Iron agar (Difco) 36 g pH should be 7.0-7.2before
Bacto-yeast extract (Difco) 1.Og autoclaving. Used in the study of
Distilled water 11 Sltepto myces.
Trcma, H.I).and F. Danp. 1958. J. Bacterial. 76:239-244.
ShirPnp. E. B. and D.Gotttieb. 1966. Int. J . Syst. Bartcriol. 16:313-340.
181. Petri hledium

Calcium nitrate 0.4 g For production of sporangia of
Magnesium sulphate 0.15 g Phytophtlrora. Place small pieces of
Potassium acid phosphate 0.15 g agar, host tissue or seed containing
Pot assiurn chloride 0.05 g mycelium into slution partly or
Water II completely immersed. In 3 4 days
at 12-20 C replace with distilled
water.
Waterhouse, Grace. 1954. Myc. Pap. 57. CS11. - Holliday, P. and W. P. Mowat (1963, ChlI
Phytopath. Pap. 5) recommend not sterilizing to produce sporangia of Phytopkthora palmivora.
Tucker,&.bf. 1931. Missouri Unh. Res. Pull. 153.
182. Pfeffu Medium

C@03)l 4H20 333 g ZnCl
MgS04 7ti20 B83 g H J ~ O ~
KK03 .083 g CuCIp 2H20
KHz PO4 083 g MnCI1 4H20
KC1 AM2 g NatHP04 2Ha0
FeCI, 6H20 Dextrose
(24 mg/100 ml - 10 ml) .24 mg Distilled H 2 0 to a 1
183. Phytone Dextrose Agar

Phytone (BBL) Good sprulation medium for
Dextrose Stemphylium boticki.
Yeast extract
ABar
Sobers, E. K. and C. P. Seymour. 1963. Phytopathology 53:1443-1446.
184. Plant Parts

B a n pods, carrot and potato slices, soybean stems, sweet clover stems are some of a number of
materials used to induce sporulation. Can be autoclaved separately in large test tubes cclntaining
cotton with 1-2 ml tJ20in the bottom (A. J. Riker and Regina S. U e r . 1936. Introduction to
research on plant diseases) or placed on water agar. An alternative method as suggested by H. N.
Hansen and W. C. Snyder (1947,Phytopathology 37;420421)is to sterilize with propylcne oxide
and incorporate s m d pieces of material into melted water agar.
185. Pontecorvo Minimal Slediurn (1953)

6.0g If abundant perithecia of ~spergillus
052 g niduhnr desired, reduced NaNOj to
052 g 1 D g and inaease glucose to 20D g.
101) mg
3s g
1Dg
101)g
Adjust pti to 6.5 with NaOH before sterilization of I0 Ib for I0 min.
Pontecorvo, G, 1953. Adv. Genet. 5: 141.238,
186. Porter Pimarcirn Medium

Glycerol Bg Useful for isolation of Actinomycetes
Larginine 23 g from mils and freeing them from
NaCl 1Bg fungal contaminants.
CaCOj 0.1 g
FeS04 7H20 0.1 g
Dipotassium hydrogen phosphate 1L)g (omitted from publication)
MgS04 7H20 0.1 g
Distilled water 11
Pimaticin 50 ~lgld
Pimaricin stock solution 1 rng per 1 ml
of N, N-dimethylformanide added before
autoclaving. hlediums' antibiotic
ability reduced after 3 days.
Pottcr, 1, N, J. 1, W h i m a d H, D.Trcmer. 1960. Appl. bfimbiol. 8: 174.1 78.
Media and Nulricnt Solutions
187. Potato A p r
Potato 20 8 Repare like Potato Carrot Agar,
Agar 20 g
Water 11
bnpron, M. 1945. Pdcis de mycologie. Chronica Botanica, Wdtham, hlass.
188. Potato Carrot Agar

Potato a g Steep sliced tissue in water for I hr
Carrot 20 g then boil for 5 min, Filter through
Agar 20 g cotton wool. Recommended for
Water 11 maintaining fungus stock cultures.
Lnngcron, M. 1945. P~'cisdc mycologie. Chronica Botanica, Waltham, $lass.
189. Potato Malt Agar

Potatoes 60 8 After the agar is sterilized and p u r e
Fleischman dry malt syrup 10 I3 place strips or squares of sterile wllitt
Difco bacto-agar 15 fi blotting paper on surface. Strips can
Distilled water to 1 I be added to media in test tube before
autoclaving. For the production of
perithecia of Chaetomium.
Amer, L. M. 1961. A monograph of Chaetorniaceae. U. S. Army Res. and Dev. Ser.
190. Potato Marmite Agar (PMA)

Dextrose 20.0 g Modifiation of N , F. Flentje P~IA
Potatoes 250.0 g (1 956. Brit. Myc.Soc., Trans.
Marmite yeast extract lag 39:343-356).Steep potatoes for 1
(Marmite Ltd. Lond) hr at 6 0 C. Fruiting of Rhizoctottia
Agar 20.0 g obtained by transferring 2-3 day old
PMA blocks to the edge of 2% wa'ter I
agar plates. Keep at room temperaturt
in diffuse light.
Whitnay, H. S. and J. R . Parmeter, Jr. 1963.Can. J. Bot. 41:879-886.
191. Pridham Yeast Malt Dextrose Medium

Difco yeast extract 4.0 g Useful in identification of
Malt extract 10.0 g Streptomyces as most isolates
Dextrose 4.0 0 produce abundant aerial mycelium#
Agar 12.0 g Adjust pH with NaOH to 7 3 , pH
after sterilization 6.8-7.0.
Pridham, T. G., P. Anderson. C. Foley, 1.A. Lindenfelser, C. W. Herscltine and R. G. Bcnedict. 1957 Antibiotics
Ann. 1956-1957:947-953.
58 Media and Nutrient Solut~ons
192. Pridham and Cottlieb Trace Uts

C S O 4 5H20 0.64 g Store at I S C but bring to room
FeS04 7H20 0,11 g temperature before using. Prepare
M d l z 4H20 0.79 g fresh solution each month. Disregard
ZnSO, 7H20 0.1Sg precip~tate.
Distilled w t e r 100 ml
Shirling, E. B. and D.Gottticb. 1966. Int. J . Syrt. Bacterial. 16,313-340.
Prunes
Agar
Water
Riker, A. J. and Regina S. Riker. 1936. Introduction to research on phnt diseases. John S. Swlft lnc. St. Louis.
194. Pseudomom 51orspnrnorum Isolation Medium

Sodium tartrate 10,O g Fungi and mny bacteria fail to
Di-octyl sodium sulphosuccinate 0.1 g grow o n this medium.
(anionic surface active agent)
NaN03 2.0 g
K 2 HP04 1.0 g
MgS04 * 7H20 0.5 g
KC1 05 g
FeS04 5H20 trace
Class distilled water II
K2HP04,sodium tartrate and the surfactant are dissolved separately in 150, 100 and 250 ml of
water, respectively, and added wlthout sterilization to the cooled sterilized solution of the mineral
salts, A few crystals of iron salt is added and the pH adjusted with IiCl to about 5.5. The broth is
distributed t o previously autoctved tubes and sterilized by bringlng the steam pressure slowly up to
7 Ib and then turning off.
Cmsse, J. R. md lfargery Bennett. 1955. Brit. Mycol. Soc..Trans. 38.8347.
195. Pyocyanh Medium

Bacto p e p t o n 20 g Colors appearing on medium varies
Glycerol C. P. 10 8 with strains of Psedomonas: light
K2SO4 (anhydrous) 10 g pink to dark maroon (latter with
MgClz (anhjddrous) 14 g strains producing Pyorubrin).
Agar 1s 8
pH 7.2
King, ElzabethO., Martha K. Ward and D. E. Rancy. 1954. J. Lab. Clin. hled. 44:301-313.
196. Radish Agar

Radish roots Steep root tissue in water at 60 C
Agar for 1 hr. Add melted agar, make up
Water to make a 1 to a 1. Used to maintain Aptlanomyfl
mphoni and for oospore production,
G h f o o r , A . 1964. Phytopathology 54: 1167-1 171.
197. Rape Seed Agar

Rape seed Wash seed thoroughly in water and
Agar boil in 1 I of distilled water for 30
Distilled water min. Filter extract tluough cotton,
adjusted to its original volume.
Adjust pH t o 7.0. A clear agar
suitable for production of oospores
of Phytophtlura.
Satour, M. M. 1917. hlycoloda 59: 161-166.
198. Rautelb and Cowling Cellulose Medium

Cellulose Particles (dried, T o measure cellulolytic ability.
preparation described) Cellulose prcpared according to
Ntj4t12P04 Walseth: Put wllatman powdered
Kfi2P04 cellulose in 85% o-phosphoric acid
K2HP04 for 2 hr at 4C, wash in the cold by
MgS04 * 7 i j 2 0 repeated suspending and decanting
Thiamine IICI with distilled water and 1% (wlv)
Yeast extract Na2C03 until neutral, Sterilize in
Adenine 3 to 6 cm high amounts in 18 mm
Adenosine test tube. Inoculate with 1 2 m m
agar discs of organism and measure
Distilled water to make a I depth of clearing every 7 days for
35 days grown at 26 C.
RrutcUa,G. S. and E.B. Cowling. 1966. Appl. hlicrobiol. 14:892.898.
Wllaeth,C, S. 1952. Tappi 35:22B-233.
199. Reischer Agar ( m o d i f ~ d )

Sucrose 0.41 g All ingredients added before auto-
Lasparaginc 0.12 g claving. Medium used with soil
KH2 P o 4 3 0 ~ inverted agar technique and screened
MgS04 71f20 20 mg particles to isolate Pythium from
CaCll 056 mg mil.
MnC12 2.88 mg
ZnClz 1.67 mg
FeC12 0.10 mg
(continued on next page)

199. Rcisckr Agar (modifd) (continued)

Disodiurn salt of ethylenediamine
tetraacetic acid (EDTA) IIhOmg
Thiamine hydrochoride 0.04 mg
Endomyc in 5 q
Streptomycin sulphate 50 W
Agar (Difm) a g
Schrnitthenncr, A. F. 1962, Phytopathology 52: 1133-1 138.
200. Reynolds Medium

K2 HPO4 0.7g Used as an enrichment medium to
Kii2P04 0.3 g isolate chitindecomposing microbes.
MgS04 5H20 05
FeS0, 7H20 0,Olg
znso, 0.001 g
Chitin (powdered) 0.25% w/v
Distilled water 11
Reynolds, D. 51. 1954, J. Cen. Microbial. 11:150.159.
201. Rice Polish Agar

Rice polish 2og
Agar 17 8
Water 11
Mix the rice palish with half the water in a flask large enough to allow steeping in the autoclave.
Steam in autoclave for 15 min (without pressure). Blend the steep in a waring blendor for several
min. hlix steep and melted agar thoroughly. After autosking swirl the flask during cooling to
prevent the rice polish from settling. Used for sporulation of PuiarW or~~rue. Fazli and Schroeder
(1966. Phytopathology 56:507-509) used 30 g of Rice polish and 23 g of agar for sporulation of
Helminf hosprium oryzae.
Ubn&Frances hf. 1966. Personal communication.
202. Richard Solution

Potassium nitrate 10 P
Potassium monobasic phosphate 5g
Magnesium sulphate 25 g
Ferric chloride 0.02 g
sucrose 50 g
Distilled water II
Fahmy, T. 1923. Phytopathology 13543-550.
Medb and Nutrient Solutions 61
203. Ring Rot Medium

Difco nutrient agar 23 8 Supported goad growth of
Tryptont 5.0 g Corynebacterium sepedonicum.
Yeast extract 5.0 g Addition of sodium dichromate
Glucose 5.0 g 1:20,000 or polymyxin at
l:S,000,000 increased its isolation
SbU and Strong Solution A
Potassium hydrogen pl~osphate value.
Potassium dihydrogen phosphate
Water 250 rnl
Slution B
MgS04 7 H z 0
Sodium chloride
Ferrous sulphte heptahydrate
Manganese sulphate tetrahydrate
Water
K a t u w h n . 11. and hl. I). Sutton. 1956. Can.J. Bot. 34:48-53.
20J. Russell Raidiomycete Medium

Oxoid desiccated malt extract 308 Used for selective isohtion of
Oxoid Myoological peptone 5.0 g Basidiomycetes. Autoclaved at 10 lb
0-phenyl phenol 0.06g for 10 min. Dissolved 1 g of 0-phen]
A%ar 25 g phenol in 50 ml industrial alcohol,
Water 11 water added to bring to 100 ml.
Russell, P. 1956. Nature 177: 1038-1039. Also see Fornes amoms isolation medium.
205. Rye Seed Medium

Rye seed Steam rye seed in water for 30 min,
Dextrose filter through cheesecloth and ?4"
Water filter paper pulp, add dextrose and
dilute extract with an equal volume
of water. Fill to %" level in flasks
and autoclave for 10 min at 240 F.
Sporulation of Phytoplltlwra
iq/i?stans is usually obtained after
1 weekat 20C.
Hodgmn, W. A. and P. 21'. Gminger. 1963. Can. J. P b t Sci. 4 4 5 8 3 .
206. Saboumud Dextrov Agar

Neopeptane, Difco Generally used for isolation of fungi
Dextrose from animals. For other variations
@r consult Difco and BBL manuals.
Calcium nitrate 1.Og Extensively used with corn kernels

Dipotasium hydrogen phosphate 0.15 g or leaves to induce perithecia of
LlgS04 0.25 g Helrninthosporium.
Fmic chloride traco
QCOJ 4.0 g
Ag4r 20.0 g
Water 11
208. Slnd Soil - Tomato Root lledium

Sand (quartz) 2mm sieved 1 part by wt
w 9,
Garden loan 2mm sieved
Tomato roots (from mature plmts
17 f*
and finely ground)
Autoclave in 270 g anlounts for 45 min 15 Ib, after 2 days at 25 C moisten to 80% of its water
holding capacity with sterile distilled water and autoclave for 45 min at 1 5 lb. Used for the
production of sclerotia of Colletorrichum coccodes.
Hornby, D. 1964. The survival o f Colletonichum coccodcs in grcenhouv soils and related problems. P h S . Thesh
Unir. of Notthgham.
209. Schitophyllum Fruiting Medium

Glucose mg For fruiting of Schitopltyllum
Lasparaghe 2.0 g commune. Peptone may substitute
Thiamine hydrochloride 1 0 ~g for L-asparagine (J, B. Raper and
0.46 g Gladys S,Krongelb. 1958.
K2 I
m 4 1.0 g Mycologia 50;707-740.)
7H20 0.5 g
Noble agar 20 #
Distilled water 11
210. Sea Water Agar

KBr
NoF
SICla 6 H a 0
Minonutrients
Distilled water
4Pr
For otter media for culturing marine fungi see
tcference below. In addition, Jones, G,E,(1964.
1. Bacteriol. 87:483499) discusses value of
cheloting agents in sea water solutions.
(coptinued on next page)
210. Sea Water Agar (continued)

Microelements
Ethylene dhmine tetracetic acid 05 mglml "Concentrations refer to the final
2.5 mg/rnl ion."
1D mglml
0 5 mglml
0.1 mglml
0.05 mglml
0.02 mglml
T. W. Johnson, Jr. and F. K . Sparrow, Ir. 1961. Fungi in oceans and estuaries. Hafner,S. Y.
21 1. Sea Water Glucose Medium

Glucose 1O. g Filter freshly collected sea water
Bacto yeast extract 0.1 g through medium porosity sintered
Aged sea water 11 glass (or through cotton pads). Then
Agar 18g store in the dark in a cotton stopp~l
container for 3-6 wks.One gram 01
Phytone may substitute for yeast
extract. Useful in isolating saprobic
marine fungi.
T. W,Johnson, Jr. and I:, K, Sparrow, Jr. 1961. Fungi in oceans and estuaries, Hafner, S . Y.
212. Sea Water Glucose Peptone Medium

Bacto-peptone 0.1 g Useful in the isalation of lignialoui~
Glucose 1.0g fungi irnperfecti and some
Dibasic potassium pl~osphate 0.05 g caulicolous ascomycetes.
Ferric citrate 0.01 g
Agar 18.0 g
Aged sea water 1I
T. W. Johnson, J t . and F. K . Sparrow. 1961. Fungi m oceans and estuaries. tiafner, H. Y.
213. Shive and Robbins Nutrient Solution

Formula I Formula 11
i
KH2po4 5.9 g Dissolve separately MzP04 3.9 g
Ca(N03) 4H20 20.1 g in about 230 ml of NaN03 6.4 g
MgS04 72i20 10.7 g water. hlix together MgS04 7H20 1 0 3g
(N114)1S04 1.8 g and bring to 5 gal. CaC12 (anhydrous) 3.2 g
Trace elements
H3BQ3
MnS04 4H20
ZnSO, 7H20
::::]
0.8 g
Dissolve together
in 230 ml of water.
Add 10 ml of trace element solution
to 5 gal of either Formula I or 11,
Then add 5 ml of iron solution
FeS04 7H20 0.8 g ] Dissolve in 236 ml before adding to plants.
of water.
Shive, J. W. and W. R. Robbinr. 1948. Ncw Jnwy Agr. Exp. Str. Bull. 636.
214. Silica Gel Medium

-
Phase I
Soybean oil meal (powdered) Ludox 500 ml (commercial source
20 g
Tryptic digest of casein (BBL) 108 of colloidal silica gel obtained from
Glucose 10 g Dupont).
Sodium chloride 10 f i
DistiCed water 500 ml (1sol;ition of Thermophillic
Actionomycetes)
Nitric acid Used for pli adjustment,
NaOH 1% solution
1. All materials sterilized separately by autoclaving.
2, Cool t o room temperature and adjust phase 1 and 2 to pH 7.
3. Mix 2 phases and pour 4U ml per petri dish.
4. Put plates immediately in a 60 C force draft oven for 10-24 tu.
Uridil, J. E. and Pa A, Tetrault. 1959. J. Bacteriol. 78:243-246.
21 5. Silica Gel Medium

aphosphoric acid Mix KOH with either silica gel or
(20% aqueous cert. ACS Fisher) 4 ml silicic acid and heat to form
Silica gel potassium silicate. Sterilize separate
(powdered grade 923, 100-200 mesh solutions of potassium silicate,
Will Scientific, Baltimore) 10 S double strength growth medium and
or phosphoric acid, Mix equal amounts
Silicic Acid of potassium silicate solution and
(Reag. grade, J . T. Baker) 10 growth medium (20 ml), Add 4 ml
KOH (7% solution w/v) 100 ml of phosphoric acid. hlix and pour
immediately. Gekt ion begins in
about I min.
Funk, Hekn 8. and Terry A , Krulwich. 1964.1. Bacteriol. 88:120G1201.
216. Snake Skin

Small pieces of boiled snake skin are added to sterile soil water, considered to be best for isolation o f
Aphunomyces by Scott, W. W, 1961. Virginia Agr. Exp. Sta. Tech. Bull. 151.
217. Soft Rot Isolation Medium

Simmons citrate agar (oxoid) 23.0 g
Calcium chloride 3.0 g
Bile salts (oxoid L 55) 5.0 g
Crystal violet 0.001 g
Distilled water 11
Poctate oolution 4 ml
(As prepared by Paton, A. M. 1959.
Nature 183:1812.) See pectate gel.
bgan, C. 1963. Nature 199:623.
218. Soil Extract

Ax-dry soil Boil soil in water for 1 hr, remove
Water solids by vacuum filtration and we
resulting solution undiluted, Auto.
ckve in 20 to 30 ml amounts 15 Ib,
15 rnin.Used t o induce sprangial
formation of Phytophtlwra
pmsiticu var, niwtbnae after 3-5
days culture in potato dextrose
broth. Mehrlich, F. P. 1935. (Phytop
ology 25:432435.) found steaming
soil eliminated stirnulatory effect for
P,cinnamani.
Wills, W, R, 1954. J. Elisha Mitchell Soc. 70:235-243.
219. Soil Extract Agar (Flentje)

Soil, airdried Steam soil-wter mixture for 30 rnm
Distilled water and leave standing overnight, Pour
Dextrose through filter paper pulp, add rest of
Yeast extract chemicals and melted agar, make up
KH2m4 to 1 1 and adjust to pH 7.0. For the
ABar formation of the perfect stage of
Rhiwctonia, grow previously on
potato marmite agar.
Fkntje, N. T,1956. Brit. hlyc. Soc., Trans. 39:343-356.
220. Soil Extract Agar

Glucose
Kz HPO4
Soil extract
Tap water
Soil extract is prepared by heating 1 kg of garden soil with 1 1 of tap water in the autoclave for 30
min. A smaU amount of calcium carbonate is added and the soil suspension is filtered through a
double paper filter. The turbid filtrate should be poured back onto the filter until the extract comes
through clear.
AUen, 0. N. 195 1. Experiments in soil bactcriolqy. Burgess, Minneapolis.
221. Soil Extract Agar (Amther version)

One Kilogram of soil plus t I autoclaved at I S Ib for 30 min. The supernatant is filtered off and
water addcd to make a 1, then I5 g of agar is added.
Yilkr, J. J., D. 1, Peers, and R, W. Neal. 1951. Can. J. Bot. 29:26-3 1.
222. Soil Extract Isolation Agar
Soil extract (I kg garden soil in I I tap 25 ml Used to isolate VcrtiiilCiumdoi~lke
water, steam for 30 rnin then decant and filter) from soil. Green, R. J. and G.C.
KHapo~ 1.5 g Papavizas (1 968, Phytopathology
K2HPO4 4.0 g 58:567-570) suggests add~ng2 gll
&a$ 15 g plygalacturonic acid. >lake up
Strepwmycin
Chlortetracy cline
Chlorarnphenicol
50 rng
50 mg
SO rng
Menzks, J. D,and C. E. Griekl 1967. Phytopathology 57:703709.
I Add separately and adjust to pH 7 before
after adding.
autoclaving.
223. Soybean Meal Agar

Ground soybeans 158 Used to obtain oospores of
Agar 20 g Phyropirttwra itfesrans, grow in
Distilled water to make a l the dark at 20 C,
Smoot, J. H.,F. J. Goqh, H. A. Lamey,J. J. Eichenmuller. 1958. Phytopatholcgy 48:165-111.
224, Starch Agar

Starch 2%
Added to a satisfactory basal medium for the organism. If starch has been attacked, a clear unstained
zone will be formed around the organism after treatment with Lugol's iodine (1 g iodine, 2.0 g KI,
300 ml distilled water),
Ikwson, W. 3.1947, Plant lliwases due to Bacteria, Cambridge L'dv.Press, Landon.
225. Stan Pectate Medium

Sodium ammonium pectate 38 Dirsofve pectate in water (add slowly)
Distilled water containing %OH, containing 0.9 rnl S SaOH, 0.6 ml
CaClo and bromthymol blue 100 rnl CaCll 2H20,and 1.25 ml brom-
Ytast extract (may be added) 1% thymol blue (0.2%)).After complete
solution in hailing water bath, pH
should be about 7.3 (blw green).
Tube and autoclave, pH should be
about 6.4 (yellowish peen). Mediun;
annot be reheated.
Stur,M. P. 1947. Phybpathology 31:291-300.
Edwards, P, R. and W. H. Ewing. 1962, Identification of Enterobaderiocue. B u ~ Pub.
u Co., bfmncrpolir.
Mcdh and Nutrient Solutions 61
226. Starr Spirit Blue Agar

Tryptone 10.0 g Add eotton seed oil emulsion and
Yeast extract 5.0 g mlution of spirit blue t o other
20% cotton seed oil emulsion1 25.0 ml pevious1y disoked ingredients.
0.3% alcoholic sol of spirit blue Lipolytic organisms form a perm.
(National Aniline) Filtered 50.0 ml nent deep blue color beneath and
Asar 30.0 g surrounding colony. Store medium
Distilled water to make a I in the refrigerator. See Dowsan, W,
J. (1 957. Plant discases due to
bacteria) for an alternate medium.
' Grind 100 ml of fresh cotton seed oil, 10.0 g finely powdered gum anbic and 400 ml of warm distilled water.
Should give a smooth permanent emulsion. Wesscrn oil is satisfactory.
Stur,M. P. 1941. Science 93:333-334.
227. Starr Xanthomonas Synthetic hfedium

~ l u c o s e '(added after separate autoclaving) 5.0 g Most species of Xuntbmonas will
NII~CI 1.0 g grow in this basal medium,but somr
KH 2 P o 4 2.0 g fastidious species, with mall arnounil
H3903 5.0 pg of inoculum, may require growth
CaC03 100 c(g factors - generally 0.02 to 0.05%
CaS04 51 120 10.0 pg Difco yeast extract will sufice.
FeS04(N114)2SO4 6I1,O 100 or 500 pg Specifically, methanine, glutarnic
K1 10 c(g acid, and nicotinic acid ma)' be
MnS04 N 2 0 10 or 20 pg required.
Moo3 10.0 pg
ZnS04 7 t l 2 0 50.0 pg
Distilled water to make 1 I
pH adjusted to 6.8 with NaOli
' May purify with Norit Hutncr. S. Ii. 1944. Arch. Biochcm. 3:439444.
wish to
Slan,M. P. 1946. J. B~cteriol.51;131-143.
228. Steinberg Unit Optima Dibasdl Medium

!!?!!I
NIi4N03 1794.6 AU elements are present in minimal
KN03 191.7 quantities that give maximum yields
KH2P04 263.7 for AspergiII~rsniger. Sucrose was
MgS04 71120 1015 used at a concentration of 50 dl.
(NH4 )2S04 48.4
+micro nutrients - Fe 0.20, h10 0.02, Zn 0.1 8, Cu 0.64,Sln 0.02 mgll
Steinberg, R. A. 1945. Pknt Physiol. 20:600408.
Steinberg, R. A. 1936.1. A g . Res. 52:439448.
229. Soaosc Ammonium Tartrate and Nitrate hfedium

Sunore 201 B
KHZP04 Ig MO
M e . 7HzO 0.5 g Fe
-urn tartrate 5.0 g Cu
Nli4S03 1.Og Mn
NaCl 0.1 g Zn
CaClz 0.1 g Biotin
Distilkd water to make a 1
'ilk medim used for assay of choline and inositol with mutants ofNeurospra crassa.
BadLc.G. W. 1944. J. Biol. Chem. lS6:683-689,
Lay. V.C, H. L.Barnett. 1951. Physiology of the fund. SlcGnw.Hil1, N. Y.
230. Suaosc Proline Agar (SPA)

Sumv Used for species separation of
Protine Drechskra (includes species of
Dipatasiurn hydrogen phosphate Helminthosprium md Pyterroplrora.
Monopotassium dihydrogen phosphate Grow in the dark at 20 C for conidia,
Potassium chloride
hlagnesiurn sulphate
Farous sulphate
Zinc sulphate
M a n p e r e chloride
Agar
-
Sh~cmJ;cr,R.A. 1962. Can. J. Bot. 40:809-836.
Dextrose Useful in the identification of

Mucorales.
KH2W4
Mgs'34
Tbiamine chloride
Agar
warn
232. T a b y Medium
Prune extract Simmer 50 g chopped prunes in a 1
Loaore of Water until soft, strain through
Difoo yeast extract muslin,filter and make up to a 1.
Agar Can be stored sterile and cold until
DiFtihd water to make a 1 needed. Used to aid in the identifi-
ation of Verticilliurna004rum
and K &hliae.
Tabny, P. W. 1960. Phnt Path. 9:57-58.
233. Tetrazolium Medium

Peptone One ml of stock 2,3,5 triphenyl
Casein hydrolysate tetrazolium chloride sol to 100 m] of
Glucose cooled liquid medium, stock solution
Agar of TZC = 05 g11W ml water, auto-
clavcd separately. For detection of
virulent-nonvirulent isolates of
PKudomnus soh~ncemum.For use
of TZC with Envirtb m r o v o r a see
B. A. Friedman 1964. Phytopar hOloly
54:494495. For P. plnseolitoh see
B. C. Small and J. F. Worley 1956.
Plant Dis. Reptr. 40:628.
Kelman, A. 1954. Phytopatholqy 44:693495.
Kelman, A. 1958. Phylopatholqy 48:155.165. (Photographs of viruknt-nonviruknt colony types.)
234. Thornton Medium

K2HP04 Phosphate, nitrate and asporagine art
MgS04 a 71120 dissolved in water and the hlgSO,,
CaCI2 CaCI2, NaCI and FeC13 added from
NaCl standard solutions in this order. Add
FeClj agar, melt and filter at 100 C through
KN03 $4" absorbent cotton. hlannitol is
Asparagine dissolved in filtrate cooled at 60 C
Mannitol and reaction checked against Brom
Agar thymol blue to pH 7.4. For
Water t o make a 1 isolation of soil bacteria.
Thornton, Ii. C. 1922. Ann. Appl. Biol. 9:241-274.
235. Tochinai Solution

Peptone TO increase inoculum of Fusarium
KH2P04 oxyspntm,
MgSQ4 a 71j20
Maltose
Water
Tochinai, Y. 1916. J. COILApr. Hokkaido Imperial Univ. 14:lf 1-236.
236. Tomato Juice Salt Agar

Tomato Juice Agar (either from BBL or Used for isolation of storage fungi
Difco) made up as directed on label from seed.
NaCl
Water
237. Tomato Juice Salt Agar (modified)

Tomato juice agar (BBL or Difco) Used for isolation of storage fungi.
112 strength
Agar 1sg
NaCl 100 g
Water II
Christensen, C. M. Personal cornrnu~cation,
238. Tomato Pwtedatmeal Agar
I
Heinz baby oatmeal 20.0 g
Contadina fancy tomato paste 20.0 g Add to 500 ml boiling tap water.
Difco agar 15.0 g
Mix tomato pasteoatmeal decotion with melted agar, then steam for 10 rnin. Dispense and sterilize,
Final pH varies with brand of tomato paste. Recommended for maintenance and determination of
morphology and spore color of S ~ e p t o n ~ y c e s .
Pridham, T,G., ct al. Antibiotic Ann. 1956/1957:947.953.
239. Trace Salts Solution

FtS04 7H20 0,l g Usually used at a concentration of
MnQ2 4H20 0.1 g 1 ml/l.
ZnS04 7H20 0.1 g
Shirling, E, h a n d D. Gottlieb. 1966. Int. J , Syst, Bzcteriol. 16:313-340.
240. Trione Dwarf Bunt Synthetic

Sucrose 20 g Adjust pH to 6.0. Teliospores of
I,-a sparagine 3 !I Tilletiu contraversa produced on
KHz% 613 mg this medium, with or without agar.
M@04 * 7iIz0 246 mg
K2m04* 3fj20 1 1 4 ~
CaCll 55.5 mg
Sodium ferric die thylenetriamine
pentaacetate 20-
ZnS04 7H,O 3.52 mg
C U S O ~ 5f120 038 mg
MnS04 H20 0.031 mg
Na2 MOO, * 21I2 0 0.025 rng
Thiamine HCI 5mg
241. Trionc Mixed Cereal Agar

Mixed cereal (mixture of corn, wheat, Teliospores of Tilletia wntraversa
oats and barley made by Gerber) 50 8 produced on this medium,
Sucrose 15 8
Agar 15 a
Thiamine HCI 5-
Trions, E. 1.1964. Phytopathology 54:592.596.
242. Tris EDTA Algae Medium

3 20 ml O.IM Add to 800 ml deionized or glass
Na21fPO4 10 ml 0.1M distilled water. Add 1 ml each of
MgS04 a 7H20 3 ml 0,IM the 4 stock sol and bring to 1 1,
CaCll 2 H 2 0 1 ml 0.1M pH will be about 8,8 and may k
Tris (hydroxy me thy1 adjusted downward to 7.0 with
amino methane) 25 ml 0.2M 1 N HCI.
Stock Solutions
1. EDTA May be suitable for the cultivation
Kori (85%) of a large number of algae.
Distilled water
2. H3B03
Distilled water
3. FeS04 71l20
Acidified water
(999 ml water,
1 ml conc. H2S04)
4. ZnS04 71120
MnClz * 411 2 0
Md3
CuSO', * 5t120
C O ( N O ~ )* ~6 H 2 0
Acidified water
Smith, R. 1,and V. E. Wiedernan. 1964.Can,I . Bot. 42;1582-1586.
243. Tryptone Glucose Yeast Agar

Tryptone pH adjusted to 7.0 suitable for the
Yeast extract maintenance of a majority of
Glucose industrially important bacteria.
K21iPOi
Agar
Tap water
Hayncs, W. C., 1.J. Wickerham and C.W. HessoHint. 1955.AppL MiaobioL 3961-368.
Media md Nutrient Solutions
244. T r y p t o ~Yead Extract Broth (Shirling & httlieb No. 1)

Bacto-tryptone (Difco) 5.0 g pH should be 7,O - 7.2 before auto-
Bacteyeast extract (Difco) 3fig claving, Used to reconstitute lyophil
Distilled water 1I cultures of Strepton~yces.
Piidham,T, C. and D,Gottlieb. 1948. J. Bactcriol, 56:107.114.
Shirling, E. B. and D. Cottlieb. 1966. In!. J. Syst. Bactcrid. 16:313-340.
345, Tyrosine Apr (Medium 7, Shirling and CottIieb)

Glycerol Adjust pH to 7.2-7.4 for
Myrosine (Difw) characterization of Streptomyces.
Lasparagine (Difco)
KzHP04 (anhydrous basis)
MgS04 7H20
NaCl
FeS04 7H20
Distilled water
Trace salts sol. no, 239
Bacto Agar
Shinobu, R . 1958, 3fc.m. Osaka,Lniv. B, Sat. Sci. 7: 1.76. (modilicd)
Shirling, E. 0, and D. Cottlieb. 1966. Int. J. Syst. Blcreriol. 16:313-340.
246. Tyrosine Cadn Hydrolyszrte Medium
Sucrose pH 7,2-7.4, bacterial cultures examined

Casein hydrolysa te up to 8 days for tyrosinase activity-
Ltyrosine reddish-brown discoloring of medium,
KaHP04
MgS04 7H20
Agar
Crow, 1, E. and Constance hl. E,Guntt. 1963. J. Appl. Bactcriol. 26: 159.177.
247, Tyrosine Caseinate Nitrate hfedium (TCh')

Sodium caseinate 25 8 Tho medium inhibits bacteria and
Sodium nitrate 108 favors the production of a dark
LTyrosine ID8 brown pigment around eolonies of
Agar 1sg Streptomyces saabies,
Dissolve sodium caseinate with gentle heating in 500 ml tap wrter, add other ingredients and make
up to a 1, sterilize 20 min at 10 Ib.
Yentier, J. D.d C m h e E. Dde. 1959. Phytqahokw 49:457458.
Medlr and Nutrient Solutions
248. V-8 Juke Agar

-
V-8 juice contains juices of tomato,
carrot, celery, beet parsley, lettuce.
spinach, watercress, a product of the
Campbell Soup Co. a ml
CaC03 3g
Agar 15 g
Water 800 rnl
Aids sporulation of many organisms, extremely useful.
D i m e , U. L. 1955. Phytopatholggy 45: 141-145.
Miller, P.M. 1955. Phytopathology 45:46146?.
249. V-8 Juice Agar

V-8 juice Used to produce sporangia of
caco,, Phytopttthoro positicu and
Agar P. prlmiwra.
Distilled water
250. V-8 Juice Agdr

V-8 juice 300 ml to start Clear V-8 juice with CaC03 and
caco3 43 g centrifuge at 3000 RPll, 200 ml of
Agar 15g this is made up to one 1. Used to
Water to make a 1 produce sexual bodies of
Phytoplttlwra infestans.
Romero, S. and bf. E. Cdkgly. 1963. Phytapathology 53:899-903.
2Sl. V-8 Juice Agar (modified) (VDYA)

V-8 juice For the isolation of funpi from mil.
C&03 Sterilized at l l Ib 15 min.
Dextrose
Yead extract
Oxgall
As=
Sodium propinate
Chlortetracycline Add to agar at 48 C after sierilization
Streptomycin
P8pnius.G.C. and C. B. M y . 1959. Soil 5ci. 88:112-117.
252. V-8 Juice A p r (modified, VDYA-PCNB)

V-8 juice Used for isolation of Tl~iebviopsis
CaC03 hsiwb from soil. Filter and adjust
Cluoose pH to 5.2.
Distilled water
PCNB active ingredient
Oxgal Made up in aqueous solutions and
Nysta tin added to autoclaved media at
Streptomycin sulfate 50-55 C.
Chlatetr acycline HC1
Pynvizru, C. C. 1965. Phytopathology 54: 1475.1478.
253. Van Gundy and Walker Medium (modified)

KH aP04 03 g Used in protease study of
K2t1P04 1.2 g Ps@udom~nas
kt duyrnans.
MgS04 71i20 0.3 g
Fc 1 PPm
Cu 1 PPm
Zn 1 PPm
Mn
Sucrose modification of Keen et a!.
Glutamic acid
Van Gundy, S. D. and J, C. Walker. 1951. Phytopathology 47 :6156 19.
Kcen, N. T.,P, H. Williams and J . C. Walker. 1967. Phytopathology 57:263-271,
254. Vqctable Oil Nitrate Agar

Vegetable oil (such as Wesson oil) 0.5-2 ml Agitate agar gently before pouring
NaNOj 13s to disperse oil. Most species of
KH,po4 lag Pythium and Phytophthura will
MgSO, 71i20 0.5 g produce sporangia or oospores
Thiamine-HCI (1000 ppm sol) 2 ml around the drops. I have found
Aegr 17g very limited growth with
Distilled water (adjust water to pH 6 Phytophthora megasprma var.
before autoclaving) 11 sofae on this medium.
Hondtk. I. W. 1967. I n Solrrce book o f laboratory exerciser in pbnt pathobgy. W. H. Freeman & Co., San
Fmnciroo.
Hcsrdrh,S. W. md J. L. Apple. 1964. Phytopatholqy 54:987994.
2SS. Vopel Medium N

Najcitratc 5% H 2 0 lSOg Add 2 ml of chloroform as a
KHzPOl anhydrous 250 g preservative. For use, dilute medium
NH4N03 anhydrous lwg 50 fold with distilled water, pH 5.8
M@04 7 H 2 0 10 g
CaC12 2 H 2 0 5ti
Trace ekmen! sol 5 ml
Biotin sol 2.5 ml
Sucrose 20 g/l Or 10 g sucrose and 10 g glycerol/l,
Agar if desired I5 g/l
Trace element sol
Citric acid 1110 Dissolve successively, add 1 ml of
Z ~ S O711~0
~ chloroform for storage at room
Fe(Nl1,)2(SQ4), 6H2O temperature,
C S 0 4 51120
MnS0, 1120
HamBanhydrous
NazMoO4 2 I i 2 0
Distilled water
Biotin (hlerck) solution Stored frozen,
Distilled water 51)rnlm g ~
50
To prepare "complete medium" add 0.5% yeast extract and 0,5% N-Z case (Sheffield). A useful
growth med'iurn for Neurospora.
Vogel H. J. 1956. MicrobioL Gen. Bun. 13:4243.
256. Wsrcup Medium

Czapekdox + 0.5% yeast extract acidified with phosphoric acid to pH 4.0.
Warcup, J. H, 1950. Nature 166:117-118.
257. Warcup M d u m (modified)

Modified to increase its use in isolating fungi from soil, Add 5.0 g oxgall, 1.0 g sodium propionate
and 35 mg of chlortetracyline, the latter after sterilization, pH 6.3-sterilized at 11 Ib for 15 min.
P a p h s , G. C. and C. B. Dmty. 1959. Soil Sci. 88:112-117.
258. Water Agar

1sg Claimed superior t o common media
II for imhting Actinomycetes,
Amount of agar depends on the brand, purity and intended use of medium.
259. Wheat Straw Agar

Sodium nitrate 3g auto-Sprinkle chopped wheat straw
Magnesium sulphate 1g chve previously sterilized with propylene
Agar 20 g and oxide on surface of agar before it is
Water 11 pour set.
It is claimed that Pyrenocltaero is the only fungus known to produce a pink pigment on this agar
making this a useful diagnostic medium.
Watson, R. D. 1961. Plant Dis. Rrptr, 45:289.
260. White Nutrient Solution 1963

1. Set out four 1-1 flasks of double-purified water (1,11,111, IV).
to I add Ca(NO,), 4H20 12.0 g According to Street, H. E,(1957.
KNOB 3.2 g Biol. Review 32: 1 17-15 5 ) a
KC1 2,6 g eomplexed form of iron for
t o I1 add MgS04 * 7H20 30.0 g Fe2 will decrease "staling."
Na2S04 8.0 g
to I11 add Na1i2PO4 Cf20 0.76 g
t o 1V add MnS04 * 7 H 2 0 0.20 g
ZnS04 71120 0.12g
H3B03 0.06 g
KI 0.03 g
CUSO. SH2O
I 0.0004g*
Moo3 0.0004 g*
* These two elements may be added by making up a c~ncentratedsolution consisting of 40 mg
CuS04 SH20and 4 mg Mooj in 100 ml of water and adding 1 ml of this stock to flask IV.
When the four (1, 11, 111, IV) are dissolved, they are mixed slowly to provide stock I , which should
be stored in the dark or in black or non-actinic bottles.
2. In 100 ml of water, dissolve 300 mg glycine, 50 mg nicotinic acid, 10 mg thiamine, and 10 mg
pyridoxine, Stersze by filtration, draw off into test tubes, and store frozen,
3. Dissolve I00 mg chlorophenol red in 25 ml N/100 NaOH; then add water to make 250 ml, Adjust
to pH 6.0, filter, and store in tubes in the refrigerator.
Stocks 1 and 3 will keep indefinitely if not allowed t o become contaminated; stock 2 should not be
kept longer t h v l 6 0 days unless frozen (-1 5' C).
One 1 of nutrient is prepared by dissolving 20 g sucrose and 2,s mg F C ~ ( S Oin~ 889
) ~ ml of water,
then add 100 ml stock 1 (salts), 1 ml stock 2 (vitamins), and 10 rnl stock 3 (indicator). The pH
should be about 5.5 (a pink color); if it is too yellow, adjust with IN KOH. Distribute to containers.
For a semisolid substratum, the same procedure is followed except that only 389 ml of water are
used. Five grams of agar (Difco "Noble" agar or its equivalent) are dissolved in 500 ml of hot water.
When thoroughly liquified, the two are combined, distributed, and au toclaved. This basic nutrien t is
suitable for many sorts of tissues although there are many modifications.
White, P. R. 1963. T k cultivation of M i m J and plent ceb. Ronald Pnu, N. Y.
261. White Solutiun (Modified by Hildebriindt, Riker and Duggnr)
Tobacco tissue Sunflower tissue
Nutrient ingredient medium medium
No2S04 800.0 mg 100.0 mg
Ca(N03)2 4H,O 400.0mg 800.0 mg
MgS04 7H20 180.0 mg 720.0 mg
KNO 3 80.0 rng 160.0 mg
KC1 65 B rng 130.0 mg
NaH2P04 t120 33.0 mg 132.0 mg
Fe~(~4fi406)3 40.0 mg 5.0 mg
MnS04 4 I I 2 0 4.5 mg 4.5 rng
ZnS04 * 71i20 6.0 mg 3.0 mg
H3 903 0375 rng 3.0 mg
KI 3.0 mg 0375 mg
Glycine 3.0 mg 12.0 mg
Thiamine 0.1 mg 0.1 mg
Pyridoxine 20.0 rng
Sucrose 20.0 g
A%ar 5.0 g
Water 1I
tlildebrandt, A. C., A. J. Riker and 8. hi. Duggar. 1946. Amer. J. Bot. 33:591-597.
262. White Medium (modified)

White's inorganic salt solution and:
Sucrose 2% Used to culture apical meristems
Cocanut milk 10% of strawberry.
Agar 0.7%
Indoleacetic acid 4c, mgn of medium (added just before sterilization)
Belkcnpen, R. 0. and P. U'. hfiller. 1962. P l a t Dis. Reptr. 46:119-121.
263. Wieringa Pectate Gel (lhwson's modification)

(1)Soil extract or lap water 500 ml Adjust to pH 9.0 and autoclave at
NaCl 0.5 g 121 C for 15 min. Pour enough to
CaCI, 2.5 g cover bottom of petri dish and
Yeast extract 0.5 g place in desiccator to dry (about
@r 10.0 g 2 days).
(2)Sodiurn polypectate
(Sunkist Inc. product No. 24K12) 2%
Prepared by adding powder dowly to 90 C distilled water. Add 4% bromothymol blue and adjust to
pH 8.Q by N KaOH. Autoclave at 1 15 C for 2 min. When cold, pour enough t o cover the surface of
the agar and allow t o set for about 6-8 days. hiedium demonstrates soft rot organisms as they leave
circular depressions in medium. For another method of preparing pectatc'gells see A. M. Paton.
1959.Nature 183:1812-1813.
Ikmsa, W. 1.1927. Pkat diseases due to bacteria. Cambridge Univ. Pnu, London.
Peptone
K2 m 4
h@O4 7H20
Sucrose
Agar
Dirtillcd water
Roib, H 1965. Phytopathology 55:317-319.
265. Will Mineral Salts Medium

KC1 pH should be on alkaline side to
KH2rn4 induce sporangial formation of
MgS04 7Hz0 Phytophthora. After growth on
K2C03 potato dextrose broth (mntaining
Distilled water 30 g dextrose) transfer to salts
medium,
91% W. H. 1954. J. ELisha Mitchell Soc. 70:235-243.
266. Withrow Nutrient Solutions

Recommendation
A Solution - cloudy weather and
-
high temperatures good solution
for seedlings and plant propagation
in general. Has given excellent
results in northern greenhouses in
winter.
B Solution - well adapted to average

anditions of bright weat her coupled
with moderately high tempcratuers.
I For all Probably best for average circum-
Ammonium citrate solutions, stances in the summer months or
Bins04 4H20 dissolve in in tropics or subtropics.
MnC12 4H20 acidulated
H3BO3 water, can C Solution - contains the highest
Na2B4O7 10H20 be highly level of nitrate nitrogen, used only
C S 0 4 5H20 concentrated under conditions of clear bright
ZPS04 7H20 weather and low night temperatures.
Added after pH of solution of major solutions is adjusted t o 6.8.
Wlhor, R. B. and Alice P. Wlthrow, 1948. Nuvlcuhrur. Purdue Exp. Sta. Cir, 328.
Malt extract 15 g The final pH of Difco H'ort agar
Peptone 0.78 g is 4.8.
Maltose, technical 12.75 g
Dextrin 2.75 g
Glycerol 235 g
Dipotauiurn phosphate 1.Og
Ammonium chloride 1.o g
15.08
Awm. 1965.9th Ed. Difco manual Difco hboratories, Detroit.
268. Xanthomnas Vesieatoria Isolation Medium ST

No. I G!ucose 5.Q g Make up in 150 ml amounts in 250
NHICl 1.Ogq rnl flasks and autoclave. Add 3 ml
KH2m4 1.0g of #2 (oxine) to individual flasks
K2Hm4 1.0s containing % 1, Add 1,5 ml of $3
7H20 0.2 g (triton) to individual flasks
DLrncthionine 0.1 g containing $1. Blix and pour,
A-Z Sol. 1 ml
Actidione 0.04 g
Agar 17 g
Water II
No. 2 Oxinc
Sol. A, 0.22 g Ferric WIydroxyquinoline dissolved in 4 ml abs, ethanol, add 16 ml water (sterile).
Sol. B. 0.48 g Fe2(S04)j H20in 10 ml water heat to dissolve.
Mix solution A and 9. Take 1 rnl and add to 49 ml w t e r (sterile). No sterilization necessary.
No. 3 Triton Hf-30(Sodium octyl phenoxyalkyl sulfate-anonic detergent). 0.7 ml of triton in 69 ml
water (sterile). S o sterilization necessary,
269. Xantbmonas Vesicatoria Isolation hledium X-1

No. 1 Glucose 5.0 g Make up and autoclave no. 1 in
Peptone 5.08 150 rnl amounts. Add 3 rnl of no, ?
Dhthionine 0.1 g (oxinr) to individual flasks of
Actidione 0.04 g no. (X-1). Add 1 ml of SCS solution
Agar 17 g to individual flasks of no. (X-l),
Watcr 11 Mix and pour.
No. 2 Oxine as prepared in X. vesicntorh medium ST.
No. 3 Sodium cebl glffate (SCS, anionic detergent)
0 3 g of SCS in IOv ml H 2 0
270. Yeast Extract A p r

Yeast extract 4.0 g Especially suited for Chaetomium
Malt extract 10,O g and some other ascomycetes.
Dextrose 4.0 g
Agar 15.0 g
Distilled water 11
Haynes, W. C., L. J , Wickcrham and C. \V. Hesseltine. 1955. Appl. blicrobiol. 3:361-368.
271. Yeast Extract Medium

Yeast extract Envinia amybvora grew well in
K IHP04 this medium, remained viable and
MgSO4 7H20 mostly in single cells. See also
Tween 80 modified Emerson medium no. 70.
Reinhatdt, J . F. and D. Powell. 1960. Phgtopathology 50.685.686.
272. Yeast - Glucose (Yp C)

Yeast extract (Difco) Used for production of zoosporangia
Glucose of A phanunytces sp.
K2HPO4
MgS04 71t20
Water to make a 1
Emerson, R. 1958. blycologia 50:589-621.
Disinfection and Sterilization
of Laboratory Equiptnent and hledia
Sterilizatio~~
Introduction
Requirements of most Laboratories
Methods and Uses
1. Steam under pressure (autoclaving)
Tabk 3. Recornmcndcd timo in an autoclave for sterilization o f various volumes
2. Stoam without pressure (Tyndiliz~tion)Arnold Sterilizer
3. Hot dry air
4. Gas sterilization
Table 4. Recammcndod time and t e m p e r ~ t u r rfor stcrilization with dry air
5. Disinfectants and preservatives
6. Liquid sterilization by fdtration
Table 5. Antiseptics and disinfectants and their uses
7. Air filters
Table 6 . Sornc rums that sell bactcriolopical air filters
8. Radiation
Sail "Sterilization" and Greenhousr: Practices
Literature Cited
There are several extensive treatments on sterilization (36,40,48). They should be consulted for
theoretical aspects and fuller details. If there is concern about human infection or the loosening of
harmful microorganisms, see (16) for relevant sterilization and laboratory techniques. With the
demonstration that many fungi are allcr~enicand some have toxic products in their spores more
attention needs to be given t o the prevention of inhalation of fungus spores. Culture of microorganisms
for most purposes requires consistent sterile techniques, Contamination costs time and money and
confuses results. Selection of a method of sterilization is largely determined by cost, efficiency and
applicability, To choose a method only because ofits convenience or to expend effort on an unneeded
elaborate method of sterilization seerns unwise. It is helpful to any method to prevent or reduce the
inoculum load so that minimum periods of time, temperatures or doses result in a sterile product that
is changed as slightly as the method wid permit. Recommendations of dosages and times given
subsequently are conservative, but they ensure sterility except for the presence of large numbers of
highly resistant bacterial spores.
82 Disinfection and Sterilization
REQUIREIIENTS OF MOST LABORATORIES

The following items will meet most laboratory needs.
ltem For sterilization of:
-
7
1. Autoclave heat stable liquids usually the common media ingredients, dry bulk materials
with difficulty, heat resistant instruments and equipment, glassware, rubber
products with some damage.
2. Arnold Sterilizer very few materials, of more use in the elimination of fungal contaminants from
media, etc. prior to wash up, also used in melting media and its pr'eparation.
3. Oven glassware, metal instruments resistant to high temperature and oil.
4. Gas sterilants heat sensitive equipment and substrates.
5. Liquid disinfectants
and sterilants working surfaces, instrumer~ts,tools, and air and heat labile substances.
6. Liquid filters heat sensitive solutions.
7. Air filters air for cultures and transfer chambers.
Other specialized equipment and chemicals valuable for specific needs will be
discussed later in this chapter. See for information on freeing nematodes (14) and plants (5,7, 18) from
microorganisms,
METHODS AND USES

1, Steam under pressure (auloclaving)
Autoclaving is commonly used because it is usually effective, cheap and requires relatively short
periods of time. Its liability is "heat damage" which may or may not be tolerated depending on severity
and satisfactory alternative methods. Autoclaves equipped with a jacket shorten time in the autoclave
(they bring temperature up more quickly) and they reduce the amount of moisture left on the material.
Complete air exhaustion is necessary to obtain sufficiently high temperature for efficient
sterilization ( I 20 C-250 F). Pressure does not indicate whether pure steam or a mixture of air and
steam is present. Therefore, consult the thermomeler at the bottom exhaust line and not the pressure
gauge to determine if sterilization is occurring. Temperature and time required for adequate
sterilization of liquids are given in Table 3. With experience, and if heat sensitivity of materials demand
it, time and temperature may be reduced. The recommendations given in Table 3 do not apply to
materials that are hard to heat (oil, soil, seed, and powders). These materials should be sterilized in
small amounts and for longer periods. Oil is often sterilized by dry heat and, if heat sensitive, by filter
oteribation.
Table 3. Recommended time in an autoclave for sterilization of various volumes.

(After Reddish (40))
Container' Minutes exposwe at 121-1 23 C
1 1 alcnmeyn 20.25
500ml " 1 7-22
200rnl " 12-15
125ml " 12-14
Test Tubes
18x l a m m
38 x 200 mm
'Not mom than 112 to 213 full.
Disinfection and Steirltation 83
In nutritional studies possible advpnc or beneficial effects of autoclving should k particululy
recognized. For example, the pH' is usually lowered 0.24.4 unit, and rarely as much as I unit. Glucose
may be inhibitory when autoclaved with amino acids (23, 24, 35). Xylose forms toxic furfural(3O)
Agar is hydrolized when autoclaved ;,x lirilg, thus making a mushy and sometimes inhibitory me&um
(41). Sucrose is partially hydrolized t o readily available glucose or levulose (8). Autoclaving of glucoa
with phosphate may be stimulatory (53). There may be some stimulatory effects of autoclavine
sucrose, glucose and coconut milk in the medium that is thought to be related t o a chelating effect
(47). The pH and the presence of certain other substances influence the destruction of thiamine (9). An
aqueous solution of pH 3 to 5 is best. Separate autoclaving or filter, gas or liquid sterilization of
carbohydrates, growth substances and antibiotics is often advisable.
Rewmmendations when using an autoclave
(Particularly for nonautomatic types)
1. Rely on temperature, not pressure guage.
2. Keep thermostatic trap clear of plugging substances, check trap if temperature is not being
reached.
3. Avoid filling containers more than 213 if not sealed t o prevent agar from boiling over and
plugging trap.
4. -
Do not exhaust autoclave rapidly if Liquids are being sterilized plugs win be blown and agar may
boil over.
5. -
Do not use overly tight plugs plugs may be blown.
6. Do not fill autoclave with materials requiring widely different times for sterilization.
7, Do not pack load too tight.
8. Increased time for materials that are hard t o heat or are heavily contaminated.
9. If agar has solidified, allow time for melting.
10. Do not over-wrap materials.
1 Do not leave paper-wrapped items on bottom of autoclave.
12. Investigate heat labile properties of s u b
stances t o be sterilized, relating properties
to conditions of test such as ptI.
13. Do not autoclave cellulose nitrate tubes,
they may explode (45).
14. If volume of liquids is critical, use tightly
capped containers to prevent the usual 3:;
t o 5% b s s by evaporation, but allow for a
longer sterilizing time,
15. Acid agar media will not gel properly after
autoclaving. either increase amount of agar
or acidify after autoclaving,
16. Empty and dry containers should not be
tightly stoppered and if possible mverted or
laid on side.
2. Stmm without pressure (Tyndilization )
Arnold Sterilizer (Fig. I )
Substances steamed for 15 minutes on 3
atcccssive days. Formerly used to sterilize bGlt
snsitive volumes such as gelatin and litmus milk,
F& I: -
rlrndd S t a W PPd m l y to p c p m
deoocions, melr rgu and to free roncrmaof
now usually accomplished by au toclaving for mom a~tunimtionp r i m to washup.
84 Didnfcction and Sterilization
12-15min at 121 C followed by immediate cooling (12). Also, considerable b r e d down of sugars docs
occur with tyndilizathn (33). Its main use is for freeing substances of funpl contalninants and for
and melting of a p r media.
3. Hot dry air

As dry air is involved, the process is longer than autochving, but it is preferred for glassware
because of lack of' moisture residue and perhaps less damage. Petri dishes can be sterilized and stored
without wrapping in an oven located close to their use. Table 4 gives a schedule of time and temperature
recommended for sterilization. If dishes are wrapped in paper, use the lower temperatures t o prevent
unnecessary brittleness of paper.
Table 4. Recommended time and temperature for sierilimtion with dry air.
(After Reddish (40))
Required for Sterilization
Temp. C Time in hfinutes
170
4. Gas sterilization
Considered here are three compounds: ethylene oxide, propylene oxide, and peracetic acid. See
(1 1) for a review of other gaseous sterilants. They are used in treatment of heat labile substances, plant
tissue, and bulky materials or where steam is n i t available. They may effect, however, some adverse
changes (1 I ) and are slow acting.
Propylene oxidz (PO) - Although not as efficient a sterilant as ethylene oxide, it is more easily
handled. Placc moistened materials in a jar, add 1.3 ,mlof PO (use a bulb on pipette) per 1 capacity of
the sterilizing container, seal jar (mason jar with a gaskct is suitable) and leave overnight (24). It may
not successfully eliminate all bacteria from seeds (51). Seeds obtained from the dry western areas of the
United States may be more successfully treated.
Propylene oxide can also be used to sterilize agar-filled petri dishes (50). Add 1 ml t o hardened
media in petri dishes and leave at room temperature under a h o d . After 23 hr plates may be used. Ten
ml of PO may be added t o a polypropylene plastic bag containing plastic petri dishes with ordinary agar
media. If the media is acidified or has bacterial antibiotics, 3 ml of PO may be used. Add the PO to a
cotton wad in an empty glass petri dish (54). Some fungi may be subsequently inhibited. Keep
propylene oxide refrigerated, tightly sealed, and handle carefully.
Ethylene oxide - Ethylene oxide with a carrierCOz or fluorinated methane (usually Freon)
eliminates inflammability. It usually is not handled as a liquid, but its use in this form is described at
the end of the paragraph. If the carrier is C 0 2 , the mixture is cheaper but must be kept m a heavy steel
container. Aerosol-bomb type cans containhg ethylene oxide and fluorinated methanes are available
from American Sterilizer Co., Erie, Pa., and the Wilrnot Castle Co., Rochester, N. Y. They also sell
adapters for controlled release of the material. Simple improvised sterilizing chambers, such as
polyethylene bags 5 t o 6 rnl thick or 55 gal drums may be used (42). A dosage of about 112 g per 1 of
chamber capacity for 6 hr at 25 C will sterilize heavily contaminated material (42). For ordinary
materials, in a commercial unit, time may be reduced to 2 t o 4 hr (46). Sterilizing is affected by
humidity, a range of 30 t o 50% is preferred (46); increased temperatures increased the rate of
derilization (46). Readiiy penetrated t h h layers of certain plastics, tg., polyethylene and pliafim (42),
Disinfection and Sterilization 85
allows sterilization of packaged materials; however, certain plastics, e.g., styron and tenite, however, are
damaged (49). Ethykne oxide is toxic and should be handled in an aerated room and not auowed to
contact the skin. As it temporarily leaves a heavy residue, anow 12 hr or more before handling treated
materials.
Liquid ethylene oxide sterilization (27)
1. Coal carbohydrate solution t o be sterilized t o 3-5 C using an ice bath.
2. Add 1 per k t by volume of chilled liquid ethylene oxide using a chilled pipette and agitate.
3. Kepsblutioninicebathfor 1 hr.
4. Transfer t o a water bath of 45 C and allow t o volatilize under a hood.
Peracetic acid (obtained from Becco Chern. Div.) This substance is effective in both liquid and
vapor phase. Its use in the laboratory seems to be mostly for sterilizing plastic (polyethylene, tenon,
polystyrene) chambers and connections by atomization of a 2.3% solution. It is corrosive to skin and
will attack most metals, except stainless steel and some plastics. Fumes must not be inhaled.
5. Disinfectants and preservatives

Some of the liquids used in eliminating or lessening of organisms from bench tops, instruments,
the surfaces of plants and air are given in Table 5 .
Occasionally substances which can be broken down by heat are sterilized by ether or ethylene
oxide than by filtration. The procedure for ether is as described by Shirling and Gottlieb for a
carbohydrate (43).
1. Spread dry substance in a shallow layer in a pre-sterilized erlenmtyer fitted with a loose cotton
plug.
2 Add sufficient acetone-free ethyl ether t o cover the carbohydrate (OBSERVE PRECAUTIONS
AGAISST FIRE!).
3. M o w ether t o evaporate overnight or longer under a ventilated hood.
4. When all ether has evaporated add sterile distilled water.
6. Liquid sterilization by filtration

Sterilization by filtration can be a superior way of freeing liquids
of microorganisms with little change t o the properties of the matchl.
Some investigators who culture plant tissue only filter-sterilize their
media. As most filters are easily clogged, prior low speed centrifugation
-
'$
or fiitrption through filter paper is recommended if there is debris in
the liquid. The advantages and disadvantages of some filters are
discussed next. el
Sintercd glass filter (Fig. 2) (hlorton filter, Fritted glass filter) UF
grade - It appears that littk active ingredients are absorbed by this
filter. Comvonly available as a two piece unit, the filter is ready for use
after the side arm is plugged with nonabsorbent a t t o n and sterilized in
!
an autoclave at 121 C for 20 min. As it is a little top heavy, handle it in
a heavy test tube basket. If suction (applied to the side arm) is provided
by a water asphator, use a trap between apparatus and suction. When
filtering a s d amount of liquid, place a test tube inside the flask to
catch the sterile frltratc. The filter should ordinarily be thoroughly Fig. 2: S h ~ m dg l ~ uNtcr
ckaned soon after use to prevent chemical contamination and t o (Morton bacerid fil-
maintain effiiency. Do not use cleaning solutions containing bichro-
tor) -UFgrade used-
to filter stsriiizc liq.
mates as they will permanently stain and are toxic in very anall uids. (Courtesy of
rmountt. Wash a new filter by suction with hot HCI and then rinse with ~ l o e~chnm;Co$
86 Disinfection and ~tcriliation'
Table 5. Antiseptics and disinfectants and their uses.

Usual
Chemical Concentration -
Use Procedure and Comments
Ethanol 70.95% (freshly Disinfection of Dip instruments, shake excess off,
distilled preferred)instruments h m e momentarily.
Hyamine 1622 0.1 t o .05% (in an Disinfection of Because of its noncorrosiveness
(Para d i a b u t yl phenoxyalkaline solution) bench tops and relative non toxic properties
ethyl dimethyl benzyl when used at this concentration,
ammonium chloride it can be wiped on a variety of
-
monohydrate) Rohm surfaces to lessen numbers of
& Hass, Washington microorganisms, It is more effec-
Square, Philadelphia, tive against bacteria than fungi.
Same other quaternary ammonium compounds are Purasan and Zephiran.
Mercuric Chloride 0.1% Disinfection of Less desirable than hyarnine
table tops because of human toxicity.
0.1% Isolation of Leaves a residue and may be
microorganisms adversely selective.
0.1-2.5% Isolation of Time and treatment varies,
microbial free occasionally combine with other
plants methods or ridding microorganisms.
Sodium hypochlorite I lc isolation of Plant parts submerge for a few
(NaCIO). Make up fresh, microorganisms seconds to several minutes
store stock in cool dark considered superior to mercuric
place, at ordinary tem- chloride. See chapter on
peratures. An acid pH isolation.
(about 4) more efficient
than alkaline pIi of corn-
mercial bleach solutions,
but the rate of deteriora-
tion is faster (1 0, 22).
Freeing micro- Sprayed at the rate of I 1 m1/ 1000
organisms from ft3- most effective at r,h above
air (Triethylcne 7m, Gives only a temporary
glycol is also of effect (1 5).
use (49)).
Sodium hypochlorite Isolation of Time and treatment varies,
(NaC 10) miaobial free occasionally combine with other
plants methods of ridding microorganisms.
For rnalde hyde 8%formaldehyde Sterilizing o f Will eliminate all organisms after
Alcohol in 70% isopropyl instruments, par- 8 hr exposun vegetative e l l s in
alcohol titularly those with a few minutes (35). Corrosive and
large amount of strong irritating vapors; 0.1 %
organic matter borax may reduce corrosiveness
(48).
Formaldehyde 1 part of commer- Elimination of Clean off debris, soak in
cial formaldehyde plant pathogens formaldehyde several minutes,
t o 10 parts of from greenhouse rinse off with tap water (3),
water tools
Disinfection and Sterilization 87
distilled water; pH of the last wash should be close to neutral. A general cleaning schedule is suggested
by the manufacturers.
1. Set up a trap to prevent accidental suckjng of cleaning materials into vacuum line, if other than a
water aspirator is used.
2. A mixture of concentrated H2SOq and HNO3 is sucked through the filter. Transfer this mixture
to another container for future use.
3. Flush with &stilled water.
4. Follow with acetone, if material is high in fatty compounds, also use it before acid mixture.
Never run different solvents into the same container.
Seitz filter - This filter has an advantage in that a new pad is used for each filtration, thus
eliminating laborious cleaning. It docs absorb certain materials, may add iron, may increase tho pH and,
rarely, add toxic substances (26, 34). These effects can be lessened, if sufficient liquid is available, by
discarding the first volumes passing through the filter. A gentle vacuum pressure should be used,
otherwise contaminating organisms may be pulled through.
.Membrane filter (hlill~porefllter). hilllipore filters in the United States arc almost synonymous
with membrane cellulose ester filters, available through the Millipore Fllter Corp., Dedford, Mass., but
other firms such as the Consolidated Lab., Inc., Chicago Helghts, 111.;Gelrnan Instrument, Ann Arbor,
Michigan 48106, and Schleicher and Schuell Co., Keene, H. H., also sell membrane fdters. They are
about double the cost of Seitz pads. The HA grade, pore slze 0.45 1,may be used, although a finer
grade 0.2 C( is recommended. Varlous holders are made. The pyrex filter holder,although not generally
recommended for steriliz~ngfiltration, is commonly used because it costs less than the stainless pressure
filter holder (2). There is also available an autoclavable plastic filter holder. The entire unit w ~ t hthe
filter in place can be autoclaved, although a rubber cap designed to equalize pressure when autoclaving
is recommended with the pyrex filter holder. When sterilizing the membrane it should not become wet,
Filters, if not purchased in autoclave hts, and filters and absorbent pads packaged in h a f t envelopes
should be separated by filter paper, placed in a petri dish, and then wrapped in h a f t paper. Autoclave
at 121 C for 15 minutes and do not exhaust the autoclave rapidly. The filter is placed asceptically into
the filter holder. Tlrese filters are believed to have neglipble absorption and do not contarnmate a
filtrate if the ordinary solvents - water, &lute acids and alkalines - are used. Because of these
properties and a high flow rate with relatively clear kquids, they are highly recommended. These filters
give good results when used m their own holders, successive filtration through 2 filters increases
assurance of sterlllty (39). They are also useful m analysis of water and other liquids, isolation of
specific organisms, medical and clinical analysis, and aerobiological analysis (2, 19). But they are
soluble in ketones, esters,ether-alcohols and mtroparaffins, and are attacked by strong alkalies, More
resistant filters are avallable (2).
Swimy filter adapter (Fig. 3). This filter attaches to a luerlok hypodermic needle and is of use
with both the seitz filter and the membrane filter, although the latter is preferred by me. The larger
Pig, 3: -
Swlnny filter adaptor can be 6tted with r wiQ Nlrr pad or mmbmnb type fillen.
COU-Q d MUU pon Co.
&ace swinny filter is preferred as it is easier t o use. Wrap the adapter, with either filter in place, in
b d p a p r a d autoclave it. With the membrane filter do not go above 121 C ar over 15 minuter, and
exhaust the autoclave slowly. This adapter is easily used and is fine for volumes of 20 ml or less which
relatively free of plugging materials. Be sure the adapter with the filter is screwed tightly in place to
bypass of non-sterile iiquid,
7. Air fdters
Drying tubes filled with cotton wool (nonabsorbent cotton) are usually sufficient, provided they
art kept dry, for forced aeration of cultures. For the filtration of large volumes of air in conjunction
with air conditioning or transfer chambers, commercial fdters are necessary, They usually consist of
glass fibers. Companies that supply "high-efficient" and "ultra-high efficient" filters for removing
almost aU microorganisms are listed in Table 6.
For specific recommendations of air filtration of microbial particles see (17). Portable transfer
hoods equipped with a filter and a blower are available from Air Control Inc., Narberth, Pennsylvania.
There are a number of other firms that offer similar equipment.
Table 6. Some firms that sell bacteriological air filters ( I 7).

American Air Filter Corporation Louisville, Kentucky
Cambridge Filter Corporation Syracuse, New York
Farr Filter Company Los Angeles, California
Flanders Filters Riverhead, New York
Mine Safety App. Co. Pittsburg, Pennsylvania
8, Radiation
Ultraviolet radiation. Ultraviolet lamps can effectively reduce air borne contaminants but are not
cure-alls and are somewhat hazardous. One 17 watt lamp, mounted 8" below the ceiling and shielded
to radiate upwards, substantially reduced wall and air contaminants in a small transfer room (55).
Lamps should be wiped clean (turned off first) with alcohol every 2 weeks to maintain efficiency.
To reduce the number of microorganisms entering a room, a partially shielded bank of UV lamps
positioned around the outti& of the door is suggested (55), Personnel must not loiter or look into UV
lamps, and noticeable warning signs should be posted. Safety clothing and goggles may be advisable
(13). W lamps can rehove contaminants from air streams (1,32), but filters are more generally used.
As UV rays penetrate poorly, other uses are limited.
High energy ionizing radiation. Excluding food preservation, current use of high energy by
microbiologists is small because of capital outlay and inconvenience of the operation. Several
experiments indicate the value of future use. For example, soil sterilized by electrons produce from a
Van & Gaaff accelerator more closely resembled unsterilized soil than soil heat sterilized (37, 38).
Gamma radiation, emitted from a cobalt 60 sourbe, of barley malt sprouts proved superior in the
subsequent fermentative production of lactic acid to heat sterilization (21). There are other promising
reports (21,31).
SOIL "STERILIZATION" AND GREENHOUSE PRACTICES

(Elimination of plant pathogens from soil)
A comprehens,he and highly practical manual advocating a system of handling plants (3) should
be consulted. For other aspects of soil sterilization or soil disinfestation see (29,44).
-mion and Sterilization 89
A few main points stressed in this text (3) are:

1. %tilo, for the consistent production of healthy plants, should be free of plant pathogens. Steam
treatment (soil at 212 F for 30 minutes') rs considered one of the best methods. Chloropicrin,
methyl bromide, formaldehyde and vapan may be valuable. For more details on freeing soil from
microorganisms by chemicals we (28,U).
2. If pornlle, soil should be treated in the containers that plants are to be grown in.
3. R e v m t subsequent contamination by:
Always placing containers on clean surfaces.
Avoid putting freshly treated soil near older pots and plant materials.
U s clean plant materials: treat seed. or usc stock verified clean by culture technique (1 8).
Do not unnecessarily handle stock or soil.
Keep nozzles of watering h o s e from contacting sources of contamination.
Treat tools with formaldehyde (1 pt commercial formaldehyde to 20 pt water).
Subsequent reports (4,6) suggest using aerated steam which can maintain 140 F for 30 min for removal of plant
mogcnr Soil King Ca, Redwood City, California, makes electric sterilizers designed t o sterilize at this lower
tempmature. Shurtleff et ol (41) suggests i 80 F for 30 minutes, or to turn the stcam off when the soil temperatures
lcsch 200 F. Excessive temperatures increases the chiurm of phytotoxicity due to soluble d t s , m a n p e s e toxicity,
and toxic or@ mmpoundr
LITERATURE
Anonymous. 1960. Germicidal lamps and application. LS-179,General Electric Co., Nela Park,
Cleveland 1 2,0tuo.
Anonymous. 1963. Sterilizing filtration and sterility testing. ADM-20.Millipore Filter Corp.,
Bedford, hlass.
Bakcr, K. F. (Ed.). 1957.The U. C. system for producing healthy container grown plants. Calif,
Agr. Exp. Sta. 31anual No. 23.
Baker, K. F. 1962.Principles of heat treatment of soil and planting material. J. Australian Inst.
Agr. Sci. 28: 118-1 26.
Baker, K. F. 1962. Thermotherapy of planting material. Phytopathology 52:1244-1255.
Baker, K. F. and C. M. O l x ~1960. Aerated steam for soil treatment. Phytopatholr~gy50:82
(abstr.).
Baker, R and D. J. Phillips. 1962. Obtaining pathogen-free stock by shoot tip culture.
Phytopathology 52:1242-1 244.
Ball, E. 1953. Hydrolysis of sucrose by autochving media, a neglected aspect in the technique of
culture of plant tissues. Bull. Toney Bot. Club. 80:409411.
Beadle, B. W., D. A. Greenwood and H. R Kraybill. 1943. The stability of thiamine to heat. J.
Biol. Chem. 149:339-557.
Btazis, A. R, J. E. Leslie, P. W. Kabler and R. L. Woodward. 1958. The inactivation ofBaciII1s
@bigii, and B. ortthrucis by free abxiIable chlorine. Appl. Microbial. 6:33&342.
Bruch, C. W. 1961.G a s e ~ u s t e r h t i o n . .bRev. hlicrobiol. 15:245-262.
Burnett, G. W., 51. f. Pelczar, Jr. and H. J. Conn. 1957. Preparation of media. In Manual of
Microbiological .\lethob. .\icC;raw-Hill, S. Y .
Buttolph, L. J. and H. Haynes. 1950. Lltraviolet air sanitation. Pamphlet Ld-11. General Electric
Co., Cleveland, Ohio.
Cairns, E. 1. 1960. Methods in nemrology: a review. In J. N. Sasser and W. R Jenkins (Ed)
Nrmrtology. Univ. of N. Carolina Press, Chapel Hill.
Challinor, S. W. 1943. Bacteriological obseservations on the air of occupied premises. I. Air
W b c t i o n with 'hypochlorites"; a simple practical method of disinfecting the air of occupied
pmmiur. J. Hyg. 43: 1654.
Disinfection and Sterilization
Chatigny, M. A. 1961. Protection against infection in the microbiological laboratory: devices and
procedures. Adv. in Appl. llicrobiology Vol. 3. Academic Press, S. Y .
~ e c k e r H.
, M., L. M. Buchannan, L. B. Hall and K. R. Goddard. 1962. Air fdtration of microbial
particles. Public Health Serv. Pub. No. 953.
Dimock, A. W. 1962. Obtaining pathogen-free stock by cultured cutting techniques. Phytopathol-
OW 52:1239-1241.
Ehrlich, R. 1960. Application of membrane fdten. Adv. in Appl. .\licrobiol. Vol. 2. W. W.
V
Umbreit (Ed.) Academic Press, N. Y.
Englis, D, T. and D. J. tianahan. 1945. Changes in autoclaved glucose. J. Amer. Chem. Soc.
67:s 1-54.
Gillies, R. A. and L. L. Kcmpe. 1957. Comparison of gamma radiation and heat for sterilization
of fermentation washes. J. Agr. Food Chem. 5:706-708.
Greene, C. L. 1966. Response of conidia and appressoria of Cloeosporiu~nrnlrsanrm to
hypochlorous acid. Phytopatholoy 56: 1201-1 203.
Hall, Angella hi. 1959. The culture of Pl~ytopittltorairzfesta~is. Brit. Slycol. Soc., Trans..42: 15-26.
Hansen, H. N. and W. C. Snyder. 1947. Gaseous sterilization of biological materials for use as
culture media. Phytopathology 37:369-371.
Hill, E. G. and A. R. Patton. 1947. The Malllard reaction in microbiological assay. Science
105:481482.
House, W. 1964. Toxicity of cell culture medium due to fdtration through asbestos pads. Nature
201 :1242.
Judge, L. F,, Jr. and 51. J. Pelczar. 1955. The sterilization of carbohydrates with liquid ethylene
oxide for microbiological fcrrnentation tests. Appl. 11icrobiol. 3:142-245.
Kreutzer, W. A. 1963. Selective toxicity of chemicals to soil microorganisms. Ann. Rev.
Phytopathology 1:lOl-126.
Lawrence, W. J. C. 1955. Soil Sterilization. SIac51illan Co., N. Y.
Lockwood, L. B, and C. E. N. Nelson. 1946. The oxidation of pentoses by Psetrdomonas. J.
Bacteriol, 52:581-586.
Merchant, D. I., R. D. Stewart, L. L. Kempe and J. T. Graikiski. 1954. Use of tissue culture
mediums sterilized with Gamma radiation from Cobalt 60. Proc. Soc. Exptl. Biol. Med.
86:12&131.
Miller, 0. T., R. F. Schrnitt and G. B. Phillips. 1955. Applications of germicidal ultraviolet in
infectious diseases of laboratories. I. Sterilization of small volumes of air by ultraviolet
irradiation. Amer. J. Pub. Health. 45: 1420-1423.
Mudge, C. S. 1917. The effect of sterilization upon sugars in culture media. J. Bacteriol.
2:403415.
Papavizas, C. C. and W. A. Ayers. 1964. Effect of various carbon sources on growth and sexual
reproduction of Aphunomyces euteicher .\lycologia 56:816-830.
Patton, A. R., R C. Salances and M. Piano. 1954. Lysine destruction in casein-glucose interaction
measure by quantitative paper chromatography. Food Res. 1 9 : W S O .
Perkins, 3. I. 1963. Principles and methods of sterilization. Charles Thomas, Springfield.
Peterson, G. H. 1962. Microbial activity in heat-and electron sterilized soil seeded with
microorganisms. Can. J. Slicrobiol. 8:s 19-523.
Peterson, G. H. 1962. Respiration of soil sterilized by ionizing radiations. Soil Sci. 94:71-74.
Portner, Dorothy, C. R. Phillips and R. R. Hoffman. 1967. Certification of probability of
sterilization of liquid by fdtration. Appl. Microbiol. 15:800-807.
Reddish, G. F. 1957. Antiseptics, Disinfectants, Fungicides, and Chemical and Physical Sterili-
zation. Loa and Febiger, Philadelphia.
Robbins, W. J. and Llda McVeigh. 1951. Observations on the inhibitory action of hydrolyzed
agar. Mycologia 43: 11-1 5.
Disinfection md Sterilization 91
Schley, D. G., R. F. Hoffman and C. R. Phillips. 1960. Simple improvised chambers for gas
d c d k a t i o n and ethylene oxide. Appl. .\licrobiol. 8: 15-19.
Shirling, E. 8. and D. Gottlieb. ,1966. Slethodr for characterization ofSrreptomyces species. Int,
'
J. Syst. Bactcriol. 16:3 13-340.
Shurtleff, M. C., D. P. Taylor, J. W. Courter and H,B. Petty, Jr. 1964. Soil disinfestation. Illinois
Agr. Ext. Serv. Circ. 893.
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Stierli, H., L. I. Reed and I. ti. Billickc. 1962, Evaluation of sterilization by gaseous ethylene
oxide. Pub. Health Man. So. 68.
Street, H. E. 1957. Nutrition and metabolism of plant tissue cultures. J. Nat. Cancer Ins,
393467494.
Sykes,G. 1958. Disinfection and sterilization. D. Van Nostrand, Princeton, N. J.
Tessler, J. 1961. Reaction of the sterilant ethylene oxide on plastics. Appl. ~licrobiol.9:256.
Thompson, H. C. and J. W. Gerdemann. 1962. An improved method for sterilization of agar
media with propylene oxide. Phytopathology 52: 167-168.
Tyncr, L. E. 1957. Effect of moisture in partial-sterilization procedures. Phytopathology 47:536.
(Abstr.)
Virgin, W. J. and Jean C. Sldoit. 1937. The use of triethylcne glycol vapor as an aid in the control
of airborne contaminants. Phytopathology 37:365.(Abstr.)
Ward, E. W. B. 1964. Stimulation of growth of a low temperature. Basidiomycete due to heat
sterilization of a culture medium. Can. 1. Bot. 42:283-285.
Watson, R. D., H . E.Carley and D. M. Huber. 1966. Storage of culture media for lab and field
use. Phytopathology 56:352.
Wedum, A. G., E. Hanel and C. Phillips. 1956. Ultraviolet sterilization in microbiological
laboratories. Public Health Reports 71 ~331-336.
Isolation of Bacteriophage and Plant
Pathogenic Actinomycetes, Bacteria and Fungi
introduction
Facton Invoked in Isolation
1. Ihe condition of the diseased material
2. Surface disinfectants
3. Incubation temperature
4. The medium used for isolation
Table 7. Compounds commonly added to media to inhibit fungi or bacteria or both
5. Technique of isolation
Methods of Isolation
1. Induce spomlatjon
11. Induce mycclial growth
IU.Dilution techniques
N.1solation by host inoculation
V. Isolation by baitbg
M.Dltect isolation
F m h g Fungus Cultules of Bacterial Contaminants
The Isolation of Single Cells
Isolation of fungus spores
Methods using the stereoscopic microscope
Methods using an ordinary light microscope
lwlation of single cells of bacteria
Some Tools Used in Isolating Single S p o m
bolation of Bactcrioph~gts
Literature Cited
INTRODUCTION
Mostly treated here is the isolation of microbial plant pathogens from their hosts and soil. See
(129) for details on the isolation and culture of host tissue, (24) host tissue and parasite, (31)
pathogen-free clones, (10, 34, 42, 44, 58, 61, 79, 126, 128) for detailed treatment on the isolation of
microorganisms from soil, (62, 108, 115) for the isolation of aquatic fungi, (19) for the isolation of
fungi from sewage and polluted water, (3) for the isolation of Myxomycetes, (15) for the isolation of
rctasieae, (40, 81) for the isolation of yeasts, and (14, 50, 73, 120) for the detection of antibiotic
argonism.
First, it is well to know what the obligate parasites look like to avoid needless attempts to culture
them on ordinary artificial media. The rusts, powdely and downy mildews, constitute the majority of
obligates and arc fortunately easy to recognize.
FACTORS INVOLVED IN ISOLATION
Some of the factors influencing the outcome of isolation of microorganisms from their hosts and
from other material are:
1, The condition of the diseased material. Success in isolation depends upon the proper selection
and usually rather quick use of diseased host material, Isolation from badly decayed or from oldel
portions of diseased tissue is difficult, as faster growing secondary organisms may be present or the
pathogen has died. Frequently, storage of even prime material in plastic bags at room temperature
rapidly results in an urgently disposable mess, Collections if not used soon should be refrigerated and
stored dried if possible.
2. The kind of "surface disinfectants" uscd to eliminate contaminating and superficial micro.
organisms. Several workers have shown (27, 124) that HgCI2, CaCIO, and AgNOj preferentially isolated
certain fungi. Rl~ync~iosporiurn seculis was very sensitive to HgCI2 while not t o NaCIO (102). In
addition, HgClz is not removed by washing in water (84). A 1% solution of NaCIO, although reputed to
be harmful t o the isolation of Pythiutn (47, 1171, is generally suitable. Some workers, particularly in
first isolation attempts, wash the tissue, with or without sand or detergents, in running tap water or
sterile water (47, 63) to avoid damage. Soaking tissues in solutions of antibiotics prior to plating seems
t o be discontinued in favor of incorporating thew materials into the plating medium (94).
3. Temperatures at which materials are incubated. An expected example of this is the difficulty
of isolation of a low temperature basidiomycete at normal temperatures (9, 71). There are enough
other examples (12) t o be mindful of this requirement. In general isolations.at temperatures of 20-24 C
are suitable.
4. The medium used for isolation. All media are selective t o some extent, even the brand of agar
may have same influence (76). Generally, media of pH 4 5 favor isolation of fungi, as most bacteria and
actinomycetes are intolerant of acid conditions. In initial attempts to isolate an unknown plan!
pathogen, "standard isolation media" are often used; for bacteria: beef peptone agar (nutrient agar); for
fungi: corn meal, malt agar, PDA (often acidified), and water agar; and failing, media rnade up of the
host that is being isolated from or more specialized media. bficroscopic examination of infected tissue
often indicates the "kind" of pathogen involved - fungus, bacterium or actinomycete- thus suggesting
likely media. More numbers and kinds of certain groups of microorganisms, particularly from soil, are
obtained by adding selectively inhibitory (Table 7) and or enriching materials to culture media.
Enrichment materials such as pectin (32), chitin (73) or alcohol (75, 120) arc usually the sole source of
carbon in the media and are utilized by limited numbers of microorganisms, including the desired
organism. Alcohol may be effective more for its inhibitory value than differential utilization. Inhibitors
w e d are: antibiotics, commercial fungicides, dyes, detergents and certain naturally occurring products
such as oxgall and sodium propionate. Subject to their concentration, they may inhibit some organisms
completely or slow the growth of fast-spreading fungi such as Trichoderma and Rhizopus, thus
permitting the slower growers to be detected. Commonly used materials are listed in Table 7 with
references on their value and use. Some general references are (2,69,89,90, 116,122). It is important
t o know something about their solubility, stability, compatibility and inhibitory spectrum before use.
'Zheir properties may also vary in different media, pH often being important. A list of specific isolation
media is in the chapter on media.
S. Technique of Isolation. The growth habits, the mode of reproduction, and the ecology of a
microorganism largely determine how it can be isolated. For example, as bacteria produce large
numbers of reproductive units, they are more efficiently isolated by culturing dilutions of infected
plant tissue than by culturing intact pieces of tissue. Slow growing organisms are often more readily
obtained by the direct isolation of spores than by culturing tissue o n a nutrient agar. Certain grain
storage fungi: members of the Aspergillus g h c u s group that grow under conditions of physiologicd
drought are best isolated on high osmotic media which duplicates their environment in part (8).
BETHODS OF ISOLATION
Before isolating from plant materials, particularly when seeking an organism or organisms
ipvolved in a disease, the tissue should be examined for fmiting bodies, mycelium and bacterial cells.
k t d s of procedures may be found in the section dealing with microscopic techniques. The methods of
d a t i o n and recommendations are:
I,
-
Method
Induce sporulation
Rtcommendation
Primarily for leaf spotting fungi
11, Induce mycelkd growth For deep seated infections and poorly spodsring fungi
Ill. Dilution technique For bacteria and microorganisms present in luge numbers of repre
ductive units
IV. Host inoculation Obligate parasites, badly contaminated and/or very slow growing
organism
V. Baiting Qrtain organisms, usually occurring in the soil, that are able to grow
more competitively in certain bait substrates
VI. Direct isolation Organisms that may be found in pure colonies and some slow grow-
ing organisms
I. Induce sporulation: (fungal lesion producers). Most micro fungi attacking aeri31 parts of the plant
mll sporulate on the host if kept moist at a suitable temperature, permitting subsequent transfer. One,
however, should be prepared for the occasional sterile fungus or one that produces nonviable spores.
The procedure is as follows:
1. Cut across lesions, including some healthy tissue, to give pieces of 114 t o 3/4", If a pathogen is
known to be very slow growing and there are contaminants, cutting very srndl pieces after the tissue
has been disinfected and dried on sterile filter paper is helpful (96).
2. Submerge pieces in 1% sodium hypochlorite or other disinfectant for different times from 5 seconds
t o 1-2 minutes. If tissue is hard t o wet, either dip momentarily into 7m ethanol or add a drop of
Tween 20 or 80 or a suitable detergent to the NaC10.
3. Using a forceps, shake off excess and place 5 or 6 pieces, well separated, on sterile moist fiiter paper
or on water agar, in petri dishes. The latter maintains a more uniform supply of moisture and can be
acidified if bacteria are a problem. In any case, this low nutrient medium is u x d t o encourage
sporulation and discourage secondary organisms.
4. Observe for spores-fruit bodies from the w a n d day on. If no organisms appear after 1.2 weeks, or if
the immature fruit bodiespreviously present fail to produce spores, repeat step 1 but wash the tissue
in a screened wide mouth jar under running tap water for IGtO minutes, followed by a rinse in
sterile water, and repeat step 3. You may wish to do this concurrently with S d l O treatment. In the
event of continuing immature fruit bodies special environmental treatments may be necessary. See
chapter on increase of incxlllurn for details.
5. When fruiting occurs, spores are usually transferred t o acidified PDA, PDA or V-8 juice agar (the
latter often encourages sparulation). For details see method VI (Direct Isolation) and section on
iodation of single spores. Species of Colletom'churn, Cercospom, Phorrra {Pfri~llosticta),Ascoclryta,
Stemphylium, Septorh, fflelminrhosporium.St~gonosporaare common leaf spotters and should be
familiar. Alternu&, CLadosporiurn, Epicoccum, Tnkhoderma. Choetomium and Penicillium are
commonly present, but usually they are saprophytes. (Use some caution before dismissing them.)
Some Ascomycctes, e.g. Pseudopezzia medicagrnis, Leptosphaenrlina briosiena, and many Basidio-
mycetes, forceably discharge their spores, thus facilitating their isolation. Their fruiting bodies n a y
be placed on the inside of the lid of a petri dish and allowed to shoot spores onto the agar. If plates
ue inverted during shooting, less contaminants occur. This method only works with those organisms
able to shoot spores several em. The lid is rotated to distribute spores. Light of a few hundred ft* is
necessary to induce spore discharge of many Ascomycetes.
U. Induce mycelial growth: (Deep-seated infections and poor sporulating fungi) organisms, such as
wood rotting fungi, stem and root parasites that do not fruit readily on host t i w e and/or are found
primarily in internal tissues, are generally isolated by culturing pieces of internal tissue on a nutrient
agar or on water agar. The procedures are as follows:
For Woody Tissue - Fruits. etc.
1. If roots, wash thoroughly with running tap water -- shake dry.
2. Dip material in 1% sodium hypochlorite or wipe with a cotton swab dipped in the disinfectant.
Ethanol, is often used, followed by lightly flaming the tissue. This must be done with care to
avoid ignition of tishe or overheating.
3, Using a flamed knife or razor blade, peel back outside layer of tissue. Flame instrument each time a
cut is made. With fruits some workers prefer to score with instrument and then force open by
pressure.
4. Dig out small pieces of tissue 118 to 1/2" with a flamed knife or forceps at margin of infected tissue.
Organism may have died out or be contaminated in badly rotted tissue.
5. Place 3 or 4 pieces, well separated, on acidified PDA, PDA, m d t agar, corn meal, or other desirable
medium.
6, After growth, transfer bits of agar containing mycelium from the edge of developing colonjes to test
tubes or plates. Also look for spores on plated out host tissue.
For Fine Roots and Damped-off Seedlings
1. Wash root thoroughly for 10 to 20 minutes with running tap water using a wide mouth jar fitted
with a screen top or covered with chcesc cloth.
2. Rjnsc twice in sterile water, shake dry, and plate on water agar ~ith a dry surface. Dip others in 1%
sodium hypochlorite for 10 seconds to 2 minutes, shake off excess, and place on water agar, no
more than 3 per plate, An alternative method is to cut a thin longitudinal section, place on a
coverslip, and add a few drops of water agar containing 0.2% aureomycin. Incubate on a glass ring
sealed to a slide (47), permitting microscopic determination of origin of mycelium.
3. Transfer bits of agar containing mycelium from the outermost edge of developing colonies to
separate tubes or plates of PDA. Do this 2-4 days after plating and do not acidify the medium. Also
examine for fruiting bodies in tissue. If bacteria are present, see purification procedures. Genera
most commonly isolated from damped-off ssedlings are Pythium, Rhizoctonk, Aphanorn,vces,
Fuwium, and Phytoptlrhora, particularly the first two. Apltattonlyces is more effectively isolated by
placing washed seedlings in sterile water with frequent changes for 12-24 hr, then placing firmly on
corn meal,
III. Diution techniques: (usually with organisms producing many reproductive units). Dilution
techniques favor the organisms that produce large numbers of reproductive units - heavy sporulating
fungi, yeast, bacteria and actinomycetes. Dilution also proved useful for the detection of a nonparasitic,
but pathogenic species of Pcnicillium that grew on the surface of peach roots, produced a toxin and
caused a wilt disease (30). Because dilution physically separates organisms, slower growing organisms
that normally are overrun in tissue platings occasionally may be detected by this method.
For the Isolation of a Leaf Spotting Bacterium
1, Cut out younger portions of lesions, cut one in half in a drop of water and after several minutes
examine under oil immersion. if bacteria are streaming from the lesion, proceed.
2. A surface treatment u w d y is unnecessary because the relatively fewer contaminants are left behind.
If disinfection is desirable, dip lesions briefly in either 1% NaClO or 70% ethanol and rinse 3 times
with sterile water.
3. Place several lesions in 0.5 to 1.0 ml of sterife water in a petri dish, mince finely with a flamed razor
blade and wdt several minutes.
4. Transfer several loopfuls of the suspensions t o another plate containing 0.5 to 1.0 ml of sterile
water, mix thoroughly, and repeat this for 2 or 3 more plates.
5. Add cooled molten (45-50 C) beef peptone agar or other desirable media to the 3 plates and mix
gently but thoroughly by rotation. Invert the plates after the agar is hardened to lessen the chance of
bacterial colonies running together. Select prevalent colonies for transfer.
For the Isolation of Microorganisms from Soil
Recision and perhaps accuracy of the dilution plate technique can be increased by: pooling of a
number of original samples (58, 59), immediate use of the sample or storage at I15 C for a week or less,
use of large sample aliquots, use of dilutions yielding around 30 colonies per plate (achieved by varying
amount of soil used than by varying dilution procedure (58)), use of mechanical aids to achieve
unifonn dispersion, use of wide aperture pipettes, use of fresh pipettes for each dilution (59), use of a
diluent of sufficient viscocity (57, 58). use of media that inhibit spreading organisms (SP), and use of
10 or more plates for a dilution (105).
Two simplified dilution plate methods are suggested here. For others see Clark (16), Watson
(128), and Johnson (60). Common to both are that a lo4 dilution is usually satisfactory for fungi, 10'
for actinomycetes and lo5 to 10' for bacteria. Bacteria are often isolated on a soil extract medium or
nutrient agar. Fungi with OAES, Martins modified V-8 juice or PDA-tergitol and Actinotnycetes are
usually isolated on water agar or Casein glycerol medium. The number of colonies that appear are
averaged and multiplied by the dilution factor to give the number per gram of soil. For critical work it
may be necessary to determine the moisture content for a sample of the soil and convert to dry basis
(10 g sample at 105 C for 24 hr).
The fust procedure is modified from Hornby (58) and is much less precise than his original
method.
1. Aseptically collect and pass soil sample through a 2mm sieve,
2. Add 10 g of soil t o 200 rnl of 1% sodium carboxymethy cellulose' (CMC) or 0.2% water agar
in a 16 oz flat sided screw cap bottle.
3. Shake bottle horizontally on a reciprocating shaker for 20 min.
4. Transfer 1 d aliquot to 9 rnl of CMC diluent in a 4 oz bottle and shake for 4 min.
5. Transfer a 1 ml aliquot to 49 rnl of ChlC and shake for 2 minutes on the shaker.
6. Transfer 1 ml t o each of 5 sterile petri dishes. Agitate bottles between each pipetting.
7. Add 17 ml of a suitable precooled medium (45 C) and dispense inoculum with circular and
aide to tide movements of the petri dish.
A second soil dilution technique is modified from Maciejowska (75).
1. Add boning water to the Waringt~lendorfor a minutes; treat the lid similarly.
2. Add 300 ml of sterile 0.1% water agar and 30 g of soil,
3. Blend for 1 minute.
4. Transfer 1 ml t o 99 ml of 0.1% sterile water agar contained in either milk dilution bottles or
screwcapped prescription bottles; this gives a dilution of 1: 1000 (lo3).
5. Shake bottle vigorously and repeat step 4 for a dilution of 1: 1000,000 (10').
6. Two dilutions are selected for culturing on agar. Transfer 1.0 ml from the selected dilutions to
each of usually 5 plates.
7. To obtain a 1; 10,000 (104) dilution with fewer dilution bottles, 0.1 ml instead of l,0 rnl is
taken from the 1:1000 dilution bottle and added t o a petri dish. This procedure, although
time saving, decreases precision.
8. Add cooled agar (45-50 C) (bottle barely cbmfortable when held to the cheek) and rotate. If
'
bacteria are expected, invert plate after the agar has har&ned.
Soil plate method (Warcup ( I 25))
A less complicative method which tends to yield more kinds of fungi than those obtained from
the dilution plate-technique.
1. Fashion a flattened nichrome wire into a microspatula so it will pick up about 0,005 g to 0.15
g of soil.
2. Transfer the soil to a sterile petri dish and crush and mix the particles in a drop of water. The
latter step may be omitted for light textured soils.
3. Add 8-10 ml of melted and cooled (45 C) Czapek's agar plus 0.5% yeast extract which is
acidified with phosphoric acid to pH 4. (Other media may be desirable.)
This technique usualty reveals a greater variety of fungi than the soil dilution technique. See
Garrett's (44) for an evaluation of the numerous method of isolating fungi from soil and Johnson's et a1
(61) for details of many of the methods.
N. Isolation by host inoculation: (for obligate parasites and difficult cases). When diseased materials
are badly contaminated or the organism is difficult t o isolate by ordinary means from field ,plant
material, the contaminated source of the organism can be purified by direct inoculation of the host,
This screens out the non-parasitic organisms and usually allows isolation with methods not previously
feasible. Xarrt/~omonastranshicerrs is isolated from soil by this method (7) as is Apllanomyces euteiches
from roil and host material (63). This method overlaps the following one.
V. lsalation by baiting: (for difficult cases and for organisms in soil that do not show in dilution
plates). A variety of microorganisms may be isolated from soil and water by the use of some
preferential substrate. The desired organism is able to develop on the bait to the exclusion of other
organism, thus allowing isolation by ordinary methods. Agrobacterilrm tumefaciens (4) and Thiclaviop
sis bashla (131) are isolated from soil by the use of carrot discs (for details see media section).
Rhizoctonfa with cellophane (251, a number of water molds, are isolated by the addition of boiled
hemp seed t o pond water or soil-water mixtures (36). The insertion of diseased materials (121) or soil
(13) inside thc "baitw-apple is used to isolate many but not all species of Phytophtho~.Lemon fruits
are used for the Phytophthoras that cause diseases of citrus (I 19). Planting susceptible plants in infested
soil is often considered a form of baiting. For details of baiting and other methods of isolation of
Rhiz4ctonia from soil see (25,261.
W. Direct &lation: (fungi already fruiting). If an organlsm is sporulating on diseased material, the
spores or fruiting bodies may with the aid of a stereoscopic microscope be picked up with a f i e needle,
micro-spatula, or loop. The spores can be streaked on agar or added t o 1-2 drops of sterile water on the
surface of acidified agar and streaked from the drops. Fruit bodies not oozing spores are usually
handerred through 3 or 4 adjoining drops of sterile water on a previously flamed slide to reduce
contaminants before being crushed. Spores thus released may be strcaked on agar. Frequently, fun@
arch as C m s p o r a , Septoria, and Kabatielo that are not fast growing organisms are isolated from the
host by direct isolation.
FREEING FUNGUS CULTURES OF BACTERIAL CONTAMINANTS
The contamination of funs& cultures with bacteria is frequently encountered, occurring in many
initial isolations as well as in the routine handling or increase of the organism. "Hidden" bacterial
art usually reveded by areaking the culture on a medium a p suitable for bacterial
growth - PDA or beef peptone agar. Here are same suggestions for purifying the fungus culture:
1. Make single spore isolations. particularly of fund that fruit on aerial hyphae.
2. Use bacterial inhibitors in medium, particularly broad spectrum antibiotics like streptomycin
and aureomycin.
3. Use low temperature incubation. It may allow the fungus to free itself of bacteria, at least at
the edges from where transfers may be made.
4. Use of a low pH if the fungus can tolerate it.
5. If a pathogen, in~culatethe host.
6. Use an agar cleansing technique (62, 112). This is especially good for fast growing fungi such
as&thium which cannot tolcrate a low pH.
a. Pour a 2.0-3.03water a p r plate.
b. Cut agar into several trimplar pieces.
c. Insert a piece of contaminated culture under each section.
d. Schmitthenner and Hi!ty (104) recommend shaking contaminated tissue-bearing spores of
Cercospora bericob or Seprurio glycines in a solution of streptomycin sulfate and
chloromycetin (both 25 rnp'l). Then add suspension of spores to agar surface and invert.
They also recommend isolation of certain species of Ph~~rophtl~oru, Aphunomyces and
Pythium via their zoospores by this inverted agar technique in conjunction with selective
media. Single spores can be located at bottom surface of petri dish without opening dish
and thereby decreasing the chances of contamination with nutrient agar.
e. After 48-96 hr bacteriaI-iree mycelium usually can be isolated at top surface of agar.
f. A J. Ullstrup (personal communication) prefers to put a small block of a nutrient agar on
top of water agar, allowing the fungus to grow into it. This is then transferred to another
plate.
g. Confirm purity by streaking organism on PDA or beef yeptone agar.
THE ISOLATION OF SINGLE CELLS

Isolation of shgle cells of microorpniuns, although often necessary to microbial work, may be
used uncritically. For example, as some fungi produce heterocafyotic spores, single sporing does not
guarantee genetic purity in these cases although successive single sporing should tend to do this (56).
Also, an isolate derived from a single cell my not be as representative of the original isohte of a species
u is a mass cell transfer.
Isolation of single cells, however, ensures species purity and is likely with most fungi and bacteria
to produce initial genetic purity. Obviously, there is no assurance of genetic purity in subsequent
generations as mutations may occur. If isolates are derived from single cellr, they should be numerous
a d come from various sources when information is sought on chvactnistics of the species.
Hyphal tip isolations are valuable in isolation from a mixture of clones but, as hyphal tips m y
have it large number of nuclei (101). genetic purity is not assured A piece of a r u o r blade inserted in a
g l ~ s s(68) or a wooden rod (130) used with a dissecting microscope is usually sufficient equipment to
Cut hyphal tips in agar culture.
Micromanipulators, costing from about $150 to several thousand an, when used by a M l e d
opentor, valuable in micrurgy and bngle cell isohtion giving peat precision and accuracy. h tbe
methods employed are determined by the brand purchased, no details are given here, but there is a
recent book on the subject (37). Also see extensive reviews on micrurgy and single cell technique (51,
52, 53). Isolation of microorganisms does not ordinarily require a lot of expensive equipment. Some of
the inexpensive methods are described here.
lrdrtlon of fungus spores

Because of their usual size, single spores and sporelings of fungi are generally isolated without the
aid of a rnicromanipulator, although various mechanical aids have been fitted on t o the Compound
microscope. These attachments are not necessary when using stereoscopic microscope with magnifica-
tions from 60x t o 120x.
With most methods, the spores are distributed from a drop of water on the surface of agar using a
loop, bent glass rod, or spatula, Avoid scarifying the a p r . Zoospores are easily injured and are more
safely distributed by tilting the plate to allow a dilute suspension to flow over the surface (45).
Micro-pipettes are also useful in distributing zoospores.
Hansen and Smith (46) separated spores by mixing 75-100 spores (the concentration of the
original suspension was determined by counting the number of spores carried in a 2mm loop) in 10 ml
of melted 0.5% agar. Three 90 mrn plates were poured with the 10 ml of agar.
If the spores come from natural diseased material they may be contaminated with bacteria. To
lessen bacterial contamination do one or more of the following:
1. Streak spores on as lean a medium as is necessary for germination, most fungi germinate on
23%plain water agar, acidified with 2-3 drops of 25% lactic acid per 100 ml of melted agar,
2. In the event of acid sensitive organisms, such as Pj.tlrium, Aphulanomyces, and Ply~tophr/tora,it
usually is easier to mass isolate the organism, purify, and then proceed t o single sporing. A
modified inverted agar technique as described in the section freeing cultures from bacterial
contaminations has been suggested for single spore isolation of difficult organisms from plant
material. For an alternative method of single sporing aquatic fungi contaminated with bacteria,
see (38).
3. Avoid excess moisture by pouring plates the previous day or using clay lids, and/or pouring
agar as cool as possible to lessen condensation.
4. Streak spores dry, a spatula as described by Ezekiel (41), Figure 4, is useful for this purpose.
Avoid digging into diseased material when collecting spores,
5. Use the TDO method, described subsequently.
Methods using the stereoscopic microscope
SLI (SeparateLocate-Isolate)technique
1. Spores are streaked as previously described.
2. If convenient, atlow spores t o germinate as they hold better, are more readily located, and save
wasted effort.
3. J m a t e a weU separated spore.
4. A disc of agar containing a single spore is cut with a biscuit cutter (65). Figure 5, or a
r n i c r v a r , Figure 6. Three to 4% agar holds together well.
5. A spear or a flattened needle i s used to transfer the disc to a petri dish or agar slant. 'Be sure to
avoid touching the disc or transfer tool to anything that might contaminate it.
6. The disc may be laid on its side on the agar slant, the spore side close to the test tube wall, to
allow checking of purity at intervals (41).
Modification of SLI technique for sinde sporing of rust spores (83), Puccinia recondita
1. Grow plants t o be inoculated in a room isolated from aII mst spores for 10 days before
inoculation.
2. Leaves are rubbed with frngen and sprayed with water.
3. Detach leaves and place in a divided petri dish containing 40 ppm of benzimidazole. The
divided dish is used to prevent the leaf from becoming submerged in the solution, a glass rod
my S J ~ S U ~ U ~ G .
4. Remove spores from a 10 day old pustule with a spatula-type needle.
5. Place spores in a drop of water on a glass slide and allow to dry.
6. Place petri dish containing leaf on the stage of a stereoscopic microscope.
7. U h g another microscope pick up a single spore using an alcohol sterilized glass needle. Be
sure the needle is free of alcohol.
8. Transfer spore to a minute drop of water on the excised leaf.
9. After inoculation, incubate the excised leaf overnight in the dark and then for 12 days at
1820 C with 1500 ft-c (12 hr day). If at all in doubt about contamination, run uninoculated
controls. A somewhat different technique for conidia of Erysiphe ciclroraccarunt is described
by Schnathont (106).
TDO (Touchdropoff) technique (This method recommended by Dr. Edward Butler in a personal
communication, 1933.)
1. One of these: glass needle, insect pin, size 0 is fine enough, or sewing needle is needed. The
latter two may be ground to a narrow'er point using a fine oil stone, They can be mounted in
the bore of glass capillary tubing (Heat seal or Pliabond' will keep them in place) in a wooden
dissecting needle handle, or in a match stick. Glass needles are easily made by raking 2 glass
rods or tubing 2 4 rnrn in diameter. Heat them in a flame until the ends start to stick together,
then withdraw them from the flame and pull them apart quickly. A microburncr will give finer
points.
2. Several rows of agar discs are cut with a biscuit cutter or a 2-3mm glass tube and left in place,
3. Sterilize glass needles by dipping them in fresh 70% alcohol and then either dry by streaking in
sterile agar or allow all the alcohol to evaporate. The fine points on the metal if flamed will be
gradually destroyed and will have to be renewed.
4. Pick up a number of spores on the end of the selected tool.
5. Attempt t o leave one spore per disc by lightly touching the surface of the agar with the
spore-iadened tool and indicate by some mark which discs have a single spore. Small squares of
boiled cellophane may be substituted for transfer to host surface (127).
6. Select the discs that have 2 or 3 spores and, using a sterile instrument, remove the excess ones.
7. Wait for germination, and after inspection trans'fer to petri dishes; or agar :'ants are described
in the SW method.
Methods using an ordinary light microscope
Light location method (After Prof. Xi. hloore in a personal communication)
I. Spores are streaked as previously described.
2. Remove all but the low power objective (10 x) of the microscope.
3. Locate a separate spore in the center of the low power field.
4. Raise the objective of the microscope and swing to one side.
5. flluminate through the substage condensers with a bright light source in a somewhat darkened
room.
6. Close the rnicro~copediaphragm to a small aperture.
7. The area of the agar in the optical axis will appear bright.
8. Cut around this area with a biscuit cutter.
9. Check agar biscuit and spore with a low power lens.
10. Transfer disc as described in method SLI.
P U o n d &made by FtaJoae, Inc

Attachments t o the microscope
-
Duley isolator described in Figure 7.
I. Spores are streaked as previously described
2. Flame biscuit cutter of isolator.
3. Slip isolator around low power objective.
4. Locate an isolated spore and center in the field.
5. Crank down to cut an agar disc.
6. After cranking objective up, biscuit should still be in agar.
7. Check spore and biscuit with low power.
8. Transfer disc as described in method SLI.
Larnbert isolator - It is essentially a brass plug fitted into one of the objective mounts, bearing a
centered biscuit cutter, about 314 rnrn in diameter (slightly less than diameter of low power
field). An old objective so equipped may also be used (70).
1. Spores are streaked as previously described.
2. Locate an isolated spore and center in the field.
3. Flame biscuit cutter; a microburner is desirable.
4. Rotate nosepiece of microscope moving isolator in place.
5. Lower the cutter t o makc off circle in the agar around the spore.
6. Check spore and biscuit with low power lens.
7. Transfer disc as described in n~ethodSU.
-
Keyworth isolator A machined isolator, commercidly available1, is shown in Figure 8. Because of its
low cost, less than S10, its ease of sterilization, and its precise bore of 0.5 mm, I have found it to
be one of the best attachments. It is screwed into an objective mount and is used as is the
Lambert isolator. The cutter head can be sterilized on the microscope with a microburner. A
medium contairring 3%agar is recommended (67).
Dilutionoil-slide technique - This method originally designed to isolate single cells of bacteria may be
used for fungal spores. It is described in the section on isolation of single cells of bacteria.
Jsolation of single cells of bacteria
Dilution agar plate method
McNew (78) established that 12-16 hr broth cultures of some bacteria when agitated, suspended
in cooled melted agar and poured, resulted in almost all the cells being separated in the agar. He states
that repeating the procedure 5 times, and picking single colonies after each dilution, resulted in a
prohibitive choice in favor of single-cell origin. To make this method work, one should determine
microscopically that the cells of the culture in question will separate upon agitation and remain so in
agar. Addition of 1 .WoTween 80 aids separation of bacteria. The colonies should also be picked from
plates containing few colonies and should be carefully watched and chosen to lessen the likelihood of
the mingling of adjoining colonies. This method is still challenged for a number of reasons (28,99).
Dilution-oil-slide technique
A detailed and extensive modification of Lederberg's technique (72) by DeVay and Schnathont
(28) is summarized. This technique can be modified t o isolate single fungal spores.
Equipment and materials needed:
1. SIides of Plexiglass, 7.5 x 2.5 crn and 3 mm thick. Scratch a grid of 5 mm squares on the surface
leaving a margin of about 5 mm2 They are sterilized in 7m ethanol and dried in a sterile petri dish
at 45 C. Before use, edge with a wax pencil t o keep oil on slide.
'Available from Ivor

Saint, 50 Cl;ucndon, SPA England.
ArrItbb horn the Fowr Co, Tucson, Arizona 85705.
104 Isolation
2, Dispense heavy mineral oil into a 125 tnl flask and sterilize by heating on a hot plate for 2 hr at
&out 200 C. A glass rod inserted in a cork nrakes a convenient applicator.
3, Draw capillary pipettes from glass tubing 2 mm 1D and 3.5 OD. Fire polish pieces 6" long, then heat
in the center and draw to give a capillary 4 to 5" long and 0.5 mm in diam. Then cut apart and heat
about 0.5" from the larger diameter section drawing to a smaller size in a 118" flartie. An eye
dropper with a small tip makes a convenierit burner. Capillary ends should be about 1" long and
have a bore of SO or 100 i~Plug t l ~ e ~with
n cotton and autoclave thern,
4, Rubber tubing 2' long, 3 mm ID and 4 mm OD control the deposition and recovery of drops.
5. A microscope, equipped with a 5-power objective and an eyepiece micrometer, is used when
depositing droplets on the slides. The microscope slide should be level to prevent movement of the
oil layer and the droplets.
6. Ton t o 12 hr old bacterial cultures grown irr 5 ml of nutrient broth. Make dilutions in broth
beginning with a wmplc withdrawn from thc center of the broth culture until a dilution yields 1 or 2
cells per microdrop.
-
Method:
1. Spread about 1 1n1 of mineral oil over the grid scratched on the top of the slide. This should be done
in a level place.
2. Position slide on a level mechanical stage.
3. Using a length of rubber tubinp extending to the mouth, take up about 50 p1 of the dilute bacterial
suspension with the 50 ,u bore capillary.
4. With the hand resting on the corner of the stage, position the tip of the pipette in a stationary lateral
position just above the oil layer and a square on the slide's grid. A hidl aperture objective (5X)
allows working distance and sufficient resolution to see and manipulate the tip of the pipette.
5. Dip the tip into the oil and with slight air pressure deliver a microdroplet.
6. Without moving the pipette, position another square by means of the mechanical stage and deliver
another droplet.
7. Under bright field (43X) focus through the droplets, which have settled to the surface of the slide.
They are about 50 p and spherical. If more than one cell is present per droplet, the dilution of the
bacterial suspension is adjusted t o give 1 cell per droplet, and a new series of droplets are deposited.
8. Droplets that contain a single cell are immediately recovered and transferred to a nutrient agar
surface using a capillary pipette with a bore of about 100 p.
9. Microcolonies should appear within 3 to 4 days.
SOME TOOLS USED IN ISOLATING SINGLE SPORES

Micro-spatula (Fig.4)
End flattened out of chromel wire gauge #24.
Biscuit cutter (Fig. 5 )
1. Heat chromel gauge $22 to soften (repeat heating several times while working, since working
hordens it).
2. Bend wire to an acute angle.
3. Flatten t o paper thinness, pounding will cause acute angle to open out.
4. Trim with sharp tin snips or shears so bottom edge is straight and at right angle to stem.
5. Cut tip to n x desired diameter of biscuit cutter (1-3 rmn).
6. Sharpen bottom edge with a fine file while supporting it on the edge of a hard surface.
7. Heat t o soften and let cool.
8. Wrap around a finishing nail or brad of suitable thickness. A headless nail can be partly driven in a
piece of wood. If tip of tool is as thin as it should be, this can be done with fingers or tweezers
(After M. B. Moore).
Some Tools Used in Isolating SingIe Spores (cant.)
MicroSpear (Fig. 6)
Flatten gauge #19 or #22. Then cut angle as indicated, Sharpen 011 oil stone. (After A. J . Ullstrup)
Darley isolator (Fig. 7)
1. The biscuit cutter is made as described on the preceding pagc and has a diameter of about I mrn.
2. Solder cutter to spring brass band lined wit11 soft cluth or plastic tape,
Fig. 4. Micrwpatula
Fig. 5. Biscuit cutter
Fig. 6. Micro-Spear
Fi& 8. Keyworth isolator (available from

the lvor Sdnt Co.. 50 Clarendon
Fig. 7. Dorley isolator Ave., Learnington SPA,England)
ISOLATION OF BACTERIOPHAGES
Isolation of bacteriophages, (modification of 23,43,54,64) with suggestions from DeVay (29).
1. Inoculate a flask of 50 ml of beef peptone broth with host bacterium (2 rnl of a 24 hr culture).
2. Add 100 g of coil or supernatant liquid from plant tissue. (Grind latter in a mortar or blendor
with broth.)
3. Incubate for 48 hr.
4. Centrifuge at 200 g for 15-30 minutes.
5. Filter suspension through sterile millipore filter (0.45 C( pore size) or a UF sinitered glass frlter.
(Or you may wish to kill bacteria by adding 0.1 rnl of chloroform to 5 rnl of suspension in a
stoppered bottle. Shake vigorously.)
6. Add an aliquot of treated suspension to freshly inoculated broth. If chloroform is used, be sure it
has settled out and none is transferred to broth culture.
7. After 24 hr, culture containing phage should be clearer than turbid controls.
8. Repeat steps 4, 5 and 6. Many prefer to omit the following steps and go to the soft agar
technique described later.
9. To demonstrate plaques, spread a 24 hr broth culture on solid nutrient a p .
10. Spread a smaller loopful of the Nter-sterilized suspension over the center portion of the area
coveted by the culture.
11. After 24 hrs phage-containing fdtrate is indicated by a clear area on the agar surrounded by
bact erial growth.
If unsuccessful d o the following:
1. Collect samples from where the disease occurs.
2. Repeat for a given sample steps 4 , 5 and 6, five or six times successively.
3. Adjust pH of supernatant liquid t o 7.0 or above before filtering.
4. Use the enrichment technique of C r o s s (23) as follows: Add a 48 hr culture of host bacterium
to 150 g of soil placed in a sterile glass jar, periodically mix for 48 hr, and follow procedures
listed above using decanted liquid frorn soil mixture.
To purify and quantitate phages use the soft agar technique (1,43). Use sterile technique throughout.
I. Dilute original phage stock lo4, l o s , l o 6 or other more suitable dilutions in 1% peptone
water. Can be stored at 4 C.
2. Fill small tubes with 1.5 rnl of beef peptone broth containing 0.5% agar. Maintain at 46 C.
3. Add 5 drops af 3 broth bacterial culture, 24 hr old, to small tubes.
4. Add 0.1 rnl of diluted phage suspension. (Note another 10 fold dilution.)
5. Mix thoroughly and pour immediately over the surface of nutrient agar in a petri dish. Surface
of agar should be dry.
6. Within 24 to 48 hr plaques, usually clear areas, will be distinct enough to count and to
characterize.
7. Individual separated plaques can be selected and their area rubbed with a glass needle and
transferred to a freshly (bacterial) inoculated plate of agar (23). Or you may wish to purify
by:
a. Scraping off the soft agar layer of a plaque.
b. Mixing with 5 ml of nutrient broth.
c. Centrifuge at 2000 g for 15-30 minutes.
d. Treat with chloroform as previously described.
How t o detect plant pathogenic bacteria in plant tissue using phages is reported in several reports (17,
64). improvements have been offered (9 1).
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108 Isolation
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Diagnosing the Causes of Plant Diseases
Introduction
Table 8. Diagnostic characters and associations o f same causes o f plant di-s
Table 9. Hosts m d references on culture, pests and diseases
Dbase Diagnosis Check List
Table 10, References which consider diseases of many hosts
Idcnti5cation of Fungal Pathogens (by George B. Cummins)
The Host Plant
The Pathogen
Monogmphs
The Host Index and Other Resource hlntedals and hlethods
Table 11. Culture collections
Quarantine Rcquiremants
The Preparation of Phnt Herbarium Specirnenq
Identitition of Plant Pathogenic Bactetia
Table 12. Some diagnostic features of genera of plant pathogenic bacteria
Identifying Nematodes and Diagnosing the D ~ s c ~ s w
e sh ~ c hThcy Cause (by Glenn Bergeson)
Utemture Cited
INTRODUCTION
It is often difficult t o determine the cause of a disease. Some of the different entities that cause
plant diseases may cause similar symptoms, upsetting the same physiologic processes in the plant. For
example, cenain fungi cause a wet rot of tissue, a symptom usually associated with bacterial plant
pathogens; genetic abnormalities and mineral defjciencies frequently cause virudike symptoms. Also,
several agents, successively or simultaneously involved, may be overrun by saprophytes; or infectious
diseases may be obscured or emphasized by climatic or culturd factors.
For diagnosis, it is helpful to narrow the areas of confusion by being acquainted with: (1) the
common diseases of the host in question, (2) the sorts of troubles peculiar to the locale, and (3) the
sorts of troubles expected at the particular time of year or under the prevalent weather. These facts
help one to determine the common ills, thus leaving time and effort to the more unusual. This
procedure should not encourage snap judgment and untested opinions.
It b basic t o know what is abnormal and to be able t o recognize the significance of signs and
symptoms. Knowing the common symptom types, distribution, and prevalence of diseases If used
Czmthurly, i s helpful (see Table 8). In Table 9 are references of the culture and diseases of most
economic crops. There are outstanding treatment of the diseases of some crops; others have o d y
Cursory or semi-technical compilations. General host treatments are included because they help to
integrate disease with the culture and other problems of the crop. To be a good diagnostician one must
know much more than the strict pathology of a host. The assembled team of experts is ideal but rorely
rsrlizcd
112
Chum of Plant Disuses 113
Table 8. Diagnostic characters a d associations of some causes of plant diseases.

CIpw G m d Referenm
Actinomycetes 1. Only one common disease - potato scab, causes (1 2,309,3 10)
corky lesions on tubers, several other root crops
are attacked.
Air pollution 1. Water soaking,necrotic streaks and spots, often (80, 137,212,256,
interveinal; marginal necrosis. Compare symptoms 334,347)
with photos of experimental plants found in
occompan~ingreferences. Dr. Ruth Clater
(personal communication) suggests viewing
thin sections.
2. Although there are differences in susceptibility,
many species are similarly affected.
3. Histoq of !roubles is helpful and with some
pollutants chemical tests of tissue and air are
useful.
4. Expose sensitive indicator plants and evaluate
symptoms.
Bacteria 1. Leaf spots often are watersoaked; may have (1 2,41,49,85,94,
bacterial ooze. 106,283,305)
2. Presence of bacteria in young infected tissue.
3. There are a number of bacterial wilt and gall
diseases.
Fungi (most common 1. Symptoms extremely diverse, difficult to (12,40,41,49,
plant pathogen) generalize. 271,305)
2. Often mycelium in the lesions.
3. Fmiting structures may be present.
4. Isolation from infected tissue may be
necessary; beware of secotlricYy orgat~isnu
developing on tissue killed by other causes,
Genetic abberation 1. Mosaic leaf symptoms similar to those caused (266)
by some ~~ and some deficiencies.
2. Disease does not spread in the field.
3. Appears in most environnients and locales.
4. Nontransmissible; often traceable to certain
clones or varieties.
Guttation injury 1. Tip and margin of leaf burnt. (78,149)
2. Usually a succession of warm days and cool
nights.
3. Soil abundantly moist and fertile.
Herbicide danuge 1. S y m p t o m diverse and depend on type of
herbicide, often with preemergence treatments,
injuries originate at roots. Symptoms may include
on roots,thickening, generalized necrosis extending
up the uem; on leaves, marginal necrosis, necrotic
and chlorotic areas, cupping and shoe string. There
may be also epinasty and rosetting. These latter symptoms
asw&ted with hormonal typc materials.
~crbicidedamage (cont.1 2. Pattern in field may conform to row treatment
of previous year's crop. Injury may var). with soil
type, topography and uneven application.
3. Examine other plants for sirnilar synlptoms.
figher plant parasites 1. Often stunting of or distortion of shoots. (12,40, 108, 109,
2. Presence of parasite on shoots or roots. 183,239,339)
insects and mites 1. Great variety of symptoms, Tissue (52)
malformation and "hopperburns" common.
2. Presence of known toxic insects or mites.
3. Plants usually recover after pest has been
eliminated.
Mineral deficiencies 1. Various symptoms but interveinal chlorosis (12,49,55,?02,
and necrotic spotting and marginal burning 2 8 0 , 2 9 ? , 3 15)
common (compare with photographs in
accompanying refcrences).
2. Symptomology in combination with one of
these: soil test, tissue test. Recovery after
injection or spraying with suspected deficient
element,
Mineral excess and 1. Leavts often have marginal necrosis (compare (140, 184,212,292,
spray damage with photographs in accompanying references) 31 5)
(see also saline soils) and dull brown spotting.
2. Excess mineral syrnptonls are not as diagnostic
as deficiency symptoms.
Nematodes 1. Plants often stunted and yellowed. (12,59,110,297,
2. Galls or lesions on leaves or roots: the latter 305)
may be distorted in other ways or be decayed.
3. Presence of nematodes with stylcts in or on
tissue,
Plant hormones 1. Usually profound distortions of leaves. (165,212,348)
-
2,4 D, etc. 2. Plants often recover.
3. Similar symptoms on different broad-leaved
species in the case of 2,4-D.
Saline soils 1. Obvious symptonls may be lacking even with (25,39,257)
considerable decrease in yield. Leaves, stem
and fruits are usually smaller than normal.
Leaves are characteristically deeper blue-green.
2. Damage usually occurs in arid or semi-arid
regions.
3. Presence of indicator plants that tolerate high
d i n e soils.
4. Test measuring electrical conductivity of soil
solutions is necessary to determine degree.of
salinity.
Toxic plants - 1. Midseason wilting of plants, alfalfa, tomato (8 1,170,333)
walnut trees and and potato are sensitive.
quack-grass, see also 2. Location of a walnut tree or its roots in the
toxic plant residues immediate vicinity, usually 50' or less affecting
establishment of stands of grasses, small grains
and legumes.
Toxic plant residues 1. Primary root of seedlings inhibited arld roots (237,238)
have surface lesions in some cases.
2. Excess plant residue in soil.
3, Disease often associated with heavy, poorly
aerated, waterlogged soils and relatively cool
temperatures.
4. Occurs on several unrelated species.
5. May be a gencral dccrcase in plant size.
Viruses 1, Often chlorotic and necrotic leaf patterns; (12,19,37,52, 136,
occasionally Icaf, shoot and fruit distortions 163,274,306,337)
(symptoms may be ~naskedat diffcrcnt tirtles
of season or disappear entirely aftcr shock stage.)
2. Disease often spreads in the field.
3. May be rindolnly distributed in the field or at
edges o f field near percnnial source of virus:
4. Often on a variety of sites.
5. Absence of known toxic insects.
6, Unless symptoms are clearly characteristic of a
known virus, transn~issionto indicator plants
and/or serological testing necessary.
7, With sap Iransmission, bcwarc of relying
exclusively on demonstration of local lesions,
a bacterium may bc involved (331).
Weather - frost injury 1. Correlate with local weather. (40,239)
2. Plants growing in areas of low air drainage
and on muck soil most affected.
3. Marginal and intervcinal leaf spotting; young
tender parts of plants usually affected.
4. Distorted growth occurs whcn buds arc injured
particularly true with woody plants.
Weather - winter injury I. Sides of plants exposed t o drying winds or strong (40,239)
sun often " f i r e d or killed,
2. Symptoms frequently delayed.
3. Low temperatures previous winter without sufficient
snow cover may cause considerable killing.
Weather - excess water 1. Correlate with weather and low spots in field. (23,239)
2. Particularly severe on heavy soils and high
temperatures,
116 Cauws of Plant Discaws
Table 9- Hosts and references on culture, pests md J i s e a s ~ .

Ngae (1 54,276) Guayule (SO, 247)
Abaca (3 18) Hevea (175,247)
Alfalfa (33,81,171,262) Holly (103, 1 11, 1 34,340)
Amaryllis (300) Hop (45,46,335)
ASIbIAL & HUSIASS (3,21,67) Horseradish ( 157)
Artichoke, globe ( 2 5 1) Insects (287)
kpon (1 16) Iris (1 82)
Avocado (344,345) Lcspedcta ( 1 32)
Bamboo (1 38) httuce (32,61,251,3 1 1,328)
Banana & Plantain (3 18) Mango (272)
Barley (31 , 81,285) Maple (1 74,206)
Bean (342) Mint (I 18)
Beet (61,251,311) Muskmelon (84,25 2)
Boxtree (323) Nematode (fungi that attack them) (70)
Brome (8 1,262,281) Oats(31,64,81, 185,281)
Broomcorn (296) Onion (1 56, 3 14)
Cabbage(251,311,313) Orchid (48, 152,231)
Cacao (324) 0RNAhlENTAL.S (20, 102,228,244,245)
Carnation (1 76) Parsnip (1 20)
Carrot (61,249.3 1 1) Peas (34 1 )
Castor bean (346) Peanut (8 1, 189)
Cauliflower (61,42,3 1 1) Pecan (243,259)
CEREALS(31,81,185,281) Peppcr (38)
Cherry (233) Pineapple (1 I , 65)
Chicory (25 1) Pinus srrobus (1 39)
Chrysanthemum (1 02, 150, 178) Poinsettia (2 16)
Citrus (1 64, 167) Potato(61, 160,311)
Coconut palm (7 1 ) Pumpkin (307)
Coffee (325) Red clover (8 1,98, 100, 171)
Corn (6, 168,163,281,303,304) Rhododendron ( I 8 1)
Cotton (S8,8 1) Rhubarb(61,251,311)
Crown vetch (I 15) Rico (82, 1 16,235)
CRUCIFIERS (157,211,252,313) Rubber (Hevea) (1 75)
Cucumber (42,61,252,311) Rye(31, 185)
Endive (25 1) Safflower (1 65, 166)
b r o l e (25 1) Seed (200)
Eucalypts (240) Snapdragons ( I 77)
Fig (68,172) Sorghum ( I 86,296)
Fish (269) Soybean (81,86, 153,187)
Fkx (81,226) Squash (307)
FORAGE LEGUhIE!5 (8 1,98, 171,262) Strawberry (8,209,246)
FRUIT (9,42,49,128,335) Stored grain (56,57)
Geranium (203) Sugarcane (93.13 1,144, 145,202)
Gladiolus (I 98) Sudan grass (296)
Globe artichoke (25 1) Subterrcan clover (221)
Crape (128,210,331) Sweet potato (1 27, 136,251)
cRAS!jES(72,81,171,281) Tea (I 05,125,263)
Cuu (292) Tobacco (62,141,193,213,332)
Tomsto (83, 188) VEGETABLES (42,61,311)
TREES (17,27,40,51,53, 124,133,218,239, Vetch (132A)
244,245,277,278,279) Watermelon (236)
Tall fescue (73,28 1) Weeds (267)
Tung (1 80,232) Wood decay (53.99)
TURF (72,104,143) Wheat(31,44,81,185,241,281)
Turnip (61,252,311) White pine (1 39)
A check list, which summarizes an approach to diagnosis, follows. Its purpose is to aid in the
systematic collection of the facts necesnry for an intelligent judgment. Related to this is the collection
of suitable specimens. Some suggestions are:
1. Get specimens showing young as well as older stages of the disease.
2. Get properly dug roots if there is any likelihood that the cause is initiated there.
3. Do not package specimens in tight plastic bags if several days of storage without refrigeration
is anticipated.
More details on the identification of fungi, bacteria and nematodes are given in rtparate sections.
DISEASE DI.4CNOSIS CHECK LIST

-
Plant Botanical name (origin and area of acbptation sometimes useful)
Normal appearance of species
Symptoms and signs of common diseases on plant involved
Symptoms
Internal as well as external; attempt to determine what part of plant affected fust;hidory
of symptoms
Leaves and stem(s)
Flowers and fruits
Roots (dig and examine carefully)
Signs
Presence of fungal fruiting structure (pathogenic types)
Mycelium in tissue
Bacterial ooze or bacteria in tissue
-
Insects mites
-
Nematodes do they havt stylets?
-
Higher plant parasites some are herbaceous plants that are attached only to mots
Other information
Distribution and location of disascd plants: (Pattern may help indicate cause, ep., topography
-
often related to frost and drought effects). Genetic aberration usualfy random distnlw
tion; often in new varieties; dotr not spread.
Are other species affected? If fo, may be noninfectious.
-
Ununral weather conditions flooding, frost, lightening, hail, sunscald, drought; drying win& are
causes of common trouble.
Culturd malpractices -too deep or too close cultivation, deep fdl, etc.
-
Presence of toxic p b t s are wahut trees or quackgrass in vicinity or latter abundant previous
YW-
-
To& residues excess of crop residues may affect seedlings.
-
Adverse chemical treatments m i m e of herbicides, fungicides and insecticides; d t used to meh
mow. (Previous year's treatmenu may caurc this year's troubles.)
118 Caures of Plant D i m s
SOP conditions
Fertilizer treatment
-
Nutrient levels of soil or tissue respond to spraying or injection with suspected deficient
elemen ts
pH -may make nutrients in soil unavailable, e.g., iron chlorosis
Soil type and site poor aeration, poor drainage, shallow soit
Saline soil (usually arid or semi-arid soils)
Air pollution
Smog areas
Near industrial factories
-
Gas leaks usually manufactured gas is more toxic
Activities of animals and man
Grower's remarks - (Question closely to get all the facts)
Table 10. References which consider diseases of many hosts.

Anom. 1953. Distribution, symptoms and control of some of the more important diseases. Pbnt Dis.
Rcptr. Supp 22 1.
Anom. 1956. Plant pest handbook. Connecticut Agr. Exp. Sta. Bull. 600.
Brandes, C. A,, T. M. Cordero and R. L. Skies. 1959. Conlpendiurn of plant diseases. Rohm and H a s
Co. Philadelphia.
Butler, E. J. and S. G. Jones. 1949. Plant Pathology, Macmillan, London,
Shurtleff, M. C. 1966. How to control plant diseases in home and garden. Iowa State Univ. Press. Ames,
Iowa.
-
U. S. Dep. Agr. 1953. Plant Diseases the Yearbook of Agriculture, U. S. Dep. Agr. Washington, D. C.
Walker, J. C. 1957. Plant Pathology. McCraw-Hill. N.Y.
Westcott, Cynthia. 1960. Plant disease handbook. DeVan Nostrand Co. Princeton, NJ.
IDENTIFICATION OF FUNGAL PATHOGENS

by George 8. Cummins
There is no single or simple way t o identify fungi or the diseases that they cause. Identification is
u d y easier when the disease occurs on an important economic crop bccause of the more complete
and readily accessible descriptions of pathogens and disease. Accurate identification of a disease
requires identification of the pathogen unless the disease is known so well that one may assume from
the symptoms that a particular pathogen is responsible. Such assumptions require a background of
experience that students rarely have. The procedure outlined here is that usually followed by plant
pathologists rand mycologists to identify a disease and the causal pathogen, confirming diagnosis by
d h s c symptomology.
The Host Plant. Identity of the host plant and its technical (Latin) name are essential because
they permit the uw of such short cuts as the "host index" or simply the index present in all books and
periodicals that treat of plant diseases. For crop and ornamental plants, the common or vernacular
name may be sufficient, but the Latin binomial is better because it is universal. If the "host approach"
to identirkation of the fungus is used, accurate identification of the host is required, otherwise an
incorrect answer is nearly inevitable. If the specific identity of the host plant is uncertain, the g n u s
may provide an adequate point of reference.
Knowledge of the normal structure of the host plant is desirable, otherwise abnormalities (signs
md symptoms) may not be recognized or normal structures may be misinterpreted. Some plants have
Chum of Plant Diseases 119
&in&, specialized trichomes, or conspicuous lenticels that may be mistaken for "pustules" or "sorim.
The tissue involved, e.g., phloem or xylem, is important and so may be the color of the affected tissue.
The Pathogen. Not all fungi produce fruiting structures readily, but many do. Classifications,
keys, and descriptions are mostly based on the morphology of the reproductive structures of the fungi
(mycelial characters (229, 230) and chemical tests (270) may also be used in some groups), If fruiting
structures are associated with diseased tissue, they may be sufficient t o permit identification. Such is
uarally the case with the smut, rust, powdery mildew, downy mildew fungi, and frequently with many
other pathogens. In the absence of fruiting structures, isolation and growth on specialized media and in
controlled environment may induce reproduction. Fulfillment of Koch's Postulates, or modifications
thereof, may be necessary t o establish the cause of the disease.
Given fruiting structures, (well described in (2,7, 26)) one would need answers to the following:
1. Is the mycelium nonseptate (a Phycomycete) or septate (an Ascomycete or a Basidiomycete)?
There are exceptions. Spore structure may make this question unnecessary.
2. Are the reproductive structures one of these common ones? (a) sporangia, zygospores,
antheridia, oogonia, oospores (Phycomycetes); (b) conidia on free sporophores (5loniliales),
conidia within pycnidia (Sphaeropsidales), conidia in a c e m l i (melanconiales), or ascocarps,
where the spores are born in asci (Ascomycetes); (c) basidiocarps (Hymenomycetes) or
basidia on the host surface (Exobasidium) or produced by germinating teliospores (Uredinales
and Ustilaginaceae) of the Basidiomycetes.
3. What is the detailed morphology of the sporocarp or sporophore and what is their relation to
host tissues?
4. Are the spores colored or colorless, smooth or sculptured, wptate or nonscptate, sessile or
podicellate, borne singly or in chains, etc.? How conidia are formed may have an increasing
value in separating species and genera. (1 95)
5. Using these facts, "key" the pathogen to the order or family in Martin (201) and t o the genus
in Clements and Shear (63), Bessey (26) or Sprague (281). Many plant pathologists have been
frustrated by this route. If the conidia are produced in acemli, it is only logical to start
keying in the blelanconkles (18, 26) or to check the host index for melanconhceous
pathogens. Other short cuts may be taken as you gain experience. If you know that the
pathogen is a rust fungus, you would go directly to Arthur (14) and Curnmins (76, 77); if a
smut, to Fischer (101). Most such monographs have their own host indexes. For pathogens
other tlun rust and smut fungi - of grasses, one might consult Sprague (281) immediately.
6. Monographs. You may continue to identify the fungus by its morphology, using increasingly
specialized monographs - for Alterturia (155, 227, 302, 330), Arrhrinum (Y7), Aspergillus
(253), AphP~tottytces(269), Ceplulosp9rium (88, 293), Ceratocysris (1 46, 336), Cercospora
(60), Choetomilinl (S), Cochliobolus ( I 96), Colletotrichum ( l S), Corynespow (96), Curvrtbria
(96), Cylindrocarpon (35), Cylirtdrocladium (30), Diaporthe (321), Enronwphtltora (147),
Fusunirml (34. 1 1 1,207). Gloeosporiirnr ( 13), kfeterusporiurn (1 5 l), Hj~poxylon (2 IS),
Monoclraettrr (1 19), Penicilfium (254), Perunspom ( 122), PestaQtla (1 19), Plgvsodcrnza (I S8),
Phytopltthora (301, 319), Pleospora (322), P~tltirrrrl (205, 214, 320B), Sclcrospora (320A),
Selenophoma (282), Stsn~phylilirn( 2 2 7), Sy~tcliytrium( 1 59), Taphrirta (2 17), Tltiekzvia (36),
or consult a host index t o fmd what species parasitize the host that you have.
The Host Index. The Index of Plant Diseases in the United States (306) provides a good example.
Host plmt families are arranged alphabetically, beginning with Acanthaceae and ending with
Zygophyllaceae. Genera arc alphabetized under each family and, in general, the native species are
grouped under each genus by geographical regions. Introduced species are grouped separately. There are
regular indexes to the families and genera of hosts and to common names. Under each genus and under
ocrtlin species are listed, alphabetically, the pathogens often with a statement as t o type of disease, e.g.,
powdery mildew or the part of plant attacked, and the states where each has been recorded.
' -
A ,ant publicrlion Toussoun. T. A. and P. E. N c h n . 1969. It pictorial guide to the idcntificatlon of Fuwium
species. P ~ MState Univ. Prcu. Univ. Park. Pa.
120 Causes or Plant Diseases
A less professional but often expedient approach is the "matching method." For example, if the
disease occurred on a leaf, the leaf pathogens recorded in the host index are compared with the
organism involved. Descriptions of the unfamiliar organisms listed may often be found in a treatment
a& as S p r a p e (281), Besuy (26) or Alexopoulos 17). If not thereIVtheDictionary of the Fungi (1)
may reveal what group the fungus is located in and often cite a monographic treatment. Your task is
much easier if an economic host and a common disease are involved, as well-illustrated details of fungus -
and symptoms usually are available.
After determining the genus of a pathogen, one refers to the host index to find what species of
that genus are recorded for the host plant. Next, the morphology of the pathogen in question is
compared with that of the species recorded in the host index. This may be done by collsulting Saccardo
(260), or specialized monograptis. A valuable guide to the literature of specific fungus pathogens is
Moore (220). If the Commonwealth Mycological Institute continues to publish descriptions of
pathogenic fungi, initiated in 1964, they will make an extremely useful reference. In most situations
these procedures will suffice.
Using Illustrations, It may be less "scientific" to attempt identification by looking at pictures,
but illustrations ae made to be used and much can be learned rather quickly by using them. Genera rnay
be more readily recognized from illustrations than species because distinctions are greater. But one
needs t o be sure that the fungus rei~llycorresponds to the illustration. A visual comparison should be
followed by referring to the description of the fungus and to the symptoms 01the discase that i t causes.
If you have access t o an herbarium, or if the curator is willing to send specimens (a list of herbaria
may be found in (179)), you may be able to compare with previously identified specimens or with
living cultures available from type culture collections (Table 1 I),
The Unusual Fungus. Occasionally routine procedure brings no satisfactory answer, The fungus
may be:
1. Known but previously reported only on other hosts. If a monograph of the genus is available,
compare the morphology of the fungus with that of the species described in the monograph. If
there is no monograph, solicit help from someone with special competence in the particular
group of fungi or follow the procedure detailed under number 3.
2. The pathogen may be recently introduced from some foreign area. Most areas have literature
and descriptive manuals. If these are not available, you may again consult some specialist or
follow the procedures under number 3.
3. After routine sources of information have been consulted without success, the procedure to
follow is about as follows:
A. If there is a world-wide monograph of the genus, you may assume that it is dependable. But
because of publication tine, subtract five years from the date of issue and start the
checking accordingly.
B. Consult Saccardo "Sylloge Fungorum" (260), using first, of course, the index volumes.
Descriptions are provided in the "Syllogc Fungorum."
C. Consult the socalled Petrak "Lists" (242). These publications list new species, the host,
country of origin, and the places of publication. Descriptions are not included. For theu,
the original publications must be consulted. The Petrak "Lists" cover the period
192Cb1939, thus begin before the date (1931) of publication of the last volume of "SyUoge
Fungorum."
D. Consult the Index of Fungi (lo), 1940 t o date. The Index is a continuation of the Petrak
"Listsw and is now issued twice each year.
E. Consult current issues of periodicals in the fields of mycology and plant patholow for the
past 12 months or more, abstracting journals, e.g., Review of Applied Mycology, and lbts
of titles, c.g., The Taxonomic Index.
Table 11. Culture collections.' (Some of these laboratories published catalogs.)
(All requests for pathogenic cultum must be cleared
through the Plant Quarantine Division.
See below for regulations in the United States.)
American Type Culture Collection ~ntcrnationalCollection of Phytopath~genj~

12301 Parklawn Drive Bacteria (ICPB)
Rockville, Maryland 20853 Department of Bacteriology
Centraalbureau voor Schimmclcultures University of California
Javalaan 20 Davis, California 956 1 6
Baarn, Netherlands Japanese Type Culture Collection
Cento de Cooperation Cientifica para America Nagao Institute
Latina de la U.N.E.S.C.O. Kitashinagawa, Tokyo
Bulevar Artegas 1320 Japan
Casilla de Correos 859 National Collection of Plant Pathogenic BacteQ
Montevideo, Uruguay Plant Pathology Laboratory
Commonwealth Mycological Institute Hatching Crcen
Collection of F u n y s Cultures Harpenden, Herts, England
Ferry Lane Northern Regional Research Laboratory
Kew, Surrey, England Northern Utilization Branch, USDA
Forest Products Research Laboratory Peoria, lllinois
Princes Risborough
Aylesbury, Bucks, England
Institute for Fermentation
-
454 Juso Nishinocho, llignshiyodogawa-ku
Osaka, Japan
The New Species. If you are convinced that the disease or the fungus or both are new and you
wish t o publish, acquaint yourself with the following publications (4,5, 29, 142,54,286). In as far as
possible the conclusion that a species is new should be based on a study of variation and variability of
roveral isolates or collections.
I For other culture coUcctions see Cktk, W. A, and W. Q. Loegcring. 1967. Functions md maintcnrnce of(
m l t u n colkstion. Ann. Rev. Phytopathol. 5:3 19-342.
I. C a m Out Koch's Postulates or, for obligate parasites, acceptable modifications thereof. C r o s
inoculations t o other hosts are highly desirable.
2, When describing a new species, follow the stipulations of the International Code of
Nomenclature (29), noting especially that:
A. The description must be in Latin. Two references (54, 286) are helpful. An English
description may be provided but is not required. If color is significant in the description of
the organism (in the speciation ofSrrvptunyces it is an open question (248)) consult color
standards (79, 92, 199, 224, 258, 308). An objective measurement of color using a
reflectance attachment t o a calorimeter may be vrrluable (197). The dictionary of colors
(224) is said to have plates that are light resistant and to contain a larger number of colors
than Ridgway (258). To obtain precision in terms check with standard treatments (2, 275,
295), as terms such as flask or bottle-shaped are usolcss.
B. A type specimen must be designated and it must bear the "perfect" or sexual state, unless
the pathogen belongs to the fungi It~~pcrfecti or the Phycomycetes; in any case, the state
that is described. (If an imperfect fungus, vigorous attempts to obtain the perfect stage
should be made.)
C. State tho place where the type is deposited, e.g., The National Fungus Collections (22).
Type specimens should be deposited in established herbaria where permanence is reason-
ably assured. A properly labelled and dried specimen is necessary. For details see the
section on preparation of plant specimens. Accompanying the specimen with drawings and
photographs and in some cases microscopic mounts is desirable but not required.
QUARANTINE REQUIRE>IENTS
United States Department of Agriculture
Agricultural Research Service
Plant Quarantine Division
Washington 25, D.C.
INSTRUCTIONS TO IMPORTERS OR RECEIVERS OF CULTURES

The Federal Plant Pest Act of May 23, 1957, prohibits the movement of any plant pest from a
foreign country into or through the United States, or interstate, unless such movement i s authorized
under a general or specific permit issued by this Department. lncluded in the meaning of the term
Y
plant pest" are nematodes, bacteria, fungi, other parasitic plants or reproductive parts thereof, viruses,
or any similar organisms or infectious substances which can cause disease or damage to plants or plant
products.
It is suggested that persons contemplating the importation or interstate movement of any plant
pest as defined above (including cultures thereof) or of nonpathogenic organisms, follow the
appropriate procedure outlined below:
Importation or Interstate Movement of Plant PATHOGENIC Organisms
A. Apply, in advance of importation or movement, to the Plant Quarantine Division at the
address shown above, for a pennit and official shipping labels to authorize entry or movement
of the material concerned. Application may be made on the attached form (PQ Form 26) or
equivalent information may be furnished by letter or otherwise (See 111 for exceptions).
0. Secure advance approval from the State plant quarantine official of the state of destination.
Evidence of such approval may be furnished in Section B of the application form or my be
submitted separately by letter or other written communication.
CIum of Plant Diseases 123
C. Instruct foreign shippers to forward cultures or organisms by either air or surface mail, using
the official green and yellow labels furnished with the pennit, such labels to be used in
accordance with the instructions appearing on the reverse side thereof. For interstate
shipments a different type label will be furnished for use on shipments forwarded by my
means (mail, express, freight, etc.)
11. Importation or Interstate Movement of SON-PATHOCENC Organisms
A. Inform the Plant Quarantine Division, in advance, of the names of the organim concerned
and the number of cultures to be imported or moved interstate so that official labels may be
furnished as a courtesy to effect prompt delivery of the parcels. The application for permit
form may be used for this purpose.
B. Instruct shippers to forward the parceh using the official labels supplied by the Plant
Quarantine Division. This is important since inspectors at ports of entry, post offices, and
other shipping points cannot always be expected to be able to distinguish between harmful
and innocuous organisms. Shipments moving without labels may be subject to delay in
deUvery while the pest status of the organisms concerned is being determined.
Ill. Regulations Enforced by Other Federal Agencies
A. lnterstate Movement of Certain Plant Pests under Regulations Enforced by the Plant Pest
Control Division
The interstate movement of the following plant pests is regulated under quarantines enforced
by the Plant Pest Control Division of this Department.
1. Puccinio grominis, the black stem rust of small grains.
2. Cromrtium ribicob, the white pine blister rust.
3. Hcterodera glycines, the soybean cyst nematode.
4. Witchweed (Strip sp.), parasitic plant causing a serious disease of corn and other members
of the grass family.
Persons contemplating the interstate movement of any of the above listed pests should first
contact the Plant Pest Control Division in Washington, D. C. for a permit or other
authorization as required.
THE PREPARATION OF PLANT HERBARIUN SPECLjiENS

The principal objective in the preparation of plant specimens for herbarium use and storage is to
dry the plant material quickly to prevent molding and to minimize discoloration. In most cases an
almost equally important goal is that of preserving the plant in readily recognizeable condition,
Adequate equipment is simple, consisting of: 1) a plant press and 2) a source of heat.
1. The Plant Press consists of a "Dapood Sandwich" of a folded sheet of newspaper between 2
blotters and this unit inserted between two ventilators of cormgated box board. Several such
units are assembled between rigid, usually wood, coven. Thus, starting from one cover the
sequence would be cover, ventilator, blotter, newspaper, blotter, ventilator, blotter, newspaper,
blotter, ventilator, etc. through to the other cover. Two straps or ropes rue used to tighten the
press.
2. Plants or plant parts an arranged inside the folded newspaper Beet with sufficieat care that most
leaves, flowers, etc. will be flat and not variously folded. The newspaper is then p b d between
the blotten and the press tightened Plant disease symptom material or specimens of parasitic
hngi an handled in the m e manner. Uhcn dl the material to k dried is in the press the press
ready for drying.
3, Heat Source may be light bulbs, the run, or drying ovens of vaiious sorts. Satisfactory heat is
provided by. two 100 watt bulbs above which the press may be plucd. The press should be
oriented ro that heat from the bulbs (or otller source) riser ttsough the corrugations of the box
board ventilators, thus drying the entire press more or less uniformly. The edge of the press may
be as close as two or three inches to the bulbs without danger. Turning the press occasionally
speeds drying.
4, If no heat is available most plants may be dried if the damp newspapers, blotters, and ventilators
are changed for dry ones about three times ax 24 hour intervals.
5. A standard plant press is usually about 12 x 16 inches and blotters of this size are sold by
botanical supply firms. This is the same size as standard herbarium sheets, However, if one is
collecting parasitic fungi, and hence only parts of the host plant, a smaller press is more
convenient to carry in the field.
6. When 'the plant is dry it m y be "mounted" on a herbarium sheet by gluing it to the sheet or
attaching it with adhesive tape or paper. Scotch tape is not satisfactory f i r long-term specimenr
Leaf-spots, rust fungi, smut fungi, etc. may be, and usually are, placed in paper packets. The
packets may be glued to standard herbarium sheets or some collectors fde them in drawen like
index cards. For additional details on Herbarh procedure .ee Saville (264).
IDENTIFICATlON OF PLANT PATHOGENIC B.4CT'ERIA

Identifying a bacteria1 plant pathqen without using host specifidty is often difficult as most
species cannot be separated by bacteriolu@cal determinative tests. Bacteri31 diseases of economic hosts
are usually well described in publicatiorcr on diseases of the host or in texts on bacterial diseases (85,
283). These diseases, particularly if bacteria are detected in the tissue, often can be identified by host
symptomology. Host reaction is heavily relied on for identification and edablishment of species by
many plant pathologists. Criticism of this use of host specificity are based on the claims that
pathogenicity is often unstable in culture, is changed by host passage, may k overlapping and is usually
not adequately determined. In addition host specificity is not acceptable for species differentiation by
the nomenclatural code (148,286).
Without becoming involved in a discussion of the classification of bacteria, the ability of
bacterium to produce disease is usuaIiy of major importance to the plant pathologist. This unique
ability needs to be recognized at sonv level in classification and should receive the care and
standardization that are demanded of the usual bacteriological determinative tests. Prior water soaking
of host tissue (265), of prior light intensity (265), of temperature (1 14) and of concentration of
hoculum (1 61, 162, 190) can influence qmptomology and consequently its significance. Billing (28)
emphasized careful interpretation of host reactions by stating that, ...'*lesion tests are not pathogenicity
tests as they do not demonstrate the ability of the organism to infect a plant and cause a progessive
disease, but merely show that it i n damage and produce a characteristic lesion in certain plant tissues."
Admittedly this may take a degree of professional competence but the m e is often true of
biochemical tests, As the ability to muse a disease is neither immutable nor highly mutable for all
bacterial plant pathogens, cultures shotlld be properly preserved to facilitate genetic stability and
Purity. The claim that pathogenic abililies are readily changed by host passage (89) has not been
~bstantiated (190, 265), and the selection of plant pathogenic ixrlatn from m e saprophytic
hdontonas species (289) may also be a misinterpretation of symptoms (1 83).
Bacteriophage sensitivities (234), vrological reactions (290), and biochemical tests are sometimes
helpful in identifying species. There are a number of species, Psarclomnas wlonucearm ( 223hR
Phose~firnlb(123), Pectob~cteriumcmotor9oorum (1 12), Xnnthomow vesicbtorh (222), that are
9uickly identified by serological tests using n p from infected plants. Screening out of nonpathogenic
Gum of Plant Diwws 125
organisms by the host may contniute t o success, as there are often cross reactions between species and
in some cases between different genera of bacteria.
Of the serological techniques the fluorescent antibody and bentonite flocculation tests require
little preparation of infected host material r i d are rapid (222). Microagglutination tests required more
preparation of infected host sap, and gel diffusion takes more time for the reaction t o occur (123,223,
The latter, as it distinguishes different antigens, is suggested for more definitive and taxonomic studies
(192, 225, 265). Much of the early serological work used agglutination tests and probably needs
reevaluation.
P h a p reactions help identify Xanrhomonas vesicuroriu (91, 192) or detect bacterial pathogens in
wed (Xantltonwnus phaseoli (3 17 ) P. atrofuciens (294)) or in other plant material (Coryrrebacterizrm
Lrsldiosurn (69)). Phage typing can also identify ecotypes in epidemiological studies (75). As phages are
not species specific (they may be narrower or broader in host range than a particular bacteria species
(289, 290, 291)), they are used in sets. Individually they may not distinguish saprophytic species from
pathogens (28,289).
Biochemical and physiological tests, although stressed (43), seem to be of little use in separating
species in Xanrhomonas (90) and Pserrdomnnas (298), the most populus of plant pathogenic genera,
They are of modest help in separating genera.
Suggested procedure for identifying bacterial plant pathogens.
1. After isolation purify the organism by successive single colony transfers.
2. Immediately preserve the organism by lyophilization or other acceptable means.
3. Characterize colonies on PDA and beef peptone agar, particularly'in regard to color and rate of
growth.
4. Determine gram reaction and cell morphology.
5. If possible, determine arrangement and number of flagella (255). See Chapter 8 on
microscopic technique. Carefully interpret preparations whether viewed with the light or
electron microscope.
6. If original host symptoms are sufficiently clear, compare with those described in available host
or bacterial disease treatments. See list in Tables 8 and 9. For many purposes, diagnosis may
k complete.
7. If step 6 is negative or confirmation is needed, inoculate with several levels of inoculurn
Suspensions of 10' and 10' are recommended (162, 183, 190) with and witllout prior water
making,and at two temperatures. Age of the host, variety and its condition also should be
considered.
8. Perform pertinent bacteriological determinative tests durir.g standardized procedures (66,74,
273), and with other information identify See Table 12 for diagnostic features of
phytopathogenic genera.
9. If lacking host treatments, attempt to find species in Bergy (43) and Elliott (95) relying on
pathogenicity.
10. If step 9 is negative, consider species occuring on related hosts or those organisms that produce
a similar symptom on other hosts. You may have an "old species" on a new host.
11. If step 10 reveals the chance of a species previously unreported on this host, inoculate
additional appropriate hosts and compare with inoculations of authenic cultures obtained
from culture collections (Table 11). To completc comparison, it may be necessary to
determine reactions with phage and antiserum and to run additional determinative tests if an
"old species" is proved to be on a new host report after a thorough literature search.
' S u m that you include organism with known reactions for evaluation of test (107).
128 Causes of Plant Diseases
12. If no likely species is revealed by considering Berg, (43) and Elliott (95) in steps 9 and 10, or
11, consult the indexes of the Review of Applied Mycology from 1955 to date. Look for new
species of the p n u s and new bacterial diseases of the host in question. tf reports are located,
d o step 11.
13. If no likely species is revealed by the literature search of step 12, you apparently have a new
taxon. You may wish to consider a varietal or forma status if pathogenicity is the only
distinction. For example, Sabet (26) included in X. pltaseoli a number of bacteria that caused
leaf blights of different legumes, designating most as forma spccialis.
The following additiol~aladvice is offered before a "new" species is described.
Consult a specialist of the genus. If he agrees that the isolate is 3 new taxon or his reasons for
disagreeing are unsatisfactory, fulfill the requirements of the International Code of Nomenclature
(148). It would be desirable to describe procedures and conditions of inoculation and disease
developmetlt. Include tho results of inoculation of a variety of hosts within the family of the original
host and a number of other families. Also attempt to characterize the orginistn as fully as possible in
relation to its morphological, phage, serological and biochemicd properties. If you are not competent
in these areas, attempt to cooperate with someone who is.
IDENTIFYING NEMATODES
AND DIAGNOSING THE DISEASES WHICH THEY CAUSE
by Dr.Glenn Bergeson
Positive diagnosis of nematode injury on a plant showing typical symptoms is most easily made
when the nematode can be found in or around the diseased tissue. This will normally occur if the
nematode is an end- or semi-endosporastic type. Members of this type that migrate in the plant will
often be found midway between healthy and diseased tissue. If the nematode is non-migratory, it will
be in the diseased tissue which, in this case, usually consists of modified rather than killed cells.
Nematodes within plants can usually be detected by carefully teasing apart the diseased tissue
under the binocular microscope. Non-migratory nematodes enlarge to about the size of a pin head and
have distinctive shapes that are helpful for identification. Migratory nematodes retain their filiform
shape and are harder to identify.
Diseases caused by ectoparasitic nematodes are considerably harder t o diagnose because the
association between the plant and nematode is circumstantial, i t , , nematodes are found only in the
roil. Rarely do they adhere t o root surfaces that are pulled to be examined. The presence of
microscopic root lesions gives some support for a pathogenic relationship, but is not conclusive
evidence. Even if pathogenic nematodes are found in the soil, it is dangerous to make any assumptions
if more than one plant species is present in the immediate area.
Nematodes collected from either the plant or the soil should be examined for the presence of a
stylet. If abxnt, they can be eliminated as pathogens. If present, the next step depends on how much
taxonomy the investigator knows. A person with a course in plant nematoloa should be able to key
?ny plant parasitic nematode t o genus. In many cases, this is far enough to give the worker a hint as to
the pathogenic potential of the nematode. Adequate keys or descriptions can be found in Thorne (297)
or Goodey's Soil and Fresh Water Nematodes ( I lOA). If species identification is desired, the specimens
should be placed in a 5% fortnaldehyde and sent to a nematologist.
After identification has been made, it is advisable to search the literature to find what is known
about the relationship of the nematode and the host. A nematode may parasitize some plants but cause
little or no injury, yet in other plants it may be a high grade pathogen. The nematode may also be doing
its major injury by predisposing the plant to attack by other pathogens either by wound openings or by
tnodifying the tissue t o make i t a better substrate for other pathogens. One should aim be aware that
environmental factors such as soil nutrients, moisture, texture and temperature can influence the
Wcrity of nematode diseases.
of Plant isa am
~ a u m 129
Host range listings for nematode species are available for only a few of the well-known species. A
partial host range of economic hosts can be found in (59, 110). For Little known species, it is often
necessary t o find the original species description t o get some idea of its host range. One disadvantage of
using host ranges and host indices is that nematode taxonomy has not yet stabilized. Many important
goups are undergoing almost yearly revisions; consequently, listings older than 5 years could be
misleading.
LITERATURE CITED
Adair, C. Roy et al. 1966. Rice in the United States: varieties and production. U. S. Dep. Agr,
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Increase of Inoculum
Intductlon
Facton Involved in Increase of lnoculum
Physical Environment
Temperature
Aeration
Radiation
Table 13. Some fungi that require or sue stimulated by "Light" to sponrlatc (reported since 1959)
Media
Spedal Techniques
Various Blter paper techniques
Cellophane technique
Ground and Dried Inoculurn
Ways of obtaining large amounts of inoculum
Misecllaneous
Table 14. Check list for inducing spwlation of fungi
Incrurlng Obligate Parasites
Increase of Specific Fungi
Table IS. hfedia and procedures t o induce utsual reproduction of Pyrhfum and Phyrophtkora
Table 16. Ways o f producing asexual stages of various species of Pyrhium and Pityraplirhora
Literature Cited
INTRODUCTION
Increasing inoculum means producing large numbers of infective units, usually spores in the cast
of fungi. Although mycelial fragments may be substituted (21, techniques for their use are leu
quantitative than those employed with spores. They are usually less readily separated from the medium
on which they are grown, may give a different disease reaction (120) and be l e u infective (267).
Mycelium i s often used as inoculum for soil-borne diseases and may be stiperior to spores.
Perhaps more consideration should be given to the quality than t o the quantity of i n ~ u l u mthat
a method and medium produces because the medium may significantly influence viability (52h
longevity (52), virulence (130, 230, 272, 310) and other physiolo@4 aspects (52, 222). A h , the
environment under which the organisms are increased can be important for both culturable organ*$
(140) and obligate parasites (125). Another consideration is the growth period, particularly for
bacteria, as they may die rapidly on some media. For example, species of Xantltomonus are more
short-lived on beef peptone than on PDA,and the reverse is true for PsezIdomorurs (32).
bssening the amount of media and culture products mixed with the inoculum is recommended,
as the addition of nutrients may influence subsequent host-pathogen relationships (1 I , 287) and offed
the behavior of the organism in soil (1 52). A cellophane agar technique is valuable with nompotulatinl
cultures, as it avoids the blending of agar with the organisn~s.It is described later in the chapter. Using
on inert material or one low in nutrients such as perlite, vermiculite, or sand-soil as a f i e r in a nonagar
medium lessens the nutrients when the inoculum and substrate are mixed with soil.
FACTORS INVOLVED IN INCREASE OF INOCULUM

The isolate t o be increased should be virulent and, if a fungus, usually able to sporulate. These
properties can be assured by proper preservation or recent isolation from diseased material. Old cultures
that no longer sporulate should be discarded. Selection of sporulating isolates or within cultures for
sporulating types, as accomplished with Cercospora kikuchii (141), may be useful. Transfer of spores
during increase of inoculum tends t o maintain sporulating ability which is often associated with
pathogenicity. There are diagnostic media for some plant pathogenic bacteria that help t o distinguish
virulent from nonvirulent isolates by colony and physiological characters (76, 148). Such media for
fungi have been largely unsuccessful. An elaborate procedure for increasing inoculum, such as proposed
for the fermentation industry to prevent undesired changes in the culture (171), is ordinarily
unnecessary but may be considered when the organism is hi&ly variable ,and/or if a large amount of
inoculum is needed.
The statement by Lilly and Barnett (170), 'Thus there is no universal set of external conditions
which lead t o fructification in all fungi," is amply documented (13,106, 170). A certain amount of
experimentation may be needed t o induce sporulation even where there is literature on the same or
related species under study. Some general recomtnendations and methods relating to them given below
should aid in an orderly approach to the problem. In addition, details for increasing specific pathogens
and methods of obtaining the sexual stages of some fungi are given, Sexually produced spores are
usually not needed for inoculum as most perfect organisms produce asexual spores more readily and, if
not, mycelium may be adequate. The production of the sexual stage, however, may be essential in
genetic, taxonomic, and epidemiological studies. Homothallism is perhaps commoner than hetero-
thallism in the fungi (234), but mating of isolates, particularly those of different geographical origins
(274), should be considered. Often numbers of perithecia in homothallic strains can be increased with
resultant hybridization by applying conidia of a different isolate to a full grown culture (132). Flooding
or atomizing with water alone hastens and increases perithecial formation (132).
PHYSICAL ENVIRONllENT
Light, temperature, aeration and moisture are often not measured or'precisely contrulled when
inoculum is increased because adequate inoculum is obtained with ordinary procedures and conditions.
For example, "grown at room temperature" implies n o measurement, little control, and that constant
or fluctuating temperatures are suitable. But reports indicate the importance of temperature, light, gas
content and moisture on sporulation of many fungi.
Temperature. Fungi spomlate within narrower ranges of temperatures than they grow (43,106,.
170). In relation t o growth-temperature curves the respons is variable, some sporulating towards the
maximum temperature permitting growth, others toward the minimum, and still others about the
optimum (43, 106, 170). Occasionally fungi sporulate best when grown at a temperature optimum for
growth followed by transfer t o a substantially lower or higher temperature. It is impossible to
characterize taxonomic groups in their response t o temperature although there are a number of
k m y c e t e s (Clathrospom diplospom (27 1), Pyrenophora bromi (39), Pleospora pltaeocontes (86), P.
tdchostoma (307), V e n t u inaequalis
~ (146, 246, 247)) and particularly many of the Sclerotiniaceae
(93,94, 95) that form perfect stages only after prolonged exposure tb low temperatures. Most species
of &thium and Phytophtliom form and release zoospores when the temperature under which they ore
&Qwn is decreased.
Increase of lnoculurn 143
Caution should be exercised for the following reasons when applying published temperature
rccornmendations:
1. Isolates within a species may respond differently t o temperature.
2. Media may affect response to temperature.
3. Light and temperature may interact (discussed in the section on radiation).
4. Temperature of incubation may vary with the amount of solid media as the latter will influence the
degree of transfer of heat of respiration.
Extrapolation of temperature response in artificial culture t o field situations also should be done
cautiously because :
1. Temperatures in the field fluctuate much more than in the laboratory.
2. Effects of temperature on sporulation may be as profound as they are on growth (274).
3. Biotic factors of the environment may influence an organism's reaction to temperature.
Aeration. Adequate aeration is critical in determining mycelial yields and metabolic products in
deep tank fermentation and is probably as important in liquid cultures in the laboratory (174). There
are few reports of the influence of aeration on sporulation on solid media. Some reports indicate:
decreased sporuiation of Pirimlo* otyzac due to accumulation of ammonia (1 13), decreased conidial
and sporangial production of Ciwatrephora cunrrbitanrrrl (IS), and sporophores of Schizoph)~llum
commune (220) due to carbon dioxide. Low concentration of O2 probably decreased sporulation of
Mumr hiemalir, Cylindrclcarport radicicola and P a p ~ b f i aaru~tdi~lisa(300). k c k of aeration decreased
the numbers of perithecia of Vctttrrria itueqttalis (248) and conidk of Borr)tris citterea (209). One of
these studies clearly showed the effect of relative humidity independent of gas content (14). Scarcity of
information prevents generalizations on whether sporulation is routinely inhibited by lack of aeration
in culture. Most studies have dealt with the effect of constant and artificial gas environment on growth
(203, 269), and they have shown differences in C 0 2 tolerance and oxygen requirements for different
funpi. One would expect sporulation to be more affected. With the availability of gas chromatographic
instruments, atmospheric contents of small containers can be readily determined. Also, sensors small
enough t o be kept in culture dishes permit oxygen and carbon dioxide to be monitored.
If sporulation appears depressed or absent, the following may be pert'inent:
1. Check to see that screw caps are loosely fitted on containers.
2. Milk filter discs permit more gas exchange than cotton plugs (47).
3. Evaluate the box type plastic dishes before using extensively; some apparently reduce gas
exchange more than glass dishes (248).
4. large amounts of substrate and containers with narrow openings may prevent gas exchange
and inhibit sporulation.
5. If addition of 5 % KOH in vials to culture dishes results in increase sporulation, C02 inhibition
is suggested (220).
W t l o n . Many fungi require radiation to sporulate or d o so more abundantly when exposed to
light, as shown by a 1959 compilation (1 87); Table 13 lists some species added since 1959. In addition,
some fungi are indifferent to light (6), some sporulate better in darkness (270) or require a dark period
(6, 14,40, 164, 179, 283). Some isolates within a light-requiring species do not require light (88), nor
m y d spore types of a fungus require light (14, 102, 184).
Near ultraviolet radiation (310400 mp) is of particular importance for induction of fruiting in
artGcial culture (165,168) and probably in nature. It is transmitted through pyrox better than through
window glass (313). Although shorter wave lengths may be more efficient, they may produce damage
(168) and are not transmitted through ordinary glass (316) nor are they found at the earth's surface.
Visible light, usually the bluegreen region of the spectern, is also stirnulatory (7, go), although some
of the older work is questioned because of inadequate filtering of radiation. Near UV and the short
wave kngths of the visible part of the spectrum are produced by ordinary fluorescent lamps. As
incandescent lamps produce little UV and are poor in the blue region, they arc of l e u or little value in
inducing sporulation of most fungi.
144 Increase of InocuIum
Table 13. Some fungi that require or are stimulated by "light" to sporulate
(reported since 1959).
Fw'us
Altenurio brassicae (40), A. brassicoh (40), A, raphani (40),A. chqtsarithemi (1 65, 168), A. tenuls
(1 65), A. zinniae (1 65), Ascobulirs itrttnersus (3 1 9), Ascoclrjtra catilicola ( 1631, A. pitvrodella (1 6S), A.
pisI (165, 166). Blakeslea trispom [116), Botryosphaeria ribis c77, 260), Butrytis citterea (165),
Cercosporu bericok (34), C. zebrina (la), Choa~tephumcucurbirarilinr (1 16), C. conjutlcta (1 16),
Cbchliobolics honrurphus (1 84), C. kusarroi (1 84), Cor~.tteumsp. (165). Epicoccunl nigtrrm (1 65), E. sp
(165). Fusarfum trivale (1 6S), F. oxyspunrm f. lycopersici (300), F. rosetrm (1 651, F. rofani (1 65).
Gliochdium sp. (1 65), Gulgrzardia bidbvellii (35). Helminrllosporittni avetiae (1 65), H, oryzae (1 64),
Hypomyces sularii /: cucurbitae ( 5 1 ), Leptodiscus renestris ( 196, 22 1), Leptosplurerulirta arachidicolo
(BB), L. briosiana (88), L. rrifolii (88), hlacruphomirtia plaaseoli ( 2 2 l), iMycosphaerelkr pillodes ( I 65).
Necrrh galllgena (1 76), N. glicladiulder (1 Ol), iV. peziza (1 01). Ophiobolus grantinis (1 65, 307).
Pestalotin theae (Y2), Phorna llerbantrri var. nzedicagirtis (165), P. spp, (165), P. trifolii (165),
Phomopsis juniperovora (257), Pltyllosticta sp, (1 65), Physantrrr gyruntm (7 I), Pltytoplrthura cactorum
(102), P. cupsici (102, 199), P. drechslen (199), P. er)lthroseptica (P.itinwhyensis) (102), P. heveae
(102), P. palt?rivora (7, 199), P. parasirfca (7, 199), P, nicorbtrae (199), Piricularia oryzae (165),
Pleospom herbarum (1 67), Poria arribigua (24 l), Pyrenoclzuera rerrestris ( 1 15, 16S), Pyre/$ophom
bromi (39), Sclerotinh trifoliorum (134), Scolecortichum grantittis (89), Septoria nodomnr (239),
Septoria tritici (l65), Spluerobohrs stellatics (1), Stcmpliyliutn burryosum (1 65), S, floridatiuni (l26),
S. trifolii (165). Trichoderrna sp. (201, T. lignomm (204). Verticillium albo-atrum (144, 16S), K
lateriturn (1 36). Wojitiowicica gtontinis ( 1 65)
Temperature, media and the length of photoperiod may profoundly affect the response to
radiation. At temperatures of 26 C and above, radiation may not be stimulatory but inhibitory (6).
Some media can substitute for light (5, 7, 163). The latter media often consist of portions of the host
plant or are made up of plant decotions, e.g., V-8 juice.
Suggestions for inducing sporulation using radiation include the following:
1. OK standard or cool white fluorescent lamps to give 200 f t c or more. (Many fungi will be
stimulated by lower intensities.)
2. Attempt to keep temperatures below 24 C if facilities will not permit the uw of a range of
temperatures with light. A temperature gradient plate with rapidly sporulating fungi is an
efficient way to assess temperatures effects (99, 162).
3. Use a 8-12 hr photoperiod initially, although other regimes may be better upon subsequent
testing.
4. Natural media and media of host plant parts may be desirable initially; consult lists of media
that have been valuable for similar fungi.
5. If determining whether light is required, cultures left in the dark should be aerated or the
experiment so designed that inhibitory gases do not accumulate.
a
6. Exposures of few seconds to germicidial LW with the petri dish lid off may be stimulatory
(35,55,28B), although the above procedures are usually more applicable.
MEDIA
Of the media used to induce sporulation (listed on page 4) a few have wide applicability. Some
are: alphacel, corn meal, lima bean, fdter paper yeast extract, oatmeal, malt, potato dextrose or sucrose
(either carbohydrate usually in lower concentrations than ordinarily recommended, or for mme
Increase of lnoculum 145
rscomycetes, higher concentrations (101, 108)), V-8 juice, water agar and mixtures of intact
grains. These media consist of natural materials in whole or in part. Generally media that do not
encourage excessive vegetative growth are desirable and should be tried first if there is no specific
recommendation in the literature. If these fail under varied environments, the use of host plant parts or
decotions of the host should be tried, Success may be enhanced by the use of non-heat-sterilized plant
tissue (sterility achieved by aseptic remove1 of internal tissue or by less drastic forms of sterilization
such as surface disinfection or gas sterilization). It may be necessary to tolerate some contamination.
The usual efficacy of natural media does not preclude synthetic media as some synthetics are as
good and have the advantage of precision and reproducibility. They may require more time to prepare
and be more costly. Choice ought to be determined by the intent and requirements of the experiment.
A synthetic medium that will support sporulation may be devised in various ways. However, the
literature should be consulted first for defrned media that support growth and sporulation of the
species, or related species, or fungi that occupy the same host-substrate. In testing, a natural medium
should be employed for comparison. Isolates may be more variable in their reaction to synthetic media
than to natural media. Fungi on synthetic media may require additional stimuli, such as light, to
tporulate.
Some approaches are given and may be used in combination.
1. Devise, a medium that resembles the natural substrate. Information on the nutrient composition
, of various agricultural products is available (23, 15 1, 20 1, 318). Systematically eliminate
unnecessary ingredients (26). This is a historical approach usually less followed today.
2. Test a number of partially defined media such as lactose-casein-hydrolysate, no. 1 13, Leonian
agar, no. 114 (see list page 4,6). If successful, the natural ingredients such as yeast extract,
peptone, or casein hydrolysate may be replaced by their known components. Unfortunately, the
complete composition of these materials is not known and the known components may vary in
amount. Auxanographic techniques (37, 229) and amino acid "block techniques" (263) arc
useful in efficiently screening compounds involved.
3. Test a number of diverse synthetic media (see page 6), varying in complexity, that induce
sporulation of some fungi. If successful, attemtp to refine, particularly if sporulation is not equal
t o that obtained on natural media, testing various compounds and their concentrations. A
factorial test (195) or the triangle system (314) may be used. The latter enables testing of 3
compounds to determine optimal concentrations. Carbon, nitrogen, and phosphorus, as well as
mineral salts are often evaluated by this method.
4. Add to a basal synthetic medium, extracts of the host or materials that are stirnulatory (tomato
paste, coconut milk, etc.). If successful, substitute known components of these '
materials or
attempt to separate and characterize active portions (usually a difficult task).
Without attempting to give many details on the nutritional requirements for sporulation some
nlient points are presented which should also aid in designing a synthetic medium. Further detail may
be found elsewhere (43, 106, 170).
1. Carbon sources and their concentrations are of extreme importance, Frequently compounds that
do not give maximum vegetative growth are desirable. Althou& there is considerable specificity,
some generally recommended carbon sources are: sucrose, celulpse,'lactose, maltose, and starch.
Mixtures may be desirable (108).
Nitrogen
\,(\y!
has a complex but important effect, and the C:N ratio is usually critical. Apparently
2.
t h e n are species that either require or sporulate more abundantly on an organic source of
nitrogen (142) such as an amino acid (1 1 1). In these, purity should be verified as contaminating
growth substances may be involved (106). There may be a difference in the purity of the amino
acids that companies supply.
3. As sporulation is more sensitive to trace elements than is growth, it is desirable to,@d specific
mixtures t o supplement those found as contaminants in the main constituents of the medium.
Lilly and Bamett's solution, no. 143, or a modification of the A to Z solution (the latter when
extremely pure chemicals are used) are suggested. Chelates may help to keep available important
trace elements, particularly hon, which is readily precipitated at high pH.
4. Calcium often appears to a critical clement for sporulation of some organisms (37, 319) and
should be supplied.
5. As pH may be critical, it should be measured at least at the start and end of the experiment. A pH
gradient agar plate may be useful (250). Initial adjustment of pH or periodic adjustment to assure
the desired pH may be desirable.
6. Certain kinds of fungi may have unusual nutritive requirements, such as organic or reduced sulfur
for many of the water molds (53, 226), sterols for Plrytophrkora and Pythium (63, 103, 104,
lM,l lo).
7. Amounts and kind of vitamins needed for sporulation may be specific and should be tested to
obtain maximum production of sporea 5iethods of sterilization of these and other growth
substances should be adapted to prevent their breakdown.
8. Source of water and cleanliness of glassware is important, because sporulation is often sensitive to
toxic materials. Sterilization by filtration through seitz pads may add substances inhibitory to
p r u l a t i o n (234).
SPECIAL TECHNIQUES
Various filter paper techniques for encouraging fungi to sporulate
Lukens' technique (178): Developed for Helrnintlwsporium vagans and AlternarL solaai. Bean and
Wilcoxson found it suitable for H. sativurn and H. dictyoides but not for their isolate of H. vagans
(17).
1. The fungus i s grown in synthetic medium no. 123 for several days on a shaker,
2. The mycetial pellets are fragmented with a wring blendor for 2 minutes without using sterile
technique.
3. Centrifuge the fragments and suspend in 0.02 51 phosphate buffer (pH 6.4).
4. Add 2 ml of the suspension to dry filter paper in petri dishes.
5. Keep plates in dark or alternating dark and fluorescent light, depending on the fungus.
McDonald and Martens' version (193) (used with Aftenlarin zinniae).
1. Dip fdter paper circles into V-8 juice, place a number of them in a petri dish and then autoclave.
2. Add water to agar to cover the discs.
3. Flood plates with inoculum
Many authors recommend placing filter paper, lens papcr or blotting paper on the surfacc of various
media.
Cenophane technique (73,261) (To separate fungus from agar medium)
I. Cut moisture permeable cellophane discs 85 crn in d i m e ter.
2. Autoclave discs between sheets of filter paper or in water in petri dishes.
3. Transfer to the surface of hardened agar, attempting to prevent air spaces.
4. Inoculate in the usual manner.
5. After sufficient growrh, cellophane discs are puUed off arid may be blended in water or transferred
to another container, and the spores washed off.
h u n d mnd bicd inoculum (160)(Useful for fungi that do not sporulate readily and where mycelium
may substitute for spmn)
This technique has worked with a number of f o b pathogens.
1. Add 2 parts of wheat and 1 part of oat reed t o flasks.
2. Add distilled water at intervals to keep grain submerged. After 2 hr, water level in the flask usuay
remains const&?t .
3. Autoclave for 1 hr at 15 Ib.
4. Inoculate seed and keep at suitable temperature (21 days at 15 C for Sclemtinia nifolionrm (160)
5. Spread grain on screens and air dry using a fan. (Those producing spores should be covered with
paper to prevent quantities of spores being spread around.)
6. Grind grainculture in one of the following:
A wiley mill using a 14 mesh screen.
A food chopper fitted with a coarse disc.
A bamrnermill fitted with a %" screen.
7. Inoculum can be stored at 0-5 C in open cans until ready for use, (Storage for S. rrifoliomm is as
long as 24 months,)
Ways of obtaining large amounts of inoculum Used to increase inoculum of species of Fusariurn and
Helr~tinrlrosporium(262).
1. Soak seed and vermiculite 24 hr in cold water or 2-3hr in hot water.
2, Seed mixtures are: 2:l mixture of oats and wheat ur barley and wheat or, 2:1:2 oats, wheat,
vermiculite (latter advised for soil inoculation but not for sprayins as particles may clog sprayer).
3. Fill flats )c, full with media (flat has coarse wire mesh bottom).
4. Cover flats with brown paper (Kraft) and autoclave on two successive days for 1!4 hr at 15 Ib.
5. AUow to cool.
6. Blend I or more petri dish cultures in 500 rnl of sterile water for 45 sec in a Waring Blendor.
7. Lift one side of the paper cover and pour the suspension evenly over the medium.
8, Stack flats with sterile spacers.
Large amounts of inoculum can be grown on agar media without using large numbers of petri
dishes by substituting aluminurn (173) or Pyrex cooking pans fitted with 1/8" thick "transite"
(asbestos board) lids. The lids are held in place with fold back type paper clips. Ten x 10'' spores of
Glomerelh cingulatu were obtained in 3 days on PDA in thcx containers. The method also worked well
to obtain quantities of spores of A l r e r ~ r i asolarli and Muniliru fnrficolu (240).
Miscellanmus
Multipoint inoculation of the substrate (usually on agar medium) often induces fungi to sporulate
faster and more abundantly than single point inoculation. Flooding with spores is recommended and
mycelial fragments often can be substituted successfully (282). Reasons given for the value of
multipoint inoculation conflict, but differences between fungi may partly account for thc lack of
agreement.
The main explanations that have been proposed are:
1. Rapid and uniform depletion of nutrients occurs and encourages sporulation (282).
2. Presence of stimulating substances in the spore matrix ( 1 88).
3. Presence of stimulatory substances in the spores ( 1 94).
Wounding of mycelium by scraping often encourages localized spore production, Alternod soloni
(190) and a number of other dcmataceous fungi respond well to wounding.
Other techniques may be found in Lilly and Barnett (170).
148 Increase of h x u l u m
Table 14. Check list for inducing sporulation of fungi.

(Items are not in order of approach or of importance. See text for details.)
Consult literature for recommendations on the fungus or related species. If none, or initial trials
apparently fail to induce sporulation, consider the following:
Are spores present but hidden by mycelium or buried in the substrate?
When (the time of day, the season and the associated weather) and where does the fungus sponrlatc
naturally? Are there any unusual circumstances?
Are sufficient isolates being used that at least some would be expected to sporulate if environmental
and nutritive requirements are met? If the sexual stage is sought, is the fungus heterothsllic? Are
known mating types available?
Has the organism been grown with other species?
Has the fungus been grown on a wide number of natural media under a variety of temperatures in
continuous light, alternating light, and continuous darkness?
Does the fungus sporulate on infected host tissue when taken into the lab? If so, under what
conditions? Is sporulation associated with contaminating organisms?
Has plant tissue, variously treated, been tried as a substrate?
Have decotions of host tissue been tried?
Is aeration limiting?
Has agar of various depths been used?
Has the substrate been inoculated by flooding or streaking of the inoculum?
Are the glassware, closures, water, and in~redientsof the media free of toxic compounds?
Has the mycelium been injured by scraping, exposure to germicidial UV or freezing?
Has the culture been allowed to dry and then wetted or exposed t o high humidity?
Have cultures been transferred from rich t o lean media?
Is the hngus parasitized or badly contaminated?
INCREASING OBLIGATE PARASITES

The quality of inoculum of obligate parasites may be affected by conditions under which they are
increased. It may be that the variety of the host and its age are also of significance. Included in the
suggestions given below are a number that attempt to lessen contamination. The amount of
Contamination that may bc tolerated influences the manner of increasing obligate parasites. The report
of Barr et a1 (16) with leaf rust of wheat suggests that a substantial amount of contamination may
Occur even with considerable care. If high quality and uniform inoculum is desired, procedures of
~noculation,environmental conditions, selection of host and harvesting techniques need to be evaluated
and controlled where necessary. Some suggestions for increase are:
1. If possible, increase or at least maintain the isolate on a cultivar, line, etc. that is resistant t o
most other isolates of the fungus and other troublesome pathogens.
2. Use a host that produces abundant inoculum quickly.
3. Consider using a growth inhibitor such as "Cycocel" (American Cyanamid, 100 ml of 0.3%
solution added at planting time to 4" pots) to keep plants more vigorous and juvenile (139).
4. Inoculate in chambers that can readily be freed of contaminants.
5. Attempt t o keep utensils, pots, soil, hands, clothing free of contaminating fungi.
6. Rely on properly preserved stock cultures as much as possible.
7. Check isolates on differential hosts frequently t o verify identity and purity.
8. Use uninoculated, incubated check plants as an indication of contamination.
9. Separate different cultures as far as possible.
10. Use plastic or glass cages or cylinders (not made of cellulose acetate: it is sometimes
phytotoxic) t o hold plantculturco;
i n c r e w of InocuIum 149
11. Harvest spores using a cyclone collector' or other vacuum device to minimize spore liberation
and dispersal.
12. Promptly harvest and use freshly produced spores.
13. If increase in the greenhouse is unsatisfactory because of contamination, consider using plant
growth chambers and/or detached leaf culture, the latter described in the chapter on
inoculation.
INCREASE OF SPECIFIC FUNGI

Inasmuch as possible, several alternate techniques andlor media are given. Detail about tlme,
temperature, radiation, and type of growth containers are given if reported. These recornnlcndations
may not be optimum for a l l isolates. They may be influenced by how the medium is prepared,
particularly if it is a natural mediutn, and by the kind, amount, and age of inoculum used to start the
cultures. Details of the latter are rarely given. It would be desirable to standardize procedures if
uniform and consistent amounts of inoculum are needed. How the spores are harvested may also bc
significant (1 91).
increase of Aphanomyces - Production of zoospores
Three of a number of techniques are given here, The first two have been used for A, erttciclles
For other techniques see (259,262,263) and for a recent washing solution see mediutn no. 1 1.
A (98)
1. Grow fungus on corn meal agar for 7 days at 20 C.
2. Remove rnat and wash in tap water.
3. Cover mat with tap water and incubate at 2P C for 24 hr.
B (1 72)
1. Grow fungus in SO ml of maltose (3 g/l), peptone ( I g/l) broth in acid-washed 250 ml erlenmeyer
fIasks for S days at 23-25 C.
2. Pour medium off and add 60 m l tap water - replace 1-2 hr later with 40 ml of distilled water at
24 C.
3. Aerate at 24 C using 3-4 rnm glass tubing delivering air at the rate of 300-400 mllmin or 6
bubbles/sec. Zoospores are produced afrer 6 hr, and maxin~urnnumbers at 8-1 0 hr. For a slightly
different procedure see Cunningham and Hrtgedorn (501,
c (64)
1. Inoculate 125 ml erlenmeyer flasks containing yeast glucose broth no. 272.
2. Incubate on shaker at 25 C for 2 days.
3. Divide mycelium into small tufts and wash thoroughly through 3 changes of water for 10 min
each.
Aphanomyces spp. are homothallic and commonly produce oospores on cornmeal agar (58,81),
snake skin (263). For other natural media see (263), and for other synthetic medium beside no 10 see
(224,226).
Incrarse of Ascochyta phaseolorum (49)
1. Inoculate sterilized potato cylinders.
2. Grow at 25 C for 3 4 weeks.
3. Prepare inoculurn by grinding material in a Waring blendor and straining through cheesecloth.
(Heavy sporulation also occurs on potato sucrose agar when flooded with 1 ml of spores.)
'Available: from the Instrument Shop of the P h y d o Dcpnrtrnent, Iowa State University.
Increase of Aecochyta caulicoh and A. melilota (163)

1. Inoculate autoclaved stems of .\iclilotus embedded in water agar or PDA,
2. Both species produce p y ~ n ~ d with
i a abundant spores in the dark on the ,\felllotus sterns, but A.
aoulicola requires near ultnviolet light to sporulate on PDA.
A. melitoti produces a prrfect stage - .Ilycosplzaerella lethalis on PDA streaked with mixtures of
conidia from different isolates. Culturing at 4 C for several weeks and returning to 24 C increases the
yield of perithecia.
Increase of Ceratocystis fagacearum - Oak Wilt Fungus
Production of conidia
1. Inoculate slants or plates of Sutramigen agar no. 155 (70).
2. Incubate for 12 days at 20 C (72).
An alternate method is to grow organisms on PDA for 2 to 4 weeks between 20 and 25 C (288).
A glucose phenylalanine medium no. 157, designed for isolation and quick diagnosis, encourages rapid
sponrlation (1 3).
Production of perithecia (72)
1. Inoculate plates containing 20 ml of mltox-xisamino acid medium no. 19 at 3 or 4 equidistant
points with mycelium from 10 day old colonies.
2. Incubate at 20 C for 12 days.
3. Add suspension of conidia of opposite compatibility types using loop or atomizer.
4. Fertile perithecia should be produced within several days.
For an extensive review of the orgarlisrn and diseag see (290).
Production of perithecia (245. 254) of Ceratocystis ulmi
1. Debark elm branches and cut 318" discs 1!h to 2in,'' in diam.
2. Place 2-3filter papers in petri dish, add an elm disc and 20 ml of tap water.
3. Autoclave at 15 lb for 1 hr.
4. Place mycelium and agar (pea size) on discs 1" apart of A and B compatibility types.
5. Perithecia will develop at a wide range of tcmperatures but 55 F was used by two investigators
(254).
6. After 3 weeks to 9 0 days look under rnycelid growth for perithecia.
Mycelial ConidQl increase
There seems to be no unusual requirements, the organism is grown on PDA at a wide range of
temperatures. See Kaarik (132) for effects of various media ingredients on conidial production.
Increase of Cercospora spp.
Most species of Cercospora sporulate with difficulty in artificial culture. Because of this,
mycelium is often used as inoculum. Many specialized media, usually made from decotions of the host,
have been used. The following are recommended.
1. Select and maintain sporulating types of spore transfer (207,265).
2. Inoculate pldte by flooding.
3. Use carrot leaf decotion no. 42 or V-8 juice agar no. 248.
4. Subject fungus t o varied fluorescent light.
More specific recommendations may be found in accompanying references:
C apii (206);
C. beticola (34);
C mental C. davistii, C dublo, C. physalidls. C personata (265);
C. medic~~gtnb, C. rebrim, C. davisii ( 18, 19);
C brachiata, C. carteseem, C capsici, C festuae, C kikuchii, C petmireti, C pueraticola, C.
mumi, C sorphi, C. stizlabii, C. zebniw (154);
C abelioe (231);
C nlcorianae (54).
Increase of Colletotrichum coccodes (C. atrernentarium, also C. phomoides. but sec (9))
Increase of Sclerotia for soil inoculation (128)
1. Crow organism on a sand-soil-tomato root rned~umno. 208 for 6 weeks. Cover flask opening with
cigarette paper in addition to cotton plug to prevent entrance of mites.
For acervular conidia (1 28)
Crow organism on cellulose asparagine medium no. 46.
Increase of Conidia
A (1 35)
laoculate liquid but cool PDA or V-8 juice agar with spores or rnycelid fragments so as to give
5-10 colonies per phte.
B (1 49)
Inoculate nutrient agar containing 3 g of beef extract and 10 g of pcptone/l.
c (78)
Inoculate PDA. Fulton (78) suggests that spores be washed twice by centrifugation, as he found
unwashed spores or agar discs to contain materials which produce black spots and subsequent
distortions in green fruits.
For information on synthetic media see (1 61, 175).
Increase of Colletotrichum lindemuthianurn
A (242)
1. Grow fungus on bean juice agar slant . medium no. 22.
2. Use 10 rnl of spore suspension harvested frorn bean juice slants as inoculum for grain medium.
3. For alpha and beta strains, inoculate a medium of 10 g each of wheat and barley seed, for gamma
strain use wheat and oat seed, in 5 0 0 ml erlenmeyer flasks. (Seed is previously soaked in water
overnight .)
4. Incubate at 23 C for 7 days.
5. For inoculum add 200 ml water per flask and filter through cheesecloth..
B(188)
1. Rood Neopeptone-glucose agar no. 1 5 1 with spore matrix.
2. Incubate cultures at 20 C. Light is not required.
Production of fruiting bodies of Coprinus lagapus (185)
1. Add 20 ml of medium no. 80 or no. 90 to 10 oz medicine bottles, plug with cotton, and lay on
side.
2. After inoculation grow at 25 C and at 8 f t.c supplied by continuous incandercent Iights.
lncreasc of Diplodia maydis (D. Zeae) (294)
1. Fill 2qt. fruit jars to !4 their capacity with oats and add an excess of warm tap water.
2. After 4 hr drain off water and add 200 ml of hot tap water to each jar.
3. Jars are plugged, preheated for 15 min, and then autoclaved for 3 hr at 15 Ib.
4. After inoculation, incubate for about 6 weeks in the light at 24-26C.
5. Prepare spore suspension by removing the material from the jars to a cheesecloth oack and
kneading the mass in water to separate the spores from the oats.
6. Filter through a layer of cheesecloth and make up to volume at a rate of a jar of inoculum in one
quart of water. Density should be comparable to color of India ink diluted 1: 10,000.
Increase of Fusariurn graminearurn (F. roseurn) (38A)
1. Inoculate with either a 1 rnl suspension of a macerated 7 day old PDA culture or a number of
agar.myccLial cubes.
2. Shake CMC culture medium no. 50 for 10 days, 100 rnl in 500 ml flasks. Temperature not given,
but 24-26 C is adequate. Several million spores/ml are usually produced within 4 days. Light is
not required.
3. Thc organism will usually sporulate on PDA at a range of temperatures under fluorescent light.
-
To obtain the Perfect stage Gibberella zeae (G. roseurn). (Personal communication from A. 1.
UUstrup.)
1, Inoculate soil agar slants, medium no. 221 bearing 1 or 2 autoclaved matured wheat culms.
2. Penile perithecia are usually produced within weeks under fluorescent light (104300 It*) at
22-25 C.
Incnase of Fusarium oxysporum I.lycopersici
A (1 53)
1. Inoculate potato dextrose broth.
2. Shake for 3 days and then dilute 5 times with tap water to a cell concentration of about 20 x
1o6/rnl for inoculation.
B (57)
1. Inoculate glucose casarnjno acid medium no. 83.
2. Growth in shake culture for 4 days should give about lo8 cellslml. Dilute the culture with an
equal volume of distilled water for use as inoculum.
c (8)
1. Use inoculum consisting of 1 ml spores and mycelia from fresh PDA slants.
2. Inoculate 500 rnl of Armstrong Fusarium medium no. 14 in 2 1 flasks.
3. Grow for 72 hr at 28 C, shaking flasks occasionally.
D (256)
1. Inoculate 100 xd of cerelox nitrate medium no. 47 in 32 oz prescription bottles.
2. Place bottles on side and grow at 28 C.
E
Kerr (152) considers the conventional methods of increasing inoculum as only adequate for testing
pathogenicity but not suitable for natural soil studies. He used a mil culture (no details given on
preparation) that permitted F. oxyspomm to survive much better in soil than on sandmaize meal
due, he believes, to the production of chlamydospores on the soil culture medium.
Ascording to Snyder and Alexander perfect stage of FuWurn oxyrponrrn is unknown.
Increase of Fusarium solani
F. sohni, apparently like all Fuuria, sporulates more in light (276). According to Reid (236)
microconidial production is stimulated by intensities of 2040 ftc, while macrmnidia are produced in
large numbers at 50G1600 f t c of fluorescent light.
Usually the organism is increased on PDA (276) (made from fresh potatoes), although V-8 juice
agar (236) and a synthetic containing glucose and proline give good sporulation (1 11). Temperatures of
30 C were reported to give more rnacroconidia (236) than lower temperatures, but in another study
20-24 C was better (41). See Ken's (152) comments on the value of wil culture in the section on
increase: of F. oxysponrm The notorious variability of Fusorium demands spore transfer (singlesporing
it highly recommended by some) and proper preservation to maintain sporulatiny and pathogenic types.
Production of pedthecia of Hypomyces s o h l f, cucurbitac
1. Use large test tubes and inoculate 2% PDA adjusted to pH 6.2 with NaOH or HC1 before
outoclaving (51), or use 30% V-8 juice agar, adjusted to pH 5.5 prior t o autoclaving (287).
2. Grow under continuous cool white fluorescent Light of 5G250 f t c at 25 C 2 2 C for 20 days for
homothallic, 30 days for Hererothallic culture (5 1).
3. Spermatizt heterothallic culture with a spore suspension of opposite compatability type; or if
homothatlic, spray with sterile water (1 32).
4. Examine for pcrithecia 15 days later.
According t o Tousson and Snyder (288) race 2 does not form perithecia as readily as race 1. For
synthetic media see (1 24,287).
Helminthorpodum, increase of 3 species that attack corn
Fungiis H. wrbonum (I 86) H. maydis (296) H. turcicum (1 86)
Medium V-8 juice agar or lactose PDA V-8 juice agar or lactose
casein hydrolysate no. 1 13 casein hydrolysate no. 1 13
Temperature 26 C 26 C 26 C
Time A week to 10 days A week to 10 days A week to 10 days
A summary of these diseases is in (295).
I- of Helminthosporium sorkinianum (H. sativum)
A(17) ,
1. inoculate Sach's agar no. 207.
2. Grow for 2 weeks.
B(65,181)
1. Inoculate 1% water agar containing on its surface scattered bits of coarsely ground barley straw
previously sterilized with propylene oxide.
2. Grow for 2 4 weeks.
C (1 77)
For inoculation of soil.
1. Inoculate coarse sand mixed with 5% cornmeal moistened with Czapek's inorganic salt solution.
2. Grow for 7-10 days.
3. Prior to mixing with soil,screen through fly screen.
Rodudion of the Ascomycete stages (Cochliobolus, Trichornetasphaeria) of certain species of
Helminthosporium and Curvularia
The method used by Tinline et a1 (283, 284) for the production of Cocliliobolus srrtivus, the
perfect stage of Helminrhosporium sorokblic?nrrm(H. sarivum), is as follows:
1. Soak barley seed in water for 1 4 hr.
2. Treat with HgCI,, 1 : 1000, for 5 min.
3. Rinse seed 3 times with sterile water.
4. Boil seed for 1-3 min t o kill the embryo.
5. Dip seed in an aqueous suspension of conidia from 2 compatible isolates.
6. Place seed on Sach's agar having a pH of 4 to 6.
7. lncubate at 24 C for 7 days followed by 14 days at 20 C. Light is not necessary, and in fact
Shoemaker (270) reports total darkness best. For 13. rtodubnrm, Luttrell (181) used corn seed
and barley straw previously sterilized by propylene oxide or sterilized as above. The latter was
tecommended for H. holrnii (183).
The perfect stages of H. tuicicum (182, 21 2), H. spiciferum (217) and Curnularia lunata (219),
have b a n obtained with a simpler procedure,
I. Heat barley seed in an oven at 180 F for 2 hr (2 12) (1 60 C for H. holmii (1 83)).
2. Place seed on Sach's agar.
3. Transfer small blocks of agar containing mycelium of compatible isolates to opposite sides of seed
embedded in Sach's agar (for H. turcicum add 20 g of dextrose and 10 g of ceUulose per 1 of
Slch's agar (2 1 2)).
4. lncubate at 24 C for 3.4 weeks in the dark. For Curvu&& genicubta constant artificial light is
mcommea&d by Nelson (2 18).
For H. moydis (2 10, 21 l), H. carbonum (2 13), H. cynodontis (2 16) and CurvulariP intennedio
(214) use corn leaves 1'Aw x H" that have been autoclaved for 1 hr and place on Sach's agar. .
Two homothallic s p i e s , H. hornonwrphrrs and Ii. kusanoi, produced fertile perithecia (Cochli-
obolus} on 5% V-8 juice agar when exposed to 18 hr of fluorescent light for 3 weeks' (1 84).
-
Not all species of Helmintirospon'um respond to these treatments some produce only conidia or
require prolonged periods of low temperature to form fertile perithecia. References to a number of
species may be found in the section of temperature.
Inmase of Phoma herbarum var, mediaginis (Ascochyta imperfects)
A (237)
1. Add equal volurnes of barley or wheat seed and water to 250 ml flasks.
2. Autoclave on 2 successive days at 17 Ib for 20 min. S113ke the flask to lessen caking.
3. Inoculate with IS ml of a potato dextrose broth culture.
4. Incubate at 24 C for 3 week.
5. For storage, dry the inoculum at room temperature and keep at 3-5 C or at -20 C,May be stored
for as long as 2 yr.
B (192)
McDonald used 1" layer of barley kernels in a 1 1 flask and autoclaved for 1 hr at I5 Ib (192). After
inoculation with a 10 rnl spore suspension grow cultures at 70 F for 3 weeks.
Leach reports that sporulation is increased by exposure to light (165). For information on
inoculation procedures see (1 1,238) and for host range see (62).
Inducing Asexual reproduction in Phytophthora and Pythium
Numerous methods and media are used to induce formation of sporangia and zoospores of
Phytophthora and Pytirium (Tables 15, 16). It is unlikely that one combination of medium and method
suits all species. Generally, the organism is grown briefly on a natural medium (light stimulates
sporangia formation in sornc species) and transferred to a dilute nutrient solution, or the solution is
added to the culture. To aid zoospore discharge the temperature is decreased for $4 to several hours and
then brought back t o the original. Frequent changes of the replacement medium (64), irrigation or
aeration (83) also increases the number of sporangia and zoospores, The water used is especially critical,
particularly when it is used as the replacement medium (44).Sensitivity of species of Phytophthora to
trace amounts of copper (150, 157, 324), of pH and specific ion requirements may be involved. The
varying content of copper and of other ions of different sources of tap water probably accounts for the
erratic performance. Addition of chelates (150) and the use of charcoal water (233) are probably
valuable because they tie up the copper, although there are other advantages claimed for these materials
(314). Purified water that is improperly deionized or old may have an undesirably low pH. Also,
evidence accumulating indicates that specific ions (319) as well as unknown substances present in
natural materials (44, 322, 323) my be significant in the formation of sporangia. Finally, some
cultures are recalcitrant regardless of the methods used and the skill of the investigator. Recommenda-
tions for specific fungi are given in Tables.
Sponrlation of Phytophthora infedans
Increase of Inoculum (production of sporangia) (281)
1, Stock cultures maintained on lirna bean agar, no, 115, can be stored under oil at 3 5 C.
2. Soak yellow peas overnight, drain, and then barely cover with 1" water in 250 ml erlenmeyer flasks.
Autoclave at 121 C for 40 min.
3. Transfer mycelium or a suspension of sporangia t o seed.
4. Incubate cultures at 20 C for 1 week to 10 days.
5. A water suspension, 2 gal of water plus the contents of 6 flasks,can be used to inoculate p l m t ~
Table 15.
Media and procedures to induce asexual reproduction of Pythiuni and Pltytophtltora.
(See Table 16 for recommendations for specific fungi)
A (Medium) B (Procedure) C (Replacement Liquid) q3
Grow organism on one or more After growth, on A do one of the (See section on media for details
of the following media, usually following. on preparation)
for 3-7 days (see section on media
for details on preparation).
Boiled young leaves of 1,Transfer agar discs or Tap water
Agrostis atbu or A. semi- infected tissue to a Distilled water
verticillata 1" long in C6 replacement liquid Glass distilled water
Alfalfa stems 2. Cut out colonies from agar Water deionized, then
Bean agar without injuring tips of glass distilled
Corn meal agar hyphae and transfer to a Charcoal water, no. 48
Malt-broth, no. 142 replacement liquid 1 pt pond water and 3 pt
Infected host tissue 3. Add a replacement liquid glass distilled water, Nter
Oat meal agar to agar culture so it is partly and autoclave, add young
Potato dextrose or completely submerged boiled leaves of Agrostis
(fresh potatoes) 4. Wash mycelium in sterile albu or A. semi-tferticilhta
A. Broth distilled water, then Stream water (non-sterile)
B. Agar transfer to a replacement Pond water
Lima bean agar liquid Soil extract
A. regular strength 5. Transfer mycelium to a A. Mehrlichs (1 98)
B. dilute replacement liquid B. Wills no. 218
Seeds 6. Transfer mycelium that C. Tomlinson (286)
A. ern^^ is growing on cellophane Lima bean steep, 1 g lima
Be POPPY previously overlaid on bean11 autoclave 30 min
C. petunia culture medium .Ol M KNOj (1 69)
D. Calceolaria Petri solutionJ no. 181
E. corn Humid atmosphere
F. sunflower (90-100% RH)
G. squash Machilis dilute salt
H. pea solution no. 126
I. soybean 5% peat percolate no. 175
J. oat 4% sucrose solution
V-8 Juice agar Will salt solution no. 265
A. normal strength
B. 10% V-8 juice
Vermiculite 3 pt soaked
with one pt of the following:
A. lima bean extract
B. potato dextrose broth
C. V-8 juice
Pea broth no. 172
Boiled carrot discs
Pea broth no. 1 73
Wheat root tips (continued on next page)
156 lnncuc of lnoculum
Table 15. (cont.)

1. Schrnitthener (258) reports agar concentration may significar~tlyaffect sporangium production for
different species of Phyrophthora.
2. Klein (155) found that the production of sporangia on infected host tissue is not as sensitive as other
substrates to the kind of replacement medium.
3. Waterhousc (302) suggests that soil inhabiting species should be submerged while isolates that attack
above ground parts of plants should be partially submerged.
4. Emerson (64) advises use of live seed and boiling for 10 to 30 min. The ovary coat may be ruptured
without excessive cooking by pinching gently after boiling.
5. Waterhouse (305) suggests that this medium is of particular value in taxonomic studies.
Sporulation of P. infestarrs (cont .)

Alternative methods include growing on frozen lin~abean agar no. 119, chick pea agar no. 49 (145),
non-sterile detached potato leaves (21) or potato tubers (190).
Production of zoospores
1. Use cultures grown 1@J4 days at 20 C.
2. Incubate sporangia in tap water at 4-5 C for 4-5 hr.
3. Filter through Whtman $1 paper if you wish to remove sporangial shells (321).
Black and Gallegly ( 2 1) recommend placing the sporangial suspension at 10 C for 95 to 2 hr.
Production of sexual bodies (79, 243,275)
1. Pour V-8 juice agar no. 250, frozen h a bean agar no. 1 19 or soybean meal agar no. 233 in normal
size petri dishes,
2. Inoculate in the center of the plate with intermingled mycelia of 2 compatible types by transferring
from the juncture of mycelia of paired A1 and A2 isolates (243), or do step 3.
3. Inoculate tufts of mycelium from 2 compatible types opposite each other (275).
4. Incubate at 18-20 C in the dark.
5. Oospores are produced in 3-5 days.
6. Continuous light inhibits sexual bodies and alternating light stimulates the germination of oogonia
prior to oospore formation ("asexual type of germination"). The presence of thick walls indicate an
oospore (243). Recently, the interpretation of precocious germination has been questioned.
Increase of Phytophthom megasperma var. sojae (P. sojae) (36)
1. Inoculate frozen lirna bean agar no. 119 or no. 117 that is fresh and has been stored in the dark.
2. Incubate in the dark at 20 C for 10 days.
3. T o inoculate, small pieces of agar and mycelium are inserted into the hypocotyl or epicotyl of 10
day old soybean seedlings. For an alternative method of inoculation see (1 19).
For sexual and asexual reproduction see Table 15, 16, and p. 159.
Sexual reproduction of Phytophthora and Pythium
The sexual stage is essential for positive identification of most species of P~lthiurn(200) and
Phytophthora (305, 306). Knowledge of the genetic, environmentd and nutritive factors that are
involved in the production of the sexual stage may be of value in epidemiology, breeding for
host-tesistance and in the concept of the species.
Species of Pytlrium are hornothallic and the perfect stage is fairly readily obtained. Many species
of Phytophthora are heterothallic or are stimulated by dual cultures, either of the same or of different
species. Whether the oospores (should be distinguished from oogonia) result from a fusion of nuclei and
subsequent reduction division or from a chemical or protoplasmic stimulation has not been eqtablished
in many cases of dual cultures. The development of "structural oosporcs" (96) may be of value in
indicating genetic relationships, but it is important, if not essential, ultimately to germinate the
oospores and study the progeny. Unfortunately, oospores do not germinate readily (3,91,275).
lnerrue of Inoculum 157
Table 16.
Ways of producing asexual stages of various species of bthiurn and Phytophthora.
Funkus Media and procedurrs References
Phytophthoru spp. (See Table 15 for meaning of symbols)
I, *I
A 6 o r A 3 o r A l 0 , b I c , o r d - B 1 -C12 12-20C,when
sporangia start to form replace with C1.
See this older reference for a variety of techniques for
numerous species.
Race a hemp seed (10a) on a 5 mm2 block of a 7 day A4
-
agar culture Bl(and a x e d without agar block) C14,-
4 days later transfer seeds to C6.
- -
A7, several days 7-24 C B3 Cl? lower temperature
A1 lb, 25 C, light not stimulatory.
A9a, (frozen) 20.24 C, fluorescent light stimulatory.
- -
A13 B4 C2,2448 hr, chill to 1CL15 C for zoospores.
A9a (frozen), 2G24 C , fluorescent light stimulatory.
-
AS, 4 days 25-27 C B4 - C9a 21-25 C.
-
Al l a or A8b B1- C9a 23 C for 48 hr.
-
A3,8 &yr B1- C9b, 20 C change after 48 hr,
- -
A2 B1 C1, running and well aerated at 24 C sporangia
stimulated by light (68). For large quantities of
i n d u m see (1 58).
" colocasiae Allb.
" drechsled A9a, alternating dark and fluorescent li-& stimulatory.
" crythosepticu - -
A3 02 CIS or C 1, or C9 or compost, percolate 2-3
var. pisi days at 18 C.
" hurike - -
A9a 3 3 wk B2 C7,2448 hr at 13 C.
cl **
- -
A3 u n t l isolate almost reaches edge of plate B2 C8,
10-15 C 2448 hr.
A12a,borc-B1 -CI?
- -
A7? for 2-3 wk B1 A1 0 f, h, or i autoclaved in tap
- -
water 2-3 wks Bl C1 (shallow) at 20 C, rinse daily
for es days.
A9a (frozen) 20-24 C, fluorescent light stimulatory.
See separate section.
AlS-M-C2.
-
A15,20 C, B4 C2 (sterile).
-
AlOj, 21 C Bl Cl?, sterile.
A4 day-B1 -C12 thenC2.
-
A9 (5 glima beans/l)- B1 C10,25 C 72 hr, then
c w d to 14-3 5 C for 8-10 hr for zoospore
production.
Set separate section.
A9a, fluomcent light stirnulatory .
- -
M a (fresh) 3 to 5 days, B4 C9b 20.25 C C17 or
ptrhrpr C39,
Fungus Media and procedures References
Phytophthora nicotianae -
A7,1120 days, 24-25 C B5,C l 1 mats just arrted, 6-10 83
-
days at 2426 C to obtain zoospores &er
8 C for 25 min then return to 26 C.
t o C2 at
For large amounts of inoculum see reference (1 58).

- -
A1 lb, 25 C, 3 days 8 3 C2 (sterile) alternating dark with
fluorescent light; for zoospores 3 0 min at 13 C, return
to 25 C.
- -
A7 81 C12 3 days, zoospores formed aith a drop in
temperature.
A9a, fluorescent light stimulatory.
-
A l l b , 2G31 C, 8 3 -C2 15-20 C.
-
A1 lb, fluorescent light, 31 C.
" phaseoli - -
A7,lS-20 C, 2-3 wk, or A9 B3 C16.
n w
-
A9 (ma& from frozen lima beans ) B1 t o I g of
Golden Bantam corn in 100 ml of sterile C4, 14 days,
20 C B1-C 13,1&22 C for 24 hr.
" pma~itica (According t o Waterhouse = to P. nicotiruw)
" primulae A3, 15-20 C - B1 - C9, aerated.
Pythium spp. - -
A13,5 days B4 C1 with irrigation
I, 9,
-
A4 B1- A16 (2 steamed wheat root tips in 3 ml C2 in
Syracuse dishes ) after 3 days change water.
A 1 2 a , b P o r c - B l -C?
Transfer A4 (BBL) agar discs to A1 in C6. change water.
or A 4 , U days - B6 - C3, 24 hr, 25C, k.1 hr B6, -
25-30C, 2-3 hr (after S. Grove)
- -
A9r, pour thinly B1 C1 shallow and sterile, change
at intends, 15-18 C.
As above.
As above.
A4, pour thinly, add several: small pinches o f leaf m d d
or other vegetable detritus.
Race a glass ring on a thin layer of A4 and inoculate
inride of ring. After mycelium reaches outside of
ring, place JS hemp seed with its cut side do~n on
margin of colony. After 10-24 hr transfer seed t o
sterile water.
20-28 C, diffuse light.
Sxual reproduction of Phytophthom and Pythium (cont.)

Oospores are produced on many media, but the generally recommcndcd ones are: cornmeal,
grated carrot, hempsced decotion, lima bean, oatmeal and V-8 juice agar. A synthetic medium
containing thiamine and a sterol is available (1 10). Incubation in darkness i s preferred (102).
Temperature requirements are not stringent with a few exceptions, such as Phytophthom polrnivom
1253, 293). Usually temperatures of above 24 C should be avoided, dthough high temperatures may
h e n the formulation of oospores of some species (306). Information sptd.tPic to the following species
may be found in the accompanying references: As an exhaustive search of literature has not been rmde,
0 t h references may be more valuable.
Phytophthora spp. (67, 291, 303,305, 306),P. boehmriae (96), P. coEtonim (22,63,104,278,
298). R metorum var a p p h t a = R cittiimk*k capsici (96, 102, 199). R cirrnomomi (80,97,249,
279,322), P. cir&ola (96,303,304), P. cimphthora (64, 157), P. cryptogea (279), P. drechslerf (199,
305), P. eJythmseptica (33,9 1, 96), fragarioe (46), Pa hevcae (l04), P. hlbenutlis (102), P. ilicis (31,
102), P. ntegasperma (96, 102), P. megasperma var. sojae (96, 102, 1 10, 1 1S), P. nicwtianae ( 1 99), p,
n i o o t h e var nicotianae (3, 96, 110, 138), P, palmivora (199, 253, 293), P. parasitica (96, 1991,~.
phseoii (85, 133), P. prlmuhe (286), P. quininea (48), Pythiun~spp. (64, 137, 200), P. aphanider.
rnatum (64), P. arrhenomanes (298), P. butleri (298), P. complectetts (298), P. debapanum (25 2), P,
marsipium (59), P. myriotylum (298), P, oedochilum (59), P. oligatzdnrm (60,64), P. palingenes (59),
P. pafpbcum (1 lo), P. torulosurn (298), P. ultimum (38).
Increase of Pseudomonas solanacearum
Virulent and nonvirulent strains are distinguished on a tetrazolium medium (TZC) no. 233. The
virulent colonies ate large, irregular, fluidal with pink centers; avirulent colonies are small, round and
deep red (131, 148). See former reference for photographs of colony types. There arc host-specific
strains consisting of 3 races (30) separated partly by the production of melanin pigments in the above
medium with 0.03% tyrosine (28).
Procedures
1. Streak TZC plates with a dilute suspension of bacteria.
2. Incubate at 32 C for 48 hr (1 0) and verify virulent types.
3. Tomato and potato plants are either steminoculated at the axils of leaves with a suspension of
bacteria (10) or root-inoculated. The latter gives good differentiation between resistant and
arsceptible plants of tomato (315), For an extensive review of most aspects of the disease and the
organism see (29,109,147).
Increase of Rhizoctonia solad and related fungi
A (227)
- .
Vermiculite Add water to 2W0 moisture w/v and grow
by weight
Corn meal 1wo for 28 days. Add to the soil at 1% w/v.
B (267)
Dry m d (particles 196 g 1. Autoclave for IS min 15 Ib in 200 g
that pass through amounts in 250 ml flasks.
0.1 " sieve) 2. Inoculate with four 4 m m discs from
Ground oatmeal 4g edge of PDA culture.
Water 20 ml 3. Grow for 4 days at 25 C and then screen
through a 1/10'' sieve with a rubber
pestle.
c (2W)
Sand 98% by weight
Corn meal 2% by weight
Water 2Wo W/V
M u c t i a n of the perfect stage of Rhizoctonia

Only a few species of Rhizoctonia form the perfect stage in culture. Two of them, R. soknl and
R pWcob (considered to be closely related or the same species), are usually amenable to the
techniques to be described. Both are believed homothallic (107, 159,225) although the perfect stage ir
only infrequently obtained with single spored isolates. The perfect stage of R. sotoni is now called
ThaMtephonrs cucumeris, formerly PelliculPrirr filamentou, Ceratobasidium @men tosum or Corm
Wrn vagum At least one, R. hiemulis, formsan ascomycete stage (31 1). Several methods are gijen
below. For others fee (74,272).
160 Incnase of lnoculum
Soil Methods
A (280)
1. Crow Isolate on Potato marmite agar no. 190 for 7-10 days.
2. Remove lids and cover with 1 cm of soil (previously heated by steam at 70 C for 30 min).
3. Expose to 12-16 hr light of 10440 f t e at about 4MW rh and between 20-30C.
4. Water 1-3 times daily to keep soil moist.
5. Fruiting usually occurs after 3-11 days.
B (280)
1. ,Grow isolate on PIIA no. 190 for 7-1 0 days.
2. Macerate culture with a scalpel and incorporate into soil (previously heated by steam to 70 C for
30 min), using 30 cm2 cuIture/300 g soil.
3. Place soil in waxed or plastic coated paper drinking cups and water 1-3 times a day.
4. Light and relative humidity as in method A.
Agar Technique (309)
1. Grow isolates on P31A no. 190 for 7 days.
2. Transfer a small piece of mycelium to edge of a 6 crn petri dish containing 6 ml of water agar,
3. Keep cultures at 20-24 C for 10 days in the dark. Aeration may be desirable, Several authors
consider light of consequence (75,107,159).
Increm of Septoria triticj
A (208)
1. Inoculate 2%Difco malt extract broth with conidia obtained from a yeast-like colony (I50 rnl in
500 rnl flasks).
2. Crow 4-8 days on shaker at 70-75 F.
For inoculation, dilute 1: 1 with water containing 1% gelatin. A detached leaf technique has been
used successfully (265).
B(121)
1. Inoculate slants of Elliot - V-8 medium. (Distilled water of Elliot medium substituted with the
filtrate of 1:S dilution of V-8 juice.)
Production of fruiting bodies of Schizophyllum commune
1. Place small agar blocks containing mycelia of the appropriate strains about 2-3 mm apart on medium
no. 209 in petri dishes.
2. Grow at 24 C f 2 C under a fluorescent lamp 175 f SO f t (220).
~
3. Fnriting usually occurs within 7 days.
For additional details on genetic and environmental aspects of fruiting see (235).
lacrease of Sclerotinia frutioola ( 191)
1. Inoculate PDA slants.
2. Grow at 21 C for 7 days.
3. For uniform inoculum harvest as follows:
a. Add distilled water t o sIant.
b. UghtIy rub the spores off a mbber poticeman.
c. Filter through cheesecloth.
d. Centrifuge and decant off supernatant.
-
Froduction of Apotbtcia of Various Genera of the Sclerotinhxae Botryotinia, Sclerotinia, Ciborinia
(93,94,95)
1. Crow the fungus on PDA for 2 4 days at room temperature.
2. Transfer freshly growing mycelium to petri dishes containing autoclaved soft wheat seed (8 g wheat
and 25 ml water).
Increase of f noculum 161
3. Keep at room temperature for 2-3 days and transfer t o 5 C or 14 C. Keep in dark for 4.6 weeks or
until rclerotia are well developed,
4. Place sclerotia on moist sterile quartz sand in 100 mm preparation dishes fitted with lids. Keep at 0
C in the dark for at least 1 month.
5. Transfer to 5 C in the dark for 2 weeks.
6. Spermatite (use either spermatial suspension in sterilized moist soil or water and apply to the surface
of sclerotia with a sterilized camel hair brush). In any event sclerotia should be covered with a thin
layer of moist soil.
7. Transfer t o 14 C for one month.
8. Place cultures in the greenhouse exposed t o north light and shaded from direct sunlight.
Temperatures should be at 10-14 C. Add sterile water at intervals to keep the sand moist.
Condial increase of Venturia inaequalis (312)
1. Make up the following:
4% solution of Difco malt extract.
single thickness cheesecloth, 6 by 10 cm.
2. Add 2030 ml of malt extract solution to 8 oz flat sided prescription bottles fitted with screw caps.
3, Pkce chcesecloth against inside of the front face of the prescription bottle (a rubber policeman
helps).
4, Autoclave for 20 min at 1 S Ib.
5. Inoculate with 2 ml of a spore suspension or streak frotn a PDA slant 'A" above line of medium when
the bottle is placed on its side.
6. Place bottle flat with cheesecloth surface immersed in the malt broth for 4 days at 20 C plus or
minus 2 degrees (light not needed).
7. Turn bottle on side for an additional 10 &ys.
To harvest Conidia of If. itracquolis without contamination (31 1)
1. Make up the following:
125 hl er~enrne~erflasks fitted with 6 by 6 crn cheesecloth squares covering their mouths, in turn
covered by an inverted beaker .- autoclave.
2. After 14 days of growth in wick cultures pour off and discard broth.
3. Add 20 ml of sterile water to bottles aqd discard this liquid.
4. Again add 25 rnl of sterile distilled water, tighten caps and agitate.
5. Partially lift inverted beaker and pour suspel~sionthrough cheesecloth into sterile erlenmeyer flasks,
Reduction of ascocarps of V. inaequalis
A (24)
1. Make an aqueous suspension of conidia and/or myccliurr. of 2 compatibile types.
2. Transfer t o Petri dish and add cooled PDA fortified with apple decotion, medium no. 13.
3. E. B. Williams (31 2) prefers to make a conidial suspension of 2 compatibile types, mix them with
an equal volume of double strength agar and slant in a sterile test tube.
4. Grow at 16-20 C for 10-14 days,
5. Place cultures at 8 C until asci form. This usually takes 3-4 months and does not require light.
B
Above medium can be replaced by discs of apple leaves sterilized by propylene oxide and placed
1.7% water agar (247) or by a semi-synthetic medium (246). Seal dishes with parafdm and avoid
using square plastic dishes (248).
Innease of Vcrticillium albo-atrum
(Production of Microwlerotia and conidia)
I. Cut out discs of permeable cellophane slightly smaller than size of a Petri dish.
2. Autoclave in Petri dishes containing water. .
3. Add a single sheet of cellophane to the surface of Czapek agar. If ingredients of medium a n
highly purified, it may be desirable to add 50 & biotin11 for increased numbers of spores and
microsclerotia (1 43).
4. Flood plate with 1 ml of a conidid suspension.
- conidia
For For microsclerotia (261)
1. Grow under continuous or alternating 1. Grow in partial or complete darkness
fluorescent light at about 72 F (144, 165). at about 72 F.
2. Conidia are produced in sufficient quantity 2. Microsclerotia are numerous in about
in about 5 days. 4 weeks.
Use of the cellophane technique avoids incorporation of the culture medium when soil is to be
inoculated. If only conidia are desired they are more easily harvested, making the use of cellophane less
necessary. Microsclerotia are harvested by peeling the cellophane off and macerating it in water in a
Waring blendor.
Larger sclerotia of a more natural size are often obtairled from infected potato stems in certain
areas of the U.S. (R. J. Green personal commun~cation).
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Establishment of Disease
Inoculation, Infection and Diseased Symptoms
Definition of terms - Inocultion, infection, incubation

Objeaives and Precautions in Establishing Discase
SLjor mqulrcments for Establishment of Disease
1. A susceptible host
2. A pathogenic organism
3. The host is at a susceptible stage and condition
4. The inoculum is of suficient quality and quantity
5. fYle hoculum is placed in a suitable infection court
6. Environmental conditions are suitable for infection
7. Environmental conditions an suitable for disease development and symptom expression
W c Inoculation Techniques
I. Exposing aerial portions of the host t o diseased host material
2. Application of inoculurn t o external surfaces of the host
3. AppUcation of hoculum to inaccessible infection courts
Partial vacuum
Airbut
Wounding and inwrtion
4. Infestation of soil
Some SpecZc Considerations
Quantitative inoculation
S c t t h g tower
Horizontal spraying
Pipette deposition
Cotton swab technique
Mrrrurhrg a l l concentrations
Counting chambers
Photometric counting
Moist chambers
Tab& 17. Sources of q u i p m e n t for molst chambers
D~t.chedk f ~ l t ~ r c
Litemturn Cited
DEFINITION OF TERMS
-
Inoculation Transfer of inoculum from the source of production to infection court.
-
Infection Includes germination or growth and penetration by the pathogen until it is in organic union
vith the host (or obtains nutrients from the host). This period is called incubation by Whetzel(l36)
md Yorwood (1 41).
Establishment of Disease 177
Incubation - The period of development of the pathogen in the host until disease symptoms are
expressed. (Called infection period by Whetzel(I36) and Yanvood (141).)
As it is difficult to separate precisely the processes of infection and incubation, many use
"incubation period" t o include the time between inoculation and the development of symtpoms (1,
80). However, the separate terms are useful as for many organisms the environmental requirements for
these two processes are different (141). Prepenetration, penetration and postpenetration are being used
increasingly by rust workers (1 17).
OBJECTIVES AND PRECAUTIONS IN ESTABLISHING DISEASE

The objective of an inoculation often determines the procedure. For example, if resistance is
sought, the conditions should not be so severe that plants having some resistance in the field might be
susceptible in the test, thus losing valuable sources of resistance. Limited greenhouse space may require
testing of young material (often more susceptible than older plants) and the use of large amounts of
inoculum to insure against escapes. It is always necessary to study these materials in the field under
suitable epidemics before their worth and the method can be evaluated. The interest in general
(horizontal resistance) as opposed to specific (vertical resistance) races of the pathogen (133) has
influenced inoculation procedures, and has resulted in a different evaluation of host pathogen
interaction. Instead of merely the size of lesion or reaction type, numbers of lesions, length of
incubation period, amount of sporulation (133) and ability to yield with disease (tolerance) (1 8) are
considered.
If demonstration of pathogenicity is desired, more severe conditions may be used, although
conditions should not be so severe that the significance of an organism is overestimated.
It seems important to have criteria for determining pathogenicity, particularly as genetic,
taxonomic and epidemiologicd concIusions are often drawn from tests of pathogcnicity. It is not
unusual for organisms that have never been associated with a disease of a particular plant species to
induce a visible lesion (57, 72). According to Klement and Goodman (73), probably ELU plant
pathogenic bacteria, except those causing soft rot, often induce a visible hypersensitive reaction (HR)
when inoculated in sufficient quantity, i,e., more than lo6 ceIls/ml to nonhost plants, and particularly
if inoculum is forceably introduced, But a species that is pathogenic and virulent for a given host will
produce "typical symptoms" that usually develop more slowly than HR.,Also, virulent pathogenic
bacteria will continue to multiply in the host tissue (72,73). As for fungi, the reactions also need to be
compared with those seen in the field. If the organism normally reproduces on other hosts, perhaps this
should be a requirement. An alternate or supplementary criterion would be the demonstration of
growth in or on the host by reisolation after some suitable period after inoculation. Reisolation, one of
Koch's postulates, is often neglected by investigators. These criteria, variously modified with the kind
of disease and organism involved, should in conjunction with a consideration and evaluation of:
quantity of inoculum, environmental conditions, and condition of the host, permit an assessment of the
disease-inducing abilities of the organism.
Knowledge of the conditions necessary for infection by plant pathogens, as Eide (38) points out,
may dso be a u s f u l byproduct in the control of diseases.
MAJOR REQUIREMENTS FOR ESTABLISHJIENT OF DISEASE

Many factors determine the success of an inoculation. It is obviously necessary to have
1. A susceptible host
2. A pathogenic and virulent organism
The latter, in most cases, entails the use of an isolate w h o x pathogenicity has been reasonablY
assured by recent isolation from the host or by maintenance of the culture by some method which
avoids avirulent cultures.
178 Establishment of Discare
The other major requirements are:

3. The host is at a susceptible stage and condition
As many hosts vary in their susceptibility with age, it is necessary to inoculate at the rigllt staxe
for maximurn infection and disease development. Occasionally, infection will occur, but the pathogen
will remain dormant with delayed symptoms. An example is Botryosphaeria rot of apple (1 19).
Observation of disease development in the field or inoculatio~iof plants at different ages will help
determine the most suitable stage. With field observation one needs to distinguish between environ-
mental conditions favorable to reproduction and spread of the orgunisni and the actual level of host
susceptibility. Hirst and Stedman (62), with late blight of potato, argue against Graingcr's claim (55)
that the potato itself fluctuates in susceptibility. They suggest the plant, by its increasing growth,
progressively modified its microclimate to substantially favor the spread and development of
Phytophtl~orair~festans.Exaniples of diseases where host susceptibility changes with age are dry rot of
potatoes (12)) powdery tnildew of grape (33); for others see (140). A disease having a precise period of
susceptibility to loose smut of wheat (anthesis), while the length of the period of susceptibility to
dampingaff of alfalfa and red clover seedlings by Pytirirtni debaryanunr is affected a good deal by the
enJironment ( 2 5 ) . Calhoun ( 2 8 ) , admitting that age may determine susceptibility or resistance, stresses
that environment determines the time of change.
Environmental conditions prior to inoculation may influence susceptibility of the host @re-
disposition). In some diseases predisposing conditions are alrnost prerequisites for suitable infection.
Water congestion (68, 69), excess nitrogen (44, 129) and "weakening effects" such as high temperature
and bruising (98) may be used to obtain successful infection, particularly with less aggressive organisms.
Root rots of red clover are favored by repeated clipping of shoots (42,46) and Phytophthora root rot
of citrus by periodic water IolSging of soil (1 31). Yarwood (140) widens the concept of predisposition
to include negative and positive effects on susceptibility and includes the effects of age. But he points
out that, when trea!ments such as water soaking and nutrients continue after the host is inoculated,
they are not strictly exaniples of predisposition.
For instances where nutrition is reported to affect disease susceptibility see (24, 140).
4. The inoculum is of sufficient quality and quantity
Rarely one encounters fungi such as i\fjcosphaerella which produce spermatia (specialized sex
cells (1)) that are incapable of infection. These spores are usually bacilliar in size and form and are
produced in pycnidialikc structures.
Young sporulating cultures seems to result in more infection than spores collected from older
cultures, even if the latter has a high germination (78). Bromfield (130) uses a spore germination-rate
test (hydrated spores incubated at IS to 20 C for 2 hr) to assess the potency of cul:ures of stem rust.
A minimal level of inoculum (numerical threshold according to Cauuman (47)) is necessry to
obtain satisfactory infection and disease development (41, 47, 59). Cohoun (28) speaks of the
compensatory effect where a high concentration of inoculurn may overcome less optimal environmental
conditions. High levels of inoculum will usually decrease the time for symptoms to appear (130) or the
generation time (77) (time for the organism to sporulate) but, rarely, too much inoculum may decrease
infection (47,59) and, more commonly, confuse host reaction types (32).
According to Garrett (48), root parasitizing fungi require larger amounts of inoculum than other
pathogens, but Toussoun et a1 (130) suggest that excessive amounts of inoculum in testing pathogens in
supplies nutrients that affect pathogenic behavior.
There is an increasing awareness of the need t o control amounts of inoculum (4, 104, 115,130).
5. The inoculum is placed in a suitable infection court
Some parasites attack almost any part of the plant, but others are more rpcific. Gibbcrelkr zeae
Can infect most parts of the corn plant but only at certain ages. It is often necesrary not only to pkce
the organism on a particular part of the host but to place it internally (the latter particularly applies to
bacteria). For example, Hildebrand (61) obtained infection with Apbacterium tumefacienr only by
wounding, and the greater the wound, the more successful was the chance of infection. Some bacteria
readily infect via natural openings, such as lenticcls, stomates, hydathodcs (84) and uninjured trichomes
(81), without high pressure spraying, rubbing or brushing; but most bacterial pathogens require or are
favored by these treatments (9). Some fungi are also favored (8,34), It is not clear to what degree thea
treatments assist the entry of sufficient numbers of the bacteria through the natural openings of the
plant, or if the infection occurs mainly through the injured cuticle and/or trichomes. Also, a degree of
watersoaking may result which favors bacterial development (68, 69). Vakilli suggests (132) wounding
by these treatments causes metabolic changes that are prerequisite for the infection of Xantl~omonas
ves/cator&.
A knowledge of the prevalence of stornates (37,99, 123) may be of value, as is also the stomata1
behavior of the host. Allington and Feaster (2) obtained best infection of soybeans when sprayed with
Xanthontortas phaseoli var. sojense at the time of the day (8 am to 2 pm) when stomates were open
widest. Usually later in the day is traditionally the time used to inoculate plants in the field.
6. Environmental cot~ditionsare suitable for infection
Most foliar microbid pathogens can infect overnight in a saturated atmosphere at a range of
moderate temperatures, 20-25 C. Others require specific temperatures or a longer period in a moist
chamber, The latter probably favor post-infection development as well as infection. Prolongcd periods
in a moist chamber can also have adverse effects, as it does on stem rust of wheat (109). This effect
may result from light insufficient to stimulate penetration rather than to the moisture itself. Some
powdery mildews are inhibited by free moisture (1 13). Nair and Ellingboc (97) found that conidia of
Erysiphegramkis germinated best at 100%rh followed by 60% rh, They pdstulated a mixed population
having different moisture requirements.
Suitable incubation temperatures can not always be found from the data of irr vitro growth tests,
as Dickson (35) and Leach (83) found with dampirrgdff organisms. They concluded that the relative
growth of the host and the pathogen, as controlled by temperature and other factors, determine to a
krge degree the amount of disease.
The importance of precise temperature control, at least for one disease, is shown (134) with 2
different kinds of resistance to fi~sarirtrn wilt that are determined by about a 2 C difference in
incubation (broad sense) temperature.
Light, as previously mentioned, is important in obtaining infection with many foliar bacterial
pathogens whose entrance is gained by stomates. Conversely, light may have an inhibitory effect on the
germination of spores of some fungi, e.g., Plrccinia gramitlis, (Sl), P, recondita (SO), and Glueo-
sporturn nrusuntm (52). In the first, inhibition begins at about 200 ft-c and in all inhibition is largely
overcome with time,
Brornfield (13) and Sharp et a1 (1 17), with P. graminis f. sp. ttitici, point out the series of
processes as involved in infection, and that these processes have quite different and somewhat opposing
temperature and light requirements. Thus a constant and uniform environment may be undesirable for
maximum infection. It may be that other fungal pathogens also have different requirements for their
preinfection and infection processes.
7. Environmental conditions are suitable for disease development and symptom expression
The success of an inoculation is judged by symptom expression. The most important environ-
mental factors affecting incubation are temperature and light. Humidity requirements, according to
Yarwood (141), are slight for most fungal foliar pathogens but of importance in the sporulation of
some, c.g., downy mildew and anthracnose organisms. Exa~npleswhere disease development is visibly
affected by humidity are thorn favored by high humidities (54). Somc of the earlier work (68, 69)
refers to high humidity enhancing infection, but it seems apparent that disease development by fungi
and bacteria is d s o being favored. Dirnack (36) points out that there has been little sophisticated
environment control equipment available to properly evaluate the effects of atmospheric moisture.
180 Establishment of Disease
The duration of light and its intensity may affect the reaction of Puccftiia sthiformis (P.
gZumu~mjon wheat (6). Low light intensity increased the disease severity of 3 foliar pathogens of
sweet clover (79), while high light intensity favored lesion development of Leptosphaemliana bn'osiuna
on alfalfa (128), Cercospom beticola on sugarbeet (20), and ~~lycosplurerellt musicoh on banana (19).
The quality of light may change the reaction of Helmir~rltospohur?~ gramirrezlm on barley ( 4 7 ) .
Temperature is of extreme importance in expression of disease symptoms, as it controls the time
for expression of symtpoms and may also affect the type of reaction. Data on the effect of temperature
on stem rust of cereals is especiaLly extensive (105, 125).
BASIC INOCULATION TECHNIQUES

Most diseases can be established by using one or more of the following inoculating techniques.
Difficulty encountered in obtaining infection can often be overcome by considering the individual
factors involved with dixase and by studying the disease cycle and the environmental conditions in the
field, Clayton (27) noticed, for example, the rapidity of symptom appearance and severity of the wild
fire disease on the side of tobacco plants exposed to wind and rain. He simulated this effect by spraying
the plants with water under pressure, thus inducing water congestion. By doing this before inoculation,
,
he obtained the same disease expression in the greenhouse as that which occurred in the field,
1. Exposing aerial portions of the host to diseased host material.
The hosts of many pathogens, particularly of obligate parasites, are inoculated by shaking over
them spores from a diseased host or by placing disease material close to or in contact with them. These
methods are often preliminary, where the organism is difficult to culture, sporulates poorly in culture,
or is badly contaminated. Pssudopezzia medicagitlis is a slow growing and poorly sporulating organism
in artificial culture that leaves bearing apothecia are suspended over the host enabling spores to be
ejected onto the moistened leaves (31).
Epidemics in the field are often initiated by positioning disease material or by the inoculation of
known sufccptible varieties in "spreader" rows which subsequently supply the il~oculumfor the bulk of
the material. It saves work and is believed to be more natural and desirable than when each plant is
artificially inoculated.
2. Application of inocrulum t o external surfaces of the host.
Spores or fragmented mycelium are usually suspended in oil, water, or talc and atomized or
dusted on the host. Inoculum may also be added to an irrigation system (49). Dusting a finely
pulverized mixture of mycelium and a grain substrate on the previously moistened host has been used
with success (75). With stem rust of wheat, a 50:50 mixture of oils (107), U.S.P. light petrolatum and
Socony Mobisol 100 (108), or 3lobilrol 100 or Ortho SOS-B alone (15) have beer1 used with more
success than water in some environments. Minimal application of carrier oils is necessary to reduce
phytotoxicity (1 5).
Amendments t o water such as gelatin (0.5%), agar (0.125%), sodium oleate, Tween 20 and 80,
Triton B-1956 (122) are beneficial, Some of these materials should be checked for their effects on
germination and fungus development. They decrease surface tension and some act as stickers (3). They
nrspend the inoculum more uniformly and allow a fdm to be formed on the host surface, thus
hindering the falling off of inorrulum that occurs with heavy droplets. Wiping the bloom off also helps.
Gelatin used with Septorio pusrcrini (56) and S. tririci (1 16) apparently prevents desiccation and may
~ p p l ysome nutrients t o aid rapid gemination and penetration. It was particularly advantageous in
field inoculations (56).
Brown (16) has &own that adding nutrients to the inoculum may increase infection of lettuce
with Bonytir cine= Other examples of enhanced pathogenicity have been shown with B, fabae (34),
Phoma herborurn var. mediaginis (4, 104), and Fusarium oxysporum (53). Endo and Amacher (39)
report stimulation by guttotion water on Helmfnrhosgorium wtvkinionrrm resulting in increased
infection and disease severity of bentgnu.
Establishment of Disaw 181
Relatively new is the use of Frwn 113 (trichlotrifluroethane) for inocuttion of rusts (92).
Urediospores of P. ptaminfs f. sp. tritici should be below 15% in moisture and should not be suspended
in the Freon more than 2 hr. Spraying at a distance of 18" or more from the plants will avoid toxicity.
Another fluorochemical, perflurotniutylamine (Minnesota Mining Corp.) was u ~ t do suspend the
canidia of Erysiphe graminis uith the help of a low intensity ultrasonic cleaner (17). Previously
controlled inoculation work was prohibited by short lived conidia and the 5eII.Sit~tyof the conidia to
water, although the cotton swab method of Sair and Ellingboe has merit (96).
3. Application of inoculum to inaccmible infection courts.
Special methods are necessary t o obtain sufficient infection with organisms that do not readily
reach their infection courts. These may be less aggressive organisms or bacterial pathogens which have
little motility. They usually gain entrance by natural openings, by insect punctures, and other wounds,
or rarely with the aid of other microorganisms (23,41,47,82).
A. Partial vacuum method
The host, usually see& or seedlings, is submerged in a suspension of the inoculum, and air is
exhausted from the container (a desiccator or a pressure cooker supplied with screw clamps can be
used). After this the vacuum is abruptly broken and the inoculum is drawn into openings of the
host, Examples of diseases inoculated by this method are covered smut of barley, loose and covered
smut of oats (431, corn smut (106), and black chaff of wheat ( I I).
B. Air blast method
Air, containing carborundum dust and inoculum, may be blown againrt plant parts. The stigmas
of wheat are effectively inoculated with Ustilago tritici by blowing the dry chlamydospores at a
pressure of 60 Ib into the wheat florets at about anthesis (93).
C. Wounding and insertion technique
One type of drastic inoculstion is the incorporation of carborundum (300 Mesh) into a
suspension of the firsblight bacteria which is sprayed at 15 Ib pressure on and into the floral parts
(21). Rubbing a mixture of carborundum (300 Mesh) and inoculum across leaf sur!*aces assists
infection of many bacteria and some fungal pathogens (8,9,34). Many bacterial pathogens (22,23,
85) are inoculated by pricking the host tissue with needles that are dipped into suspensions of
bacteria or by injections of suspensions with a hypodermic needle. Tissues may be scraped (bacterial
wilt of alfalfa (29)) or cut (Stewart's wilt of corn (85)) before dipping or swabing with inoculum.
Many root rotting and wilt pathogens are introduced by dipping wounded roots (includes natural
wounds and those incurred by the process of transplanting) into the inoculum
The insertion of an infected tooth pick (30, 121, 142) into the host i s used especially with
weakly parasitic organisms. In a breeding program this method must be considered carefully (64), as
corn plants that were resistant under natural epidemics in the field were susceptible when inoculated
with tooth picks.
4. Infistation of soil.
Rootparasitizing fungi and wilt organisms may be inoculated by infesting sterile soil with a pure
culture of the organism in compliance with Koch's postulates. But according to Garrett (a), sterile sod
is not suitable for the study of environmental phases of root diseases, as the most important factor of
the soil environment, the microflora, is deliberately excluded. Garrett suggests the following for m y
root disease investigations:
1. Seedlings to be used instead of older plants.
2. Nonsterile soil free of pathogens.
3. Glass or other moisture proof container.
4. Soil moisture and soil and air temperature controlled.
Chi et d(25) and Hdpin et al(58) described the results of a somewhat s-r cxpcrimental e N P
out with sterile soil or sand in determining pathogenicity of Pythium on red clover as influenced by
environment md age of the host.
182 Establishment of Disuse
To avoid the preemergence phase of damping off, khmitthenner and Hilty (1 12) placed
inoculum 2 cm below soybean seed at the time of planting.
There are several ways to infest or inoculate soil with pathogens. Most investigators consider that
large amounts of tho culture filtrate should not be used when applying inoculum, as serious
abnormalities might occur. It has been shown for a number of diseases (63), and recently with
Hetminthosporium surtvrtm (87), that the source of the inoculum can greatly influence the kind and the
amount of infection obtained. A cornmeal-sand-nutrient medium, plus the inoculurn thoroughly mived
with the soil, resulted in a more uniform and a higher infection than obtained with imperfect mixing or
the use of culture filtrates.
A number o f parasites are soil inhabitants, and once the soil is infested, hidl infection may occur
for many years. Flax grown on flax-sick soil has been selected for resistance to Fusarium wilt disease
since 1919. By this method resistance has been obtained to a number of other diseases such as club root
of cabbage and potato scab (1 27).
SOME SPECIFIC CONSIDERATIONS

Quantitative inoculation
Prescribed amounts of inoculum of fungi may be deposited on plant surfaces visa settling tower,
horizontal spraying, by pipette or by a cotton swab,
Settling tower.
Various settling towers have been described (5, 40, 71, 76, 90) and most often used with dry
g o r e s of the cereal rusts, but dry spores of Piricrthriu or3,:ae (71,76), as well as water suspensions o f
many other fungi, have been used successfully (90). The device consists of a tower, usually circular, 3-6'
high x 2-3'diam, and set on a shallow base equipped with a sliding baffle. A measured amount of spores
(5-20 mg) is propelled upward in the tower by a blast of compressed air or by the discharge of a C 0 2
pelJet gun. After a short interval, usually a minute, the baffle plate is removed and the plants, oriented
horizontally in the base, are exposed to the spore shower for 1-10 min. Evidence indicates there is a less
variability in spore distribution with an exposure of 1-2 n ~ i n(10). Cereal seedlings may be grown in
pots with vertical plastic backs so that the leaves are easily oriented horizontally. Bromfield (13)
reports substantially more infection of P. gramirris f. sp. tritici when the adaxial side of wheat leaves
are inoculated.
A recent modification permits the inoculation of flag leaves or the upper few leaves of tall plants
(40).
Horizontal spraying.
As the use of a settling tower is slow and wasteful of inoculum, some prefer horizontal spraying.
Freehand spraying with n suspension of known concentration (for ways of determining concentration
see below) until runoff is adequate for many tests. Accuracy and precision is improved by rotating the
plants on a turn table (91) while applying a measured amount of inoculum from a fuced-position
atomizer. The amount of inoculum applied is controlled by the length of spraying time or by the
amount of spore suspension in the reservior of the sprayer. Spraying devices giving precision are
described by Browder (IS), McCallan et a1 (YI), Rowell and Olien (108) and by Schien (1 11). The
latter device has a small f ~ e target
d area.
Pipette deposition.
A measured amount o f inoculu~nis deposited on a leaf surface by applying a drop of inoculum
with either a modified hypodermic needle (77) o r pipette (130). In the case of Botrytis fabae the drop
of inoculum war rubbed over the surface of the leaf by hand, thus requiring experience t o obtain
uniformity (78). tidf leaves employed in either a latin square or incomplete block design decreased
plant variation considerably (78),
Cotton swab technique.

Powdery mildew of wheat can be uniformly inoculated using a cotton swab to collect and to
distniutc the spores on the host (96). The procedure is:
1. Use plants inoculated 6 7 days previously.
2. Gently shake plants to remove old conidia.
3. 6 hr later tap leaves so that conidia will fall on a clean microscope slide.
4. Gently blow on Jidc to remove clumps of conidia.
5. Using a rnjcroscope check distribution and condition of conidia.
6. Gently roll cotton swab over surface of slide.
7. Gently roll swab back and forth on leaf.
Measuring CeU concentration

Counting chambers.
The hemacytometer is routinely used in determining the concentration of fungal - spores
. in liquid
suspension. The ~etroff-~ausser counting chamber, because it is only 0.02 mrn deep, is more suitable
for counting bacteria. Bacteria may be stained with methyl violet, or observed with dark field
illumination. These chambers often are used to calibrate other methods, but they can give erroneous
results because of inaccurate sampling and general carelessness in their operation.
Instructions for the use of the Spenser hemacytometer with the improved Newbauer ruling
follow.
The hemacytometer is a single piece of glass with an H-shaped trough forming 2 counting areas. It
has supports that hold the cover glass the proper distance above these areas. Cover glasses and the
counting plateaus are polished. The cover glass surfaces are polished to insure good contact with the
cover glass supports.
The counting chamber should be cleansed with water, alcohol, or a mild soap, and wiped dry with
lens tissue or a clean dry cloth.
The ruling consists of 9 sq mm, (see Fig. 9). The boundary lines of the Spenser improved form of
the Neubauer ruling are the center lines of the groups of three. The central sq mm is ruled into 25
groups of 16 small squares, each group separated by triple lines, the middle one of which is the
boundary. The ruled surface is 0.100 mm below the cover glass, so that the volume of liquid over a sq
mm is 0.1 cubic m m Place a clean cover glass on to the counting chamber, rubbing it into close contact
with the supporting ribs of the chamber. Place a drop at the edge of the cover glass, A wetting agent
such as Tween 80 is often necessary to insure distribution in the original suspension but do not agitate
violently, as annoying bubbles will be formed. The drop will be drawn rapidly into the space between
the cover glass and the ruled area of slidc. If any overflow into the moat occurs, do over, as spores will
be drawn into the moat. kt the preparation stand for 1 or 2 min so cells can settle on the bottom
Check t o see if cells are evenly distributed. Usually smaller fungus spores and bacterial cells are counted
in the center square mm, which consists of 25 groups of 16 small squares. Each group is 0.2 nim square.
Count the cells in the 4 corner groups and in the center one. To avoid counting a cell twice, those on a
line are counted only when on the top and left line. Total up the number of cells in all 5 groups and use
in the formula: No. counted x SO equds no. par cubic mm multiplied by 1000 gives number/ml or cc.
For larger spores or fewer spores, count spores in 5 one square mm rulings labeled A, B, C, D, E,
Number counted x 2,000 = number of spores/ml. Counts should be made on both counting areas and
usually 2 setvps are made. Wilson and Knight (137) recommend that at least 200.250 organisms should
be counted, as the standard deviation is then approximately 15 and the count is within 10-15%.
Photometric countine,
Colorimettrs (wave lengths isolated by $lass filters) and spectrophotometers (wave lengths
isolated by a prism or diffraction gratings) are used to determine numbers of cells. The procedure is to
wash the cells by centrifugation (to remove interfering substances) and to determine the wave length of
IMM
i
Fig. 9 Rulings of the Spenser Hemocytometer with the improved form of the Neubaucr ruling.
(Courtesy of the American Optical Co.)
maximum absorption. Then construct a standard curve by charting percent transmittance or percent
absorbance at the wave length of maximum absorption with known concentrations on linear graph
paper. The original suspension can be determined with the use of a counting chamber and subsequent
appropriate dilutions made. Some organisms and tho wave lengths that have been used are: Fusarium
oxyspomm f: batatas 600 rnp (45), Saccharornyccs pasrorianus 450 rng (89), Verticillium alboatnrm
650 m p (1 14), Xanthonronas vesicaroria 600 m p ( 1 26).
Moist chambers.
A moist chamber may be any container of suitable size that is relatively moisture-proof. These
containers include supported plastic bags (Figure lo), metal cylhiders set in metal trays ~ ( ~ i ~ IuI),r e
plastic and metal cans, glass chimney lamps, wooden frames covered with plastic or muslin cloth
(Figure 12), and specidy con'structed dew chambers.
Leaves can be kept wet overnight by wetting the inside of the container (may be lined with
muslin cloth) and having standing water in the bottom or moistened sand, vermiculite or fiberglass
mats. Fiberglass does not support mold growth (67). A heating coil, a warmed stone placed in the water
or adding warmed water encourages saturation of the air. If moisture is to be maintained for several
days, usually some exposure to sun or artificial light is desirable; however, such exposure makes
periodic misting necessary, Misting can be accomplished by fogging devices that spin water into tiny
particles or by water lines fitted with nozzles such as used in oil burners, fine chemical spray n o d e s , or
deflection type nozzles designed for propagation benches. Nozzles that give extremely fine spnys
require a pump to maintain elevated pressures. Excess runoff is prevented by intermittent operation.
Kteitlow (74) recommends using a model 42 humidifier (Standard Engineering Works) 15 min out of
every hour from 7 am to 7 pm, while Howett and Grainger (67) recommend using a spray n o d e ,
atomized for periods of 15 t o 60 wc at intervals t o obtain 0.7 of rain per day. Humidistats are available
md may be more deshble than timers, as the latter do not take into account fluctuations in weather.
For sources of equipment see Table 17.
Establishment of Dimar 185
Fig. 11. Metal moist chamber.

Dew chambers were pioneered by the Ft.
Detrick group. They allow dew to form in a
saturated atmosphere as a result of heat radiating
from plants to the cooled walls of the chamber.
We have built an inexpensive dew chamber (Figure
13) that, when placed in a constant temperature
room of 13 C, gives dew in the chamber when it is
heated to temperatures of 17 C and above. The
greater the temperature differential from the dew
chamber to the cooler chamber wall, the larger is
the size of its droplet.
-Junction Box
13. Homcnudr bw dumber.

Table 17. Sources of equipment for moist chambers.
Fogging device3
Spinning mechanism
Wdton Laboratories, Union, N. J. 0783
Standard Engineering Works, Pawtucket, Rhode Island
Fine spray nozzles ,
Spraying Systems Co., Bellwood, Illinois 60104
White Showers, Lansing, Michigan 48905
Spray Engineering Co., Somerville, )lass.
Deflection type nozzles
E. C. Seiger, Box 285, Harleysville, Pa. 19438
Redirnix Division, Gray slake, Illinois
Moisture Controls
Dew Chambers
Instrument Specialities Co. Inc., Lincoln, Nebraska 68507
Percival, Boone, Iowa
Dew hfeter
Used in conjunction with a Bendix Friez Hygrothermograph
Instrument Shop, Iowa State University, Ames, Iowa
Detached leaf culture

Detached leaf culture (defined as the maintenance of leaves on a liquid or agar medium, usually
for several days to weeks) may be highly useful in determining pathogenicity (loo), screening for
resistance (1 35), evaluating fungicides (66), and for genetic (94, 100) and race studies (1 18, 15) of
plant pathogens. It is also used in environmental (120), and physiological studies (102, 110). Some
advantages of detached leaf culture are: economy of space, plant material, and inoculum; easier control
of temperature and light; ease of observation, and l e a chance of contamination. The artificiality of
using detached leaves may not be as great as expected as demonstrated by the correlation of results
between intact and detached leaves (135), particularly if vigorous leaves are maintained. According to
bwings and Acha (86) PhytopI~thorainfestans grew more in excised leaves than on intact plants, but
the difference was reduced when the plants were become naturally senescent.
Some suggestions for &:ached leaf culture:
1. h b c h leaves late in the day for high leaf carbohydrateqrotein (1 39).
2. Collect only young and middle-aged Icaves, being careful not to damage them or allow them to wilt
(139). Leaves from greennousegrown plants often are more desirable than field grown plants.
3. Keep leaves in closed (open favored by some (14,95)) containers under fluorescent lights; 1OG200
f t c is often adequate (94, 100) and avoids temperature increases. Recent (20,128) reports indicate,
however, that high light intensity is important for normal reactions in some diseases.
4. In addition to water (most commonly used alone), kinetin, 0.4 to 20 ppm (65, 94), and
benztnidazole, 30-100 ppm (14, 110, 120), should be considered because of their ability to
maintain proteinchlorophyll content (10, 110, 124). The concentration of benrimidazole inn*
enced the reaction to rust (1 10) and to Septorio tritici(116).
Establishment of D~WPE 187
5. If sucrose is used, a concentration around 2% is recommended. Do not heat sterilize, and avoid
adding minerals, if possible (both favor secondary organisms) (1 39).
-
6. Skoropad (120) used 10 ppm of a u r e o s a to inhibit bacteria in a kinetin solution maintaining
barley leaves.
7. h v e s or leaf discs placed on moistened filter paper are easier t o handle.
8. Grass leaves are frequently placed in open test tuber or the basal portions submerged in a divided
petri dish placed in a slanted rack (1 16).
For the literature up to 1946 and s o m basic remarks see Yarwood (1 39).
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Establishment of Disusc
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I
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EstabliJment of Disease 191
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Preservation of Microorganisms
LnWuctim
.. of Methods
Table 18. Recommended methods of preserving various goups of microorganisms
Periodic tmldez
on overiry
F==h
A. Decptreezc
B. Gyopnic
Lyaphilization
A Rcfrrtbng
B. Centrifugarion technique
C Hlgh-v~cuurndrying
Soil arlt.Um
Ddod bust tissue and dded media cultures
Orha methods
h S i t b gel technique
B. Water prtrmation
Litn;lNre cited
INTRODUCTION
Cbangcs in virulence, culture characteristics and decreases in sporulation occur in routine culture.
To p m e n t or lessen these adverse changes and to minimize contamination, the original culture can be
prtoerved by techniques which reduce or stop growth. Preservation also eliminates the necessity of
frequent transfer. The variety of methais given here and elsewhere (15, 46) reflect the lack of one
satisfactory and convenient method for all microorganisms and perhaps the rugged individualism of
maarch workers. It may be desirable to preserve valuable cr.ltures differently, or at least to keep
duplicates in different locations. Regardless of the methods used, organisms should be grown on media
that produce large numbers of viable cells that are longlived (1 2). High post-infection temperature and
Mted plants decreased original germination of urediospores of Puccinia coronafa and influence
storabfity (28). AU cultures should be checked for purity before preservation; streaking of cultures or
growing in broth is useful in detecting contaminants. For more information on preservation see (15,46,
47) theoretical aspects (46,47,53) and needs and procedures used by culture collcction establishment5
(62). Tabk 18 lists methods recommended for the preservation of various microorganisms.
Table
Actinomyces
IS.
-
R e c o m m e n d e d methods of preserving various groups of microorganisms.
Generally Recommended Methods of Preservation
Lyophiliza tion
Oil overlay
Soil
Bacteria
Lyophilizat ion
Deep freeze
Cryogenic
Water (promising)
Oil overlay
Fungi
Most Fungi
(having spores that are relatively Lyophilization
small and nondelicate) Oil overlay
Soil
Cryogenic
Water (promising)
Mildew fungi - cleinothecia Dried host tissue
-
-
cbnidia
Rust fungi urediospores
Periodic transfer
Cryogenic
Vacuum drying
Deep freeze
-
Smut fungi tcliospores
myceiial or sporidial cultures
Lyophilization
Oil overlay
Aquatic fungi (including Oil overlay
Phyfophthora and P)*titium Cryogenic
Slow freezing
Non-sporulating fungi Oil overlay
Cryogenic
Soil cultures
Spomlating fungi that do not survive Cryogenic
lyophilization (large spore fungi,
and those having delicate spores:
B o t ~ t i sChoa~ephom)
,
DESCRIPTION OF METHODS
PERIODIC TRAYSFER (Active Culture) - A method widely used but not widely recommended.
It consists generally of transferring cultures on agar slants twice a year. After suitable growth and
Wrulation, the slants are kept at 4-5 C. Spores, rather than only mycelial transfers, tend to maintajn
btegrity of organism Weak strength natural media are advised, with no or only low concentrations of
m@r. Potatocarrot agar, V-8 juice agar, hay infusion agar, and PDA are commonly used for fun@ (62).
Some prefer t o alternate media (78). Periodic transfer works we1 with a few stable organisms, is
convenient and allows ready access and checking of organisms. It is laborious when a large number of
cultures are involved, for they invite undesirable changes of most organisms and contamination. Test
?reservation of Microorganisms 195
tube plugs "Scoated" have been used t o lessen contamination (1). Poisoned plugs (71) and capping
tubes with cigarette paper (66) prevent contamination by mites. Addition of Cyprol or Kelthane2 to
culture media gives control of mites with apparently negligible fungicidal effects (63) Screwcapped
vials hinder desiccation and mites, but Raper (57) finds more failures with them than with
cottonplugged slants. If used, I find them valuable. Cultures should not be kept tightly capped at room
temperature,
Conidial cultures of Erysiphe grarninis, like most powdery mildews, are not readily preserved aqd
arc maintained by transfer at 4-6 wk intervals keeping the culture at a low temperature of 12 C and low
light intensity (49).
OIL OVERLAY - A simple but relatively effective method, particularly valuable as an adjunct t o
other methods. With some non-sporulating organisms it may substitute for lyophilization. Allow the
fungus (bacteria are sometimes preserved by this method (34)) t o grow vigorously on slants preferably
equipped with screw caps. Cover the slant with high purity mineral oil (medicinal grade) t o a depth of 1
cm above the uppermost edge, being careful not to get oil on the necks, plugs or caps. The oil is
sterilized by autoclaving for 45 minutes at 121 C in 125 nd amounts. A large number of cultures can be
handled using a soparatory funnel connected to a bell-shaped pyrex filling attachment. Most cultures
are stored successfully for a year or more at 5 to 10 C. Mites are no problem, but occasionally species
of Penicillium give trouble, as they can grow in the oil at these temperatures. Members of all classes of
fungi have been stored by this method (8). Pyrhitrrn and Phyruphtliura are amenable (73) (1 have not
been successful with some species) as are most of the water molds (32) which are not readily
lyophilized. It was rated poorer than lyophilization and storage in soil for preservation of filamentous
molds (1 5).
-
FREEZING May be arbitrarily separated into L'deep free~ing" - temperatures t o -100 C,
although temperatures usually are from -10 to -60C, and "cryogenic" temperatures from -100 to -196
C. These techniques are valuable, but they require ~naintenanceof continuous low temperatures.
Thawing and refreezing is harmful. Cost for refrigerators maintaining temperatures of -60 C and below
may also be prohibitive. Generally, culturable microorganisms are suspended in a 10% glycerol solution
t o lessen freezing damage and are recovered on a complex medium.
A. Deep freeze - Certain pathogens, particularly bacteria and some obligate parasites, are
maintained by freezing infected plant parts. Xarrrltort~omspkaseoli var. sojense survlved in infected
minced bean leaves for 2 years at 0 to -20 F (33) as did Albugu occirlenralis and Peronospora effusa in
infected spinach leaves at -10 C for 5 t o 6 months (52). Some other organi,ms prearved in frozen tissue
are: Phytupilrhora phaseoli (77), Co~~nebacterirtm insklionrm (37) and Psertdumunas glycinea (1 7,36),
Freezing fungus cultures on agar appears useful. Carmichacl (10) recommends that aluminum
capped vials fitted with rubber gaskets bc used. Caps were loose during growth of organism and
tightened when placed in the freezer, approximately -20 C.
Freezing bacteria in a phosphate buffer with glycerol appears superior to lyophilization in
maintaining high viability for 2. years of storage. This technique, as developed by Tanguay (70) and
others (65), preserves a number of different bacteria. The technique is as follows (70):
1. Inoculate 100 ml of media with 10 ml of a 16-1 8 hr culture.
2. After 24 hours centrifuge and wash culture in MI15 pkosphate buffer pH 7 (KH2P04 7 9 mg,
K 2 W 4100 mg, distffled water 100 ml), volume for volume.
3. Suspend washed cells t o 407i original volume in phosphate buffer containing 15% glycerol.
4. Fill ampoules % full.
5, Carefully heat seal ampoules.
6. Place in deep freeze at 4 0 C.
7. Thaw out inoculum in water bath a t 25 C.
' 1.8%
1.18%pynthrln and 11.87% piperonyl butoxide
af d).(pchlorophenyl) tdchloromethylcubinal
196 R c ~ m t i o nof Microorpnismr
A rimplified version used by Quadling (56) for Xanthomonas pluseoli is as follows:
1. Mix equal volumes of sterile broth containing 30% W/V of dycerol with culture in the late
logarithmic phase (10' ml).
2. Place 1 or 10 ml aliquots in a screw-capped vial and tighten.
3. Store at -20 C.
4. Thaw culture in air of 25 C.
He a h suggests that small colonies can be picked directly from agar and placed in small volumes
of glyc~rolbroth. Clement (11) suggests aqueous glycerol (15% W/V) and rapid prefreezing before
storage at 4 0 C.
Schein reported (60) substantial viability of urediospores of Vronyces phaseoli var. t,pico after
almost 2 years at -60 C in gelatin capsules, without amendments or pretreatment. After storage,
hydration of spores (24 hours in a moist environment or spores floated on water for 2 hours) increased
germination considerably.
B. Cryogenic Storage - Cryogenic storage is increasing as small commercial liquid nitrogen
refrigerators are now available costing from $250-300(Lindc Division, Union Carbide, Tonawanda,
New York and Cryogenic Engineering Co., Denver, Colorado 80216). They require filling about every
month. Mechanical refrigerators now generally available approach the cryogenic range, -96 C. Cryogenic
storage is suggested particularly for organisms not successfully preserved by lyophilization (29,30) or
where there is a concern that the usually low percentage of survivors by lyophilization may not
represent the original genetic population.
Muggelton (51) suggests the following for cryogenic storage of living cells.
1. Cultures should be kept in small volumes: 0.25rnl in 2.0ml thin glass ampoules.
2. One of these: Glycerol (10-2m), dimethyl sulfoxide (It%), glucose or sucrose (Im) usually
aids survival.
3. Freezing should be "slow", about a 1 C decrease per minute down to -20, or -35 C (30). Linde
and Canal Industrial Corp., Bethesda, Maryland, have equipment to accomplish this process,
but it can be roughly approximated by putting ampules at -20 C in a deep freeze for 1-2hr (7).
Species of Pythium and Phytophthora and certain other organisms seem to require this rate.
Cultures should then be quickly transferred without thawing directly to the liquid in
refrigerator (7).
4. When activating cultures, thawing of cultures should be rapid. Generally they are immersed in
a water bath at 38-40 C for 1-2 minutes (20, 30). Hwang (30) has, using essentially t h e a
recommen&tions, successfully stored spores or mycelia of a number of organisms for 5 yr.
A p r slants (75) have also been successfuUy stored,
Tht preparation of bacteria for cryogenic storage is about the same as for "deep freeze"
temperatures. Some workers prefer to wash cells and to quick freeze and t o thaw quickly. Storing test
organisms for microbiological assays in the concentrated assay medium appears feasible (64).
The original percentage germination of uredoispores of fucciniagrarninis f. tririci was maintained in
liquid Nitropn without amendments (44), and the induced "dormancy" broken by thawing at 40 t o 50
C for 2 minutes (43). According to Loegering (40), a vial of spores may be stored for less than 10 cents
per year permitting its use by the American Type Culture Collection. See (41) for details of the type
culture collection of mst fungi and (39, 42) for systems for rapid location and retrieval of frozen
cultures
Lagering et d (42) stresses these safety precautions in using liquid N.
1. Do not work or store liquid, N in a nonaerated room.
2. ~ eimprfect
t in ampoules by placing them in a dye solution for H hr prior to freezing.
3. Bt extremely cautious when removing ampoules from refrigerator as they may explode. use a
fb mask.'
4. Handle objects kept in refrigerator with asbestos gloves.
1 hvo found r pdytrtct f j , Scotch Pak (Kapak induotricc.,Minneapolb, M i n 55416)
~ to be a qfa subrtitutc. It is
rutoclnrabb md cpn k hest reded. Tuite, J. 1968. Liquld nltropn storwe of fungi scaled m a p o l y c ~ r ~ ~ .
Mycolofi 60:591494.
-
LYOPHILIZATION A highly recommended and increasingly popular-way of preserring many
microorganisms, particularly for long periods (77% of fungi tested survived 17 yeard(27)). Almost all of
the large culture collections depend on this method (45). The majority of bacteria., actinomycetes (25),
and many fungi, including yeasts(9), respond well to lyophilization, although total numbers of surviving
cells may be relatively low. Fungi that do not sporulate or have spores that are delicate or unusually
large respond poorly.
Lyophiiization usually consists of drying spores or cells from the frozen state and storing in
vacuo. There are three methods in general use: ( I ) prefreezing, (2) freezearying centrifugation, and (3)
vacuum drying (where freezing may not occur). Drying from the frozen state is believed to be
advantageous because removal of water from the rigid framework that freezing maintains does not
allow proteins to come in contact with high concentrations of other proteins or salts which would lead
to protein denaturation (18, 21). Meryrnan (47) mentions the importance of prevention of shrinkage
and maintenance of solubility by freezing, but also that freezing is incidental to reduction in
temperature, which slows the adverse chemical reactions responsible for death.
Reprdless of the method used, one of the newly lyophilized tubes for each organism should be
checked for viability t o avoid "dead" storage.
U s u ~ ythe spores or cells or the organism are suspended in a colloid, e.g., 10.12% skim milk
(14), beef serum, or in a solution of 1% lactose, glucose, or sucrose. "hlist desiccans" (3 part serum,I
part broth, and 7.5% glucose) is a popular menstruum. Spores of Puccinia coronata, however, did not
survive lyophilitation when suspended in a number of these media, but they did survive well alone (61).
P. strwormis and ~llelamspornlhi (1 6) are also preserved successfully without media (31). Hemin is
reported t o aid preservation of spores ofPuccinio gramirlis (69). Twenty per cent sucrose or glucose was
superior for Aspegillus nker (24) to the beef serum generally recommended. Certain species of
Pyrhiurn were preserved only by lyophilization of invaded wheat straw (67, 68). Thus, the particular
suspending medium appears t o be important and needs more study. A list of suspending media used for
bacteria may be found in Heckly (26).
Whatever the suspending vehicle, its original sterility should be verified by streaking on broad
spectrum media. Beef serum is usually sterilized by filtration, and Weiss (74) advises autoclaving 5 4 ml
of skim milk for 1 3 minutes at 115 C to avoid caramelization.
Lyophil cultures are best stored at a temperature of 0-4 C (26, 38). They can be opened with a
minimum of danger to the culture and the
worker by the following method. Wipe a large - Outlet to
area of the tube near the top with 70% ethanol, Vacuum Pump
makc a deep mark with a file, cover mark with a
Refrigerant
gcnerous piece of sterile cotton and apply prec
Chamber
sure. There are ampoules available marked with a
gold ring that are prefcored and open readily.
Bacterial cultures are usually reconstituted by
adding r portion t o a broth culture and streaking Freezing Trap
a portion on a complex agar medium
A. Prefreezing - According to Raper and
Alexahder (58) (SRRL Method), this method
uses an apparatus that permits simultaneous tem-
perature treatment of organism. Tubes contain-
ing suspensions of microorganisms hang down
from a g h manifold which can be raised or
lowered above a cooling bath. The procedure Ampoules
follows yith various modifications pertinent t o
this and the three following procedures.
198 Pmcw@ionof Microotpnbms
1. Make a dense suspension of spores or bacterial cells in a witable menstruum.

2. Using a sterile Pasteur pipette (pluged with cotton) add 0.05 ml of suspension to a sterile pyrex
tube 4" long and 6 mrn in diameter. Heckly (26) recommends that the tube or ampoule be about 10X
the volume of the suspension.
3. Push a sterile cotton plug 54" down the tube and burn'tov off.
4. Lower manifold to immersed tubes into a bath containing dry ice and methyl cellosolve (-30to 4 0
c).
'5. Within a few minutes add cellosolve at 0 C t o raise the temperature from -10 to 0 C and start
evacuation. Heckly (26) advises keepins the temperature between -10 and -20 C.
6. When the preparation appears dry, raise the temperature to that of the room. Continue drying 'A hr
at a vacuum of 200 t o 500 p Hg. Xiany prefer to dry for 6 hr or overnight (26), xaling at 50 to 150
p HG (74). Vacuum is often determined by a .\lcleod gauge, although it does not give a continuous
reading and may possibly give off mercury fumes (26). A cold trap (containing dry ice and acetone)
between the gauge and organisms should prevent the latter. Two cold traps are often used to assure
complete trapping of water by condensation t o prevent moisture from reaching the vacuum pump,
particularly if large numbers of cultures are being dried together, it also assists drying.
7. Seal off tubes with a cross-fire torch (air-gas type b suitable). Portable butane torches are also
suitable. It is best to apply a hemostat to rubber connector before sealing off tubes. If your
"sealing" technique is good, this is unnecessary. "Quick seal valves"' are a convenient substitute.
Rotate tube when heating pulling away gently to complete seal. Avoid leaving a fragile tip of glass.
8. Check vacuum with a high frequency spark coil tester (gives a violet discharge inside the tube with
suitable vacuum) immediately and after several days. Do this quickly as pin holes in the glass may
develop. Also vacuum testing adversely affected Puccinia striiformis, decreasing infection (31).
Because of these complications some Laboratories omit this procedure.
The following procedures used with the equipment in figures 14, 15 is different from the previous
method in these respects:
1. Tubes of microbial suspensions and menstruum are frozen by submersion in one of the following
ways: Dry-ice-acetone, dry-ice-methykellosolve, dry-ice-95% ethanol, separate from the manifold.
2. Tubes are either attached successively, time being allowed between tubes to re-establish level of
vacuum, or the tubed suspensions are all quickly attached after freezing and evacuation started or
continued. This method is recommended if adequate vacuum - 500 ~ c g- can be achieved before
thawing, usually less than 5 minutes. "Qu~ckseal valves" permit the latter in most systems.
In the method used by Weiss (74) for the American Type Culture Collection, the frozen
spore-bearing vials are placed in an evacuation chamber, and after drying the vacuum is broken and the
tube placed inside another vial and then evacuated and sealed. The double tube prevents contamination
that may occur when the vacuum is broken prior to culture of the organism. This reference (74),
together with (26), has a considerable amount of useful detail about the technique of lyophilhtion.
There are some commercial lyophilitation models which are not very expensive that use
mechanical refrigeration to condense moisture out of the system and permit freezing of cultures. The
elimination of dry ice with its inconvenience and continuing small expense may recommend these
machines t o those who are doing a considerable amount of lyophilitation.
-
B. Centrifugation technique F r e e d r y i n g occurs when evaporation is extremely rapid and the
material cannot absorb its latent heat of evaporation from its surroundings (19, 21). Frothing and
spilling that may accompany freeze drying is prevented by centrifugation (24). Aftenvards, centrifu-
p t i o n tubes have t o be evacuated prior t o sealing. In a comparative study with one organism (24) this
method was more successful than methods Al and A2, although these results are challenged (15).
Commercial models are available, one from W. Edwards & Company, Lower Sydenham, London, S. E.
Available from the Y i i r Ca.Gardiner, N w Yark

Resewation of Microorganisms
VACUUM
QAUQP CHAMBER
DRY ICE FREEZE-DRYER
Pi#. IS. A m o w IyophiUzatiun apparatus manufactured by Virtir Co, (Courtesy of Virtis Co.)
Xn) Prarerv8tion of Microorganisms
26, England, and also from companies in the United States. Apparatus is not expensive. This proadurn
has worked well for me and it obviates the need for dry ice,
-
C. High-vacuum drying Spores of rust fungi without a suspending medium or prior freezing
survived well after 1 to 3 hours of vacuum of 5 0 to 100 C( lig. They did not survive well with a snap
freering. It i s not known whether freeze drying occurred (61). hfe&t?tpsoralitli uredospores have
mrvived 9 yr of storage in sealed tubes containing CaC12 at 3 C (16). They were evacuated for 15-30
rnin before they were sealed. To avoid possible heat injury the bottom 1 cm of the tube was immersed
ia cool water while sealing.
A gradual hydration of the spores was beneficial to lyophilized rust spores (61), cultures of
fithiurn (68), and spores of Ustilago averiae (38).
SOIL CULTURES - The numbers of fungi preserved in soil is considerable (4, 22). The soil is
generally used to absorb the organism. Stcrile loam soil is inoculated with a suspension of the organism
t o bring the moisture t o about 25% of the water l~oldingcapacity (WIiCX3). (To determine WHC place
a known weight of oven dried soil on a moist fdter paper in a funnel, add water slowly until a drop of
water appears at the apex of the cone of paper. The amount of water is approximately equal to the
WHC.) Five g of a soil is added to test tubes and autoclaved for 1 hr at 121 C. Tubes may be stored at
room temperature or refrigerated, but they should be protected from dust and mites. This method has
the advantage of one tube supplying a continuirlg source of inoculum.
DRIED HOST TISSUE AND DRlED MEDIA-CULTURES - Many plant pathogens survive long
periods when in contact with host tissue. Diseased plant material such as leaf tissue may be dried in a
plant press and kept for a year or more. The fungi causing northern and southern leaf blights of corn
are efficiently preserved in this way (72). Cleistothecia of Erysiphcgrarrtitlis f. sp. hordei on leaves were
viable after 13 yrs at 10 C (SO). Leaf rust collections on wheat leaves placed between blotter paper,
dried over calcium chloride for 3 0 days in test tubes at 2.4 C, and stored in manilla envelopes were kept
viable for 7 months (79). Bacterial pathogens, obl~gateparasites, and organisms usually difficult to
maintain are often successfully stored by freezing the infected host tissue as mentioned earlier. Bagga
(2) reports 19 fungi and 4 species of bacteria survived sealed storage at 1&13 C for 12 montl~swhen
a p t cultures or rice seed cultures were first dried between sterile blotting paper and over CaC12 at 20 C
or lower (2a).
OTHER METHODS - These techniques are included because of their simplicity and their
effectiveness, at least, for the few organisms so far tested.
A. Silica gel technique (after Perkins (54))
1. Small test tubes are preferable with screw-caps and are 'A fded with non-indicating (no dye) silica
gel, 6-1 2 mesh.
2. Sterilize at 180 C for 1% hr (use immediately after cooling or keep in a tightly sealed container),
3. Add 0.5 ml of sterile water to a fresh agar culture to be preserved - loosen spores.
4. If only mycelium present, transfer mycelium t o a sterile test tube containing sterile water and
crush with a glass rod.
5. Add an equal amount of sterile skim milk to water suspension.
6. Using a Pastcur pipette (drawn out of 4 mm glass tubing) add 0.5 d of suspension per 4 g silica
gal, drop by drop. Tap particles to one side and add suspension starting at bottom. Distribute
b p s uniformly. Sitica gel tubes can be chilled or kept in an ice bath t o lessen heat production.
7. After 1 week ample by shaking a few gel particles on agar medium.
8. If viable, screw cap on tightly and wrap tubes in pliafdm and store at 5 C in a tight container.
-
B. Water preservation Consists of placing small pieces of mycelium, spores and agar or 3-5 loopfuls
of bacteria into small capped test tubes containing water. The fungi tested include dermatophytic fungi
(6) m d Ascmhyta and Colletotn'chum (55). There were kept refrigerated while virulent isolates of
Puudomnns w & ~ c e m msurvived for 18-24 months at 22 C (35). The latter is remakable but well
docunrented. B V a y and Schnathorst (13) stored their bacterial ruspenrrions at 10 C.
Prcwmtion of Microorganisms 201
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Resenation of Microorganisms 203
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Person, L. H. 1961. A method of maintaining viability and ability to sporulate in isolates of
Ascochyta. Phytopathology 5 1 :797-798.
Quadling, C. 1961. Preservation of Xa~rthonwtusby freezing in glycerol broth. Can. J. Microbiol.
6:475477.
Raper, K. B. 1962. General methods for presenting cultures. fn Culture Collections: Perspectives
and Problems. U. of Toronto Press.
Raper, K. B. and D. F. ~lcxander.1945. Preservation of molds by the lyophd proses. Mycologia
371499-525.
Rowell, J. B. 1956. Rehydration injury of dried uredospores of Pucciniu graminis var. tririci
Phytopathology 45:24. (Abstr.)
Sfhein, R. D. 1962. Storage viability of bean rust uredospores. Phytopathology 52:653-656.
Sharp, E. L. and F. G. Smith. 1957. Further study on the preservation ofPucci~riauredospores.
Simmons, E, G. 1962. Fungus cultures: Conservation and taxonomic responsibility. In Culture
Collections: Perspectives and Problems. U. of Toronto Press.
Smith, R. S. 1967. Control of Tarsonemid mites in fungal cultures. Mycologia 59:600-609.
Sokolski, W. T. and E. M. Stapert. 1964. Liquid nitrogen freezing in microbiolgoical assay
systems. Appl. hlicrobiol. 6: 178-184.
Squires, R. W, and S. E. Hartsell. 1955. Survival and growth initiation of defrosted Escherfchia
cob as affected by frozen storage menstrua. Appl. hlicrobiol: 3:4045.
Snyder, W. C. and H. N. Hansen. 1946. Control of culture mites by cigarette paper barriers.
Staffeldt, E. E. 1961. Observations on lyophil preservation and storage of Pythium species.
Phytopathology 5 1 :259.
Staffeldt, E. E, and E. L. Sharp. 1954. Modified lyophil method for preservation of Pythiunz
species. Phytopathology 44:213-215.
Stewart, D. M. 1956. A vacuurn drying process for preservation of Pucciniu graminis Phytopa-
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Tmguay, A. E. 1959. Preservation of microbiological assay organisms by direct freezing. Appl.
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Waterhouse, Grace M. 1960. The treatment of important pathogenic PhycomycetesIn Herb. I. M.
L Handbook. Commonwealth Mycological Institute Kew, Surrey.
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Wellman, A. M. and D. B. Walden. 1964. Qualitative and quantitative estimates of viability for
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Wernham, C. C. and H. J. Miller. 1948. Longevity of fungus culture under mineral oil.
Wester, R E., C. Drechder and H. Jorgensen. 1958. Effect of freezing on viability of the lima
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Werterdijk, J. 1947. On the cultivation of fungi in pure culture. Antonie Van Leewenhoek. f.
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Microscopic Techniques
Observing Host-Pathogen Rebtionships
U n c h d intact host tissue
Epidermal impression-coIloidin technique
C k h g of host tissueand staining of fungus mycelium
Fm h m d sections
Mounting and Staining Media for Semi-permanent Slounts of fungi
Nuclear staining
Cram stain or gram method
Staining bacterkl flngelh
Cement recipes for sealing liquid mounts
Ways of Prcparinp Fungi for Slicroropic Examination and/or Herbarium Prevwation
Microculturr techniques
Doubk cover slip technique
Cellophane mounts
Dried reference c u l t u ~
Cellophane method
Literature Cited
Included here is information on handling disease host tissue and microorganisms for viewing
under the light microscope. Preparing material for sectioning with microtomes is not included.
OBSERVING HOST-PATIIOGES RELATIOXSHIPS

Much information on host-pathogen relationships, as well as diagnosis of plant diseases, can be
obtained by simple techniques. Examination of free-hand sections, intact tissues or colloidin
impressions can yield information on these relationships that paraffm sections cannot. In addition,
fiehand sections are often useful in the identification of fungi that produce fruiting bodies. Paraffin
sections or other mechanically sliced sections, however, are essential in any detailed host-pathogen
relationship study.
Uncleared intact host tissue
Some investigators have obtained a surprising amount of information using intact uncleared
tissues by careful manipulation of the light and fine adjustment of the microscope.
Rowell (81) by the following viewed urediospore germination and formation of infection
structures of Puccittio graminis:
1. Spray leaf surfaces with a mixture of 2 mg cotton blue, 5 rng acid fuchsin, 0.2 ml glacial acetic
acid, 35 ml95R ethanol and 23 ml water.
2. Mount with cellophane tape.
3. Examino the surface immediately with bright reflected light.
Microscopic Techniques 205
Epidermal Impression - Colloidin Technique

This technique was originally used t o study the stornates of plants. It is very adaptable to the
study of spore germination on the host and gives information on how the pathogen enters the host. It is
more easily learned than stripping epidermis from leaf surfaces.
Fasten ends of flattened leaf to an index card using scotch tape.'Dip a glass rod into commercial
colloidin (may be thinned with 1 pt abs alcohol and 3 pt ether) and spread in a thin layer over the dry
surface of a leaf. A piece of cellophane placed at one end of the leaf and covered by the colloidin will
assist subsequent removal of the film (71). M o w the material to dry about 30 seconds and then gently
peel off the colloidin strip and place in a drop of lactophenol containing 0.05-0.1% cotton blue or in
lactofuchsin. Place a cover slip on the mount gently to eliminate trapping air bubbles. The colloidin
strip will include spores, fungus mycelium and an imprint of epidermal tissue. Cellulose acetate is
similarly used (70) or is sprayed on (7 1).
Other materials such as acrylic plastic (44) and silicone rubber (82, 95) are used to make
epidermal imprints. They, unlike colloidin, are believed not to injure the host, thus permitting repeated
application, and they also give greater structural detail. The techniques are also more involved.
Clearing of Host Tissue and Staining of Fungus Mycelium
Epidermal and sub-epidermal relationsllips can be more readily determined using cleared tissues,
as the rernoval of pigment from the host facilitates obsewation of fungus mycelium. Many techniques
are available.
Significant amounts of phenol and dioxan are only boiled under a hood, as the fumes are very
harmful (40).
1. Stemphylium solani in tomato, acetic acid-alcoholsotton blue (29). Put leaves in equal parts of
glacial acetic acid and 95% ethyl alcohol for about two days, then 4 tc, 8 days in 7540% lactic acid.
Remove the material and place on a slide in a drop or two of 0.1% cotton blue in lactophenol; after
5-30 min the excess is removed arid clear lactophenol is added.
2, ~ e n t u r i ain apple, acetic acid-alcohol-trypan blue/3!). Fix and clear leaves in acid-alcohol mixture
(as in 1) overnight. Stain with 0.5% solution of trypan blue UI 40% acetic acid and mount in
glycerine or lactophenol.
3, kscochyta in clover, acetic scid-ethanol, chloral hydrate-lactophcnol~ottonblue (57). Place leaves
in a hot solution of acid-alcohol (as in I ) and gently boil for a few minutes until the leaves lose their
chlorophyll and become opague. Clear 30 minutes in a strong chloral hydrate solution. Stain leaves
for several hours on slide in lactophenol containing cotton blue.
4. Gymnoconia in blackberry, alcohol-lactophenol-cotton blue (68). Boil pieces of tissue in 95%
ethanol until the chlorophyll is lcst and then boil in lactophenol containing a very small amount of
cotton blue until tissue is clear.
5. Puccinia graminis in wheat-lactophenol-cotton bluechloral hydrate (84). This technique, although
used for wheat rust, is claimed to have potential in diagnosis. Infected leaves 314" in length are
immersed in 10-15 rnl of 1 pt lactophenol cotton blue to 2 pt 95% alcohol. Lactophenol cotton blue
contains:
Phenol a log
Glycerine 10 ml
Lactic acid 10 ml
Aniline blue 0.02 g
Distilled water 10 rnl
Bring to boiling and simmer for 1 min. After leaves sink, bring to boil again for H rnin. Keep them in
stain for 48 hr at room temperature, then rinse in water, place leaves in chloral hydrate (5 g chloral
hydrate, 2 ml water) for 3G50 min and mount in glycerine, If leaves do not clear, boil in
alcoholic lactophenol followed by clearing in chloral hydrate. Leaves can be kept in alcoh0Jic
206 Microscopic Techniques
lactrophenol cotton blue solution for several weeks. The authors found this method especially useful
for phase microscopy.
6. Erysiphe graminis in barley, lactophenol-cotton bllre (93). Fresh specimens are immersed in 10 ml of
lactophenol containing 17A.l% cotton blue and then brought to boiling over a low bunsen flame
and allowed to simmer for 3 min. The solution is allowed to cool, leaves removed and gently rinsed
in clear lactophenol. According t o White and Baker (93), the material can be kept without
deterioration for at least 6 wk.
7. Cercospora in banana, lactophenol-trypan blue (38). Place free hand sections in trypan blue-
lactophenol (see in mounting and staining media), gently heat, then replace with clear lactophenol.
The mycelium of Cercospora is stained while the host cells remained unstained.
8. ~ o t r ~ in& onion, chloral hydrate-lactophenol-acid fuchsin (83). Clear leaf fragntents in chloral
hydrate for 12 to 24 hr and stain in 0.05% acid fuchsin in lactophcnol for 12 hr.
9. Stemphy/ium in lotus, chloral hydrate-acid fuchsin-bismark brown (30). Place 4 or 5 leaflets in
stender dish 113 full with solution A (see below) for 4-5 days (until leaves are transparent), Transfer
leaflets t o solution C for 2-1 2 hr until staining is satisfactory (younger more succulent tissues are
harder to stain). Transfer to solution A and allow to destain until good differentiation between host
cells and pathogen is obtained. Counter stain in solution D until the stomata and geminating spores
are stained.
Dehydrate leaves by passing through a series of 85%, 95% and absolute alcohol, IS min in lower
grades and 3 changes in absolute, 10 minlchanges.
Clear leaves in synthetic oil of wintergreen for 5-15 min. Leaves are then mounted in damar balsam
or Permount. Spores, germ tubes, and stomata are stained brown, and tile fungus hyphae and the
leaf cells stained pink to red.
Solution A. Chloral hydrate 250 g
Water 100 ml
Solution B. Acid fuchsin *%
used to make up solution C
70% alcohol
Solution C. 95%alcohol 8 ml
Solution A 12 ml
Solution B 1 ml
Solution D. Bismark Brown 2%
70% alcohol
10. Hyphae in roots, chloral hydrate-acid fuchsin (36). Boil roots in a saturated solution of chloral
hydrate containing 0.01% acid fuchsin, and examine in clear lactophenol. Also good fur detecting
nematodes, but sl~ouldbe destained by autoclaving in clear lactophenol for 10 min at 121 C (2).
11. Hyphae in various hosts, chloral hydrate-lactophenol-cotton blue or aniline blue. Place material
overnight in 5 pt chloral hydrate, 2 pt water ana u.15'0 of aniline biue. Airer s~uningmount the leaf
pieces in chloral hydrate for several hours. According to Bell (6) better clearing and staining was
obtained by twice subjecting the leaves to vacuum in the staining solution followed by about 4 hr at
60 C.
12. Bremia lactucae in lettuce (62).
Necrotic tissue whole mount
Place tissue for 30 rnin in 7% NaOH.
Transfer to 5% NaClO for 15 min or until brown disappears.
~ o u n int water or in 0.4% KOH. No stain is needed.
L M ~tissue -
R whole mount
Place tissue in one pt glacial acetic acid and 3 pt of 95%ethanol for at feast 3 hr.
Transfer to lactophcnol containing 0.01% orseillin BB (Allied Chemical Corp.) that is, at 95 C.
Seedlings in the cotyledon stage are left in the solution 4-5 min, ~cedlingswith 2 t o 3 leaves
require 6 7 min.
~ o u n tissue
t in lactophenol. If over-stained, soak the tissue in hot lactophenol for several min.
Fresh sections (about 5 0 p thick)
Mount sections in lactophenol-orseillin BB (as above).
Cover with a dip and keep on a 95 C hot plate until all bubbles cease to boil from the specimen.
OrseiUin BB stains the fungus mycelium bright red and host cell walls light pink.
-
13. Ustlhgo nuda in barley embryo &OH-sodium silicate-trypan blue-lactophenol (64,65,73),
Extraction and staining of embryos
Using a 1 1 beaker vigorously boil 6 W 7 0 0 barley kernels in 500 ml of water containing 25 g of
NaOH, 70 ml of liquid glass (sodium silicate), 1 drop of liquid detergent (prevents foaming), and
0.5 t o .25 g trypan blue, for 45-60 min, stirring occasionally. Add water to replace water
evaporated.
-
Pour the hot gelatinous mass on a series of screens of 4, 6, 8 and 14 mesheslsq inch spraying
warm water gently on the mass to aid separation.
Transfer embryos from lowest and finest mesh screen into a pan, then into a beaker decanting off
excess water.
Embryos are further separated by centrifugation or by flotation,
A. Place embryos in 50 ml tubes rnd fill 314 full with a 50% aqueous liquid glass solution. Spin at
4500 RPhi for 5-15 min in an International Clinical Centrifuge with a 4 bucket head. The
embryos form a layer at the top of the 5Wa liquid glass. Pour off embryos and wash twice in
water.
Or do B.
B. (51) Transfer embryos and debris t o a 5" high 250 ml jar containing sufficient sodium silicate
to float the embryos t o the top - usually SO rnl of sodium silicate is added to 200 ml of water.
They may then be poured off, skimmed off, picked up by a perforated spatula or by a pipette
with a rubber teat (63).
Clearing
Transfer embryos t o petri dishes 112 to 213 full with lactophenol: 1:1:1 :5-lactic acid, phenol,
glycerine, and water.
Boil several minutes until water has evaporated and fuming of remaining liquid commences. Do
this under a hood or very well aerated room. Remove quickly from heat, allow to cool and
examine at 15-60 X. The mycelium will be bright blue. High magnification may be desired to
confirm that the mycelium is typical of the loose smut fungus (73).
For alternative procedures without heat or stain see (63, 67), and for using pressure cooker (73)
or autoclave see (5 1).
Vstihgo nu& in plant tissue (65)
Boil tissue in 5% SaOH for about 20 A n and pour into an enameled pan. Separate nodes from
oheathes with tweezers. Place nodes in lactophenol containing 0.001-0.005% trypan blue.
14. Haustoria of Peronsporales, Resorcin (35). Place sections cut free hand or with a freezing microtome
in 1% aqueous Resorcin blue. Sections can also be treated with a 2% solution of "Pectoenzyne," a
commerEia1 enzyme in citrate buffer at pH 4 prior to stain for 4-6hr so that the relative of
-
the fungus and host elements are more readily ken.
15. Hyphae in various hosts, dioxan-propionic acid-haemgum mountant (47). Place leaf actions in a
mixture 95:s of dioxan and propionic acid (reagent grade). Allow 5 ml of solution per 5 mm2 of
time. Leave at 60 C until leaves are colorless.
Be careful of vapors. Wash in distilled water at least 3 hr. Transfer specimens to slide and add a few
drops of haemgum staining mountan t (46).
16. Hyphae in oak, holly and birch Litter, sodium chlorite safranin (41). This technique was used with a
aurabet of species of trees including oak, holly and birch t o observe fungal hyphae in and on leaves
208 Mkroropk Techniques
from litter.
Cut 0.5 g of leaves into pieces 0.5-1 cm across.
Bleach in r solution of 0.3 g sodium chlorite in 40 ml water acidified by the addition of0.S ml of
10% acetic acid for 4-6 hr at 60 C.
Pour off bleaching solution and dehydrate with SO, 70,95% and absolute alcohol (3 changes) under
reduced pressure of 1/3 atmosphere, Twenty min in each is enough.
. Replace absolute alcohol with methyl salicylatz; the material will be transparent in a few minutes.
Stlin m a t e d by placing it in 0.25 rafranin in methyl salicylate for I5 sec.
Warhin methyl salicylate and mount in Canada b a l m
17. Hyphae in wood, safranin picrmniline blue (13). Sections are usually 15 to 20 p thick, u u r d y cut
on a sliding microtome.
Stain the section in 1% aqueous safranin for about 30 sec. Wash off the superfluous stain in water
leaving the section slightly over-stained.
Cover with picro-aniline blue (25 ml of saturated aniline blue, 100 ml of saturated aqueous picric
acid) and warm gently to the point of simmering.
Wash out all the blue that comes out easily with water.
Wash several times in alcohol, 70 and 95% and dehydrate is absolute,
Clear in clove oil, wash in cedar wood oil or xylol and mount in Canada balsam. The lignified cell
walls are stained red and the fungus mycelium a clear blue.
Free Hand Sections
Elderberry pith or carrot root can be used for free hand sectioning. Elderberry pith can be kept in
70% ethyl alcohol prior to sectioning. Enclose small portiom of the materials to be sectioned between
split halves of the pith or carrot root. Section using a new razor blade across the m a t e d . Transfer
suitably thin sections directly to a drop of mounting media on a microscope slide.
Sections can also be made by rhe "chop method," with much less skill, advises Cummins (18). A
mall portion of a leaf is placed on a slide, and u s i q a razor blade in a chopping manner a luge number
of sections are made. Usually some of these will be thin enough for viewing. If a stereoscopic binocular
is available, materials can be sectioned while being vjewed through the microscope. This technique is
particularly useful when fruiting bodies are present on firm host tissue.
Several machines that section tissue with little or no tissue preparation are now available and
appear very pr~mising.
MOUNTING ASD STAISIZI'G JIEDIA

FOR SEM.IPER.\.fASEST31OUYTS OF FUNGI
These materials allow the preparation of microscopic mounts of fungi that are sembpermanent.
Water mounts usually reveal the truest colors and permit obrervation of living material the latter, of
particular value with phase microscopy. Some advantages of other mounting media are:
I. They evaporate less readily than water.
2. They can be sealed for long term presentation as such faatives or preservatives: lactic acid,
acetic acid, picric acid, alcohol, and phcnol lessens deterioration.
3. Stains are more suitably incorporated allowing more critical obsenration and identif~ationof
-c substances.
These materials, however, generally do not preserve materials for a year or more, many much less.
In addition some are not suitable for many organism. Picric acid -aniline blue, for example, cannot be
used for more delicate fungi as they are plasmolped.
Those media that do not harden require sealing to lerten evaporation and to facilitate itandlink
'Ihorc that haden often profit by ringing (for materials see section on sealants). As it b advisable not to
Microscopic Techniques #)9
have any mountant outside of the coverslip t o ensure a good seal, use minimum amounts. Use c l e ~
dissecting needles as well as clean glasmvare t o get a presentable slide. A preliminary mounting in 7Wo
alcohol combined with separating out masses of mycelium tends to eliminate air bubbles when the final
mountant k subsequently added.
MEDlA THAT REMAIN LIQUID

-
AmaM's mounting fluid Lactophenol An extensively used mounting medium. Be-
Phenol crystals (Dissolved by gentle heating) 20.0g cause its refractive index of 1.45 is close to the
Lactic acid (sp. gr. 1.2) 20.0g hyphae and spores of many hyaline fungi, 0.01
Glycerine (sp. gr. 1.25) 40.b to 0.1% cotton blue or acid fuchsin is corn-
Distilled water 20.0g monly added. For a number of variations see
Maneval(61) who suggests that rnl may be
equally substituted for g.
Bask fuchsin-tannic acid stain (27)
According to DeSilva (27) stains the spore walls of fungus spores.
lo!%aqueous tannic acid Mix and centrifuge at least at 2000 rpm
1%basic fuchsin for 15 to 45 rnin. Use supernatant.
Put material A stain, then add Sm glycerol, add cover slip and blot dry. May be sealed with fingernail
polish.
Chitin Test (Jensen (48))

1. Autoclave the cells at 121 C in 23.M KOH converting the chitin to chitosan.
2. Wash the cells and place them on a slide with a few drops of IKl in 1% H2S04.
3. Chitosan if prexnt will stain violet. Treat a control with acetic acid as chitosan is soluble in acetic
acid m d cellulose is not. Cellulose can be dissolved in undiluted commercial ammonium hydroxide
with as much copper hydroxide dissolved in it as possible.
Glycerinealcohol mixture (1 8)
50.50 mixture of 95%alcohol and glycerine
Spores may be cleared in a weak solution of chloral hydrate prior to mounting (18).
KOH
A freshly prepared solution 2 to 1Wo KOH filtered to remove the flocculent precipitate is
recommended. It is generally used when mounts are t o be made from dried specimens. PREVENT
CONTACT WITH LEKS OF MICROSCOPE. IF THIS OCCURS, CLEAV OFF THOROUGHLY AT
ONCE (3).
-
KOH Phloxine
Cooke (17) prefers to use with the sphaetopsidaceous fungi.
7Wo alcohol 1. Make up these 3 separate solutions.
5% KOH 2. Tease bits of mycelium - fruiting structures in a drop of alcohol to
05%aqueous phloxine remove air bubbles.
3. Remove m o a of alcohol by blotting or by evaporation.
4. Add a drop of KOH.
5. Add a drop of phyloxine avoiding contamination of dropper 4 thK
6. Add a cover slip.
7. A Kmi-permanent mount can be made by replacing with lactopbm
210 Miaoscopie Techniques
Acid fuchsin 0.1 g Gives brilliantly stained specimens. Seems to be incompatible with
Lacticacid 100ml fmgernail polish. If mixed half and half with Gurr's water mounting
medium, will harden.
Nigrosin 0.2 g in 10 ml saturated Picric acid

~&arol 20 g To remove considerable background, after 51 min blot away stain and
M c acid 10 g replace with lactophenol.
Phenol crystals 10 g
Mmeval's acid fuchsin (61)
Phenol 5% aqueous 30 ml Used to stain nuclei in Helminthosporia.
Glacial acetic acid (20%) 8-1 0 ml Add 1-2 ml of 1% aqueous acid fuchsin after 30 seconds
Ferric chloride (3w) 4 rnl to 3 min, drain off the excess, flood with tap water 3-10
sec (may repeat 1-3 times) and mount in lactophenol.
Mula's iodine stain (25)

Potassium iodine 1.5 g Gives considerable information about hymenial com-
Iodine 0.5 g ponents of ascomycetes and fleshy basidiomycetcs.
Chloral hydrate 200 ml
Distilled water 20.0 ml
Patterson's or Shear's mounting fluid
A. (14) OR B. (28,96)
%Potassium acetate in H20 300 ml Potassium acetate 10 k
Glycerine 120 ml Ha0 500 ml "standard"
95%ethyl alcohol 180 ml 95%ethyl alcohol 300 ml mycological
glycerine 200 ml mounting media.
Graham (39) suggests a buffered Shear's mounting fluid for taxonomic studies on smut
tetiospores. Mount spores in 2 drops of the buffered medium, add a coverslip and heat the slide over an
alcohol lamp t o remove volatiles.
2% Potassium acetate in pH 8 McIlvaines buffer
(19.45 ml,0.2M Na2HP04 and 0.55 ml of 0.1M citric acid) 300 ml
Glycerine 120 ml
95% ethyl alcohol 180 ml
kitacid aniline blue (88)

Saturated sol. of picric acid in 95% alcohol Add picric acid solution to aniline blue until a
.r n n de blue w w
medium green solution is obtained, filter before
uring
Typur blue (8)
chick staining solution Heating hastens staining with either solutions. The walls
Trypan blue 45% acetic acid 0.14.5% of the fungus ate stained, although not all fungi respond;
Sbw shining solution nor are spore walls stained, according to DcSilva (27),
Trypan blue lactophcnd 0.10.5% who suggests a basic fuchsin-tannic acid stain.
Microrcoplc Techniques 211
Zinc chloriodide stain for Cellulose (48)

1. Place sections of mycelium into several drops of zinc chloriodide (50 g zinc chloride, 16 g KI, 17 ml
water; add an excess of iodine and allow to stand for several days, and then store supernatant in a
brown dropping bottle).
2. Walls containing large amounts of cellulose will stain blue, and yellow t o orange if they contain large
amounts of chitin. Some hemiceUulo~ will stain blue (48). Ullstrup (92) used this stain to
demonstrate Sclerophthora rnacrospora in fresh sections of corn. Intensity of stain for various
species of Plryfophrlroru varied with the culture medium (9). According to Aronson (5) cytochem-
ical tests for ceUulose are not always reliable.
MEDIA THAT HARDENED
Aquamount (Edward ~ u r r ) ' '
A moderately quickdryirzg, clear, colorless mounting medium.
Cleatcol (H. Willard lark)^

A water soluble, rapid hardening, clear mounting medium with a refractive index of a p
proximately 1.4. It is also ntiscible with glacial acetic acid (96). Directions: First mount in water
(precede with alcohol to avoid air bubbles), glycerine or "Amann's lactophenol," drain or blot, replace
with Clearcol; seal with a cover glass. Normally Clearcol will become hard in froin 1 to 5 hr.
Glycerinejelly (49)
Gelatin 1.0 g Blue label Karo according to Johansen, D. (49).
Glycerine 7.0 g Makes a good substitute.
Water 6.0 rnl with the addition of 1% phenol
Glycerine-jelly-methyl green (79)
Methyl green 3%in SWGethanol Saturate glycerine jel!;! with methyl green by
Patterson's mounting medium A adding the latter dlopwise to melted jelly.
Glycerine-jelly (see above)
Mount material in a drop of Patterson's medium. Heat gently over a flame until the water and
alcohol have evaporated. Place a small block of GJMG on the material and melt over a flame. Stir the
fungus with a needle and add a cover slip. After the glycerine jelly is removed from the outside edges of
the cover slip, seal with fingernail polish, balsam or a synthetic resin.
Hoyer's mounting medium (1)
Distilled water 5 0 ml Soak arabic gum in water for 24 hr. Add the chloral hydrate and let
Arabic gum, lump 30 g the solution stand until all the material dissolves. This may require
Chloral hydrate 200 g several days before the glycerine is added and the solution is ready tu
Glycerine 20 8 use. For mounting Myxo~nycetesor Ascomycetes, wet the specimen
with absolute alcohol for about one minute, treat with 2% KOH for
a similar period of time, wash in 70% alcohol and place in a drop of
mounting fluid.
Edwud Gun. Ltd. 42 Uppet Richmond Road West, London, S. W. 14,

*Commetcirl preparation avlllobb from H,Willard Clark, 33 South High Street. Mekolc 76, Mas&
212 Minwcopic Techniques
be's Staining Mountant (46)

Combined fixing, staining and mounting media that becomes permanently hardened.
pistilled water 50 ml Dissolve finely ground arabic (wlgentle heating if necessary) in a
Formic acid 85-90% 10 rnl solution of 1 0 ml of formic acid in 50 ml dist. water. Grind 'A g
Gum arabic 20 g hematoxylin in a mortar with 1.5 g of Ferric alum and 0.5 g of
Hematoxylin 0.5 g chrome alum to a fine powder and dissolve in gum arabic solution,
Ferric alum 1.5 g then keep at 60 C for 24 hr. Bismark brown is added after cool-
Chrome alum 0.5 g ing to room temperature, (Do not heat after addition of Bismark
Bismark Brown Oq15 g brown). Lots of precipitate at this time. Centrifuge at 10,000
Glycerol 20 ml gravity for 45 min and at 25 C. Decant preparation from the
Chloral hydrate 30 g centrifuge tube, incorporate 25 ml of glycerol and chloral hydrate
Methyl green excess by stirring. Do not shake. Solution should be deep chestnut. Take
10 ml aliquot and add Methyl green excess, mix in slowly with
main preparation until neutral tint is obtained, check with a neu-
tral density filter. Keep mountant in a stoppered bottle and pre-
pare s m ~ l quantities
l at n time*Thin slides were kept for 2 yr.
Nuclei clearly distinguished in all but the apical cells.
T and N Water Mounting Medium (90)

Gum arabic 20 g Place gum arabic in water. After it becomes a uniform gel, add
Chloral hydrate 10 s chloral hydrate and dextrose and warm slightly until uniformly
Dextrose 10 g viscid. Add glycerine and mix well.
Glycerine 3 ml
Materials can be stained in cotton blue or acid fuchsin in lactophenol and slightly warmed until
proper differentiation is obtained. Drain off excess and add water mounting medium. Slides are then
kept at 40-45 C for 48 hr and usually have I~ardened.Cover glass ringed with Canada balsam dissolved
in chloroform.
Wallerite (21)
Clear gelatin 10 0 Dissolve phenol in acid without heat, digest gelatin until com-
Phenol crystals 28 8 pletely dissolved (takes about 2 days), add 10 drops of glycerol
Glacial acetic acid 28 g and incorporate thoroughly by stirring. Store in brown bottle,
may be thinned with glacial acetic acid. This medium will harden
pemanently after 24 hours. For very wet prcp2rations dehydrate
fust in gIacial acetic acid for a few minutes, then drain off.
NUCLEAR STAINING
Location and enumeration of fungus nuclei may be done through careful phase microscopy, but
m y prefer staining of f w d material. Good results have been obtained with a variety of stains
preeeded by a variety of fwatives. For details involving iron hematoxylin see (26,74,75) Giemsa stain
(45, 52), acetorcein (26), basic fuchsin (Feulgen reaction) (26), propionocarmine (60), trypm blue
(55). The details of a method recommended to me by Roth (80) using basic fuchsin is given here. It
Vwus that certain details of nuclear division in fungi will be more likely unequivocally resolved by the
electron and not the light microscope.
Staining of Fungus nuclei (J. Roth (80))

1. Crow fungus on thin agar film on new glass slides in a petri dish lined with moist filter paper.
(Aseptic technique throughout.)
2. Submerged slide in Carnoy's solution for I5 min. May be kept in this solution for several days.
3. brain off excess.
4. Hydrolke in 5N HCI at room temperature or in 1N HCI at 60 C, for 1 0 t o 15 min. Add HCI
dropwise.
5. Drain off excess and rinse in water.
6. Add Shiffs reagent dropwise and leave for 45 min, can be left on for several hours.
7. Drain off Shiffs reagent and submerge the slide in 0.5% sodium metabisulfite for 4 min.
-
8. Two separate rinses in water takirig several minutes.
9. Flood slide with absolute alcohol, repeat 3 times.
10. Add either diathane or euparal and seal with a cover slip or replace alcohol with xyol and add
Canada balsam and seal.
Camoy's fluid: 3 pt absolute ethyl alcohol

1 pt glacial acetic acid
Shiffs reagent; (keep refrigerated and tightly stoppered in smaU bottles.)
basic fuchsin
distilled water 100 ml ] Bring to a boil and cool at 50 C.
Add 1 g of sodiumor potassium metabisulfite and 5 ml of IN HCI. Leave solution at least 4 hr. It
will be decolorized to watery pink. Add '/4 g norite and shake, filter through Whatman no. 1 filter
paper. If the solution is not completely clear, add norite and filter again.
GRAM STAIN OR GRAM METHOD (87)

Prepare a smear from a fresh culture - less than 36 hr old and fu: by heat. Cover the smear with
crystal violet and leave for 1 min. Wash in a beaker for 3 or 4 seconds with tap water which should be
running at a rate of about 30 rnl per second into a 250 rnl beaker. Rinse with Burke's iodine and leave
the iodine on for 1 min. Wash with water for a few seconds.
Hold the slide in a position where the smear is clearly visible against a white filter paper laid on
the edge of the sink. Then apply 95% alcohol, drop by drop, on the top edge of the smear until no
more color runs out of the lower edge of it. The decolorization time is usually about 10 t o 20 seconds.
Wash, counterstain with 2.5 percent safranin for 30 seconds. Wash, blot, dry, and examine.
Result: A blue color indicates Gram positive; red, Gram-negative.
Note: The presence of a number of red cells among a mass of blue usually indicates the presence
of dead, unstainable cells.
Mature spores are not stained by the Gram method and will appear as clear areas in the blue or
red rod.
Reagents for the Gram Stain:
Crystal Violet
Crystal Violet. ................... 2.0 g
Ammonium oxalate .............. 1.0 g
Distilled water .................. ,100 ml
Burke's Iodine
Iodine ......................... 1.0 g
Potassium iodide ................. 2.0 g
W e d water .................. ,100 ml
Microscopic Tcchniqua
Aqueous Safranin
Safranin 0, 2.5% solution
in95%ethanol ............... 10ml
Distilled water .................. 100rnl
STALNING BACTERIAL FLAGELLA
The determination of the number and the arrangement of bacteria flagella are important
taxonomically. Regardless of whether bactcrid e l l s are viewed by the electron or light microscope,
careFul interpretation is necessary, and bscterw species of knoun flagella arrangement should be
included for comparison in every determination (42). Becaure of the apparent difficulties encountered
in successful staining of flagella, several techniques are included here. Two of them are similar.
Common to all is the agreement with Leifwn (58, 59) that cultures must be in the log or early
stationary phase usually grown at temperatures of 20 C, and slides must be scrupulously clean. Rhodes
(76) also stresses the importance of focusing and centering of the microxope condenser. (For derails of
staining fungus flagella see Koch (S3).)
Silver impregnation stain (Blenden and Goldberg (7)).

Reagent A Reagent B (must be uxd within 4 hr)
Distilled water 100 ml 2% Silver nitrate 90 rnl
Tannjc acid 5.0 g Ammonium hydroxide
Ferric chloride 1.5 g 1. Add ammonium hydroxide dropwise to AgN03 until
15%Formain 2.0 ml the heavy precipitate that is formed is dissolved.
1% Sodium hydroxide 1.O rnl 2. Then add 2% AgY03 to this until a slight clouding
appears and persists.
3. Adjust pH t o 10.0 with NH4N03 and AgN03.
Procedure
1. Slides are alcohol cleaned.
2. Place a loopful of distilled water on slide.
3. Place a loopful of culture or faintly cloudy suspension just touching the distilled water so that the
two diffuse together.
4. Allow slides to air dry.
5. Cover smears with reagent A for 2 4 rnin and rinse in distilled water.
6. Add reagent B for about 30 sec.
7. Immediately wash smears with distilled water, air dry and examine under oil immersion.
Modifmtion of Fontana's met hod for staining spirochaetes-Silver Plating Method (76).
PC& trnnate mordant

10%w n i c acid (W/V) 10 ml Add first 3 compounds in order, dissolve curd formed
Saturated sol. potassium alum 5 ml by shaking. After adding ferric chloride allow black
Saturated sol. aniline I ml sol. t o stand for I0 min before using,
5%for& chloride (WV) 1 ml
Ammodd silver nitrate solution

5% &A0 90 ml Add ammonium solution slowly to AgN03 until brown
Coaoeatmted ammonium SOL precipitatesjust redissolves. Add drops of AgN03 until
(rp. gf. 0.880) solution remains faintly cloudy even after shaking. Store
in th4 dark, remains ruble for several weeks.
Microscopic Techniqua 215
Procedure
1. Add 1-2 ml of sterile distilled water to slant usually 18-24 hr old.
2. Two 4 mrn loopfuls are inoculated into 5 rnl of sterile distilled water and left for 1 hr at 25 C.
3. Transfer drop to slide cleaned in a chromic and sulphuric acid mixture. bifson's suggests (59)
ckaning o ~ r n i g h with
t a hot s l u t i o n - 71180 C or at least several days at room temperatures.
Wash slides thoroughly with tap followed by distilled water.
4. Allow drop to dry and cover with iron tannate reagent for 3-5 min.
5. Wash very thoroughly with distilled water.
6. Add silver reagent (almost boiling), leave for 3-5 min.
7. Wash thoroughly and examine directly or under cover slip sealed with Canada balsam
Exposure to air causes disintegration of these silver plated preparations after about a week.
A modification of Leifson's method (58.59). (Ladner, Brown and Tischer (56)).

1. Make 4 wax pencil moats about 1.3 x 2.0 cm on the surface of a thoroughly cleaned microscope
slide so that the inside areas of the moats are nearly equal.
2. Place one side of the slide so it will be about 80' to the horizontal plans.
3. Place a small loop of the bacterial suspension at the top of each rnoat allowing it to run down.
4. Absorb the excess liquid from the bottom of each moat with a small piece of paper toweling to
avoid the washing away of the WLX-pencilmarking.
5. When the smear has air dried, plzzc the slide flat on a dissecting microscope and examine with
reflected light and low power so you cur see more precisely the precipitate forming.
6. Add about 5 drops of prepared flagella stain (BBL 04-104) at a temperature of 5 C. A 5 rec
interval between additions is recommended. See (59) if you wish to prepare your own flagella
stain.
7. If the areas of the moats and the volumes of &in are equal, a fine precipitate will f m t appear in
the fust moat.
8. When precipitation occurs in the second moat, immediately ring all the stain off gently with
water. Do not pour the stain off before rinsing.
9. M o w the slide 'to dry and esamine under an oil immersion objective. Optimum staining
condition should occur in one of the moats. This procedure can also be done with 4 separate
slides.
CEMENT RECIPES FOR SEALING LIQUID MOUNTS
A water miscible (commercial preparation of a mixture of plastic and solvents, hardens in 1 to 5

hr) available from H. Willard Clark, 33 Sauth High St., Melrose 76, Mass.
Fingernail Polish
Dade (20) considers fvlgernail polish the best sealant for lactophenol. For best results it must be
applied t o a dry slide. Most moisture can be removed by laying a piece of blotting paper over the slide,
holding it down to prevent shifting the cover slip and then running a finger above the edges of the cover
slip and pressing down gently (19, 20). Dade suggests using a no. 3 sable brush, kept flexible by
suspending in acetone vapor, for application. A colorless polish for the f i coat followed by the same
or cotored polish is recommended
trctophenol Cum (22)
D i l v e 38 g of pure gum vabic in 50 ml freshly distilled water; add 5 g glucose and 6 8
lactophenol. Filter solution through gkss wool. It is used cold, dries quickly and Kalr laaophmol
mounts well. It dots not work with lactofuchsin and tends to crack with age.
216 LCiaowrrpic 'fcchniques
Udoserl(Edward Gurr)'
A quickdrying, synthetic, transparent, colorless cement for sealing lactophenol, glycerine and
other aqueous mounts. It sets within a few s w n d s and is hard within a few minutes.
Wder's cement (2 1)
Melt refrned bees-wax and damrnar separately in a glass container on a water bath. Pour melted
wax slowly into dammar stirring thoroughly. Five percent gold size may be added and thoroughly
stirred in. The mixture is placed in ointment thins and may be applied with a heated bent brass rod
3/32" in diameter. A more elaborate tool is described by Dade and Wder (21).
ZPt (Thorne's ccment) (37'91)
Archer Daniels Midland 100, polymerized oil 31.75 ml
Industrial methylated spirit 4.76 ml
Butyl acetate 20.41 ml
Toluol (sulphur-free) 20.41 rnl
Use butyl acetate as sr thinner. A tinted zut suitable for ~ g i n coverslips
g is made by adding a
little oil-soluble pigment, such as Scarlet R. (37). Zut is best applied in two layers. It can be purchased
from George T. GUK' or Bennett Paint and Glas Co., 2131 S. 2nd St., Salt Lake City, Utah.
WAYS OF PREPARING FUNGI FOR MICROSCOPIC EXAMINATION

AND/OR HERBARIUM PRESERVATION.
MICROCULTURE TECHNIQUES
These techniques usually involve growing fungi in a mall area such as on a cover dip whereby the
o r m s m can be conveniently examined with the compound microscope with little fear of con-
tomination. These techniques also give a potential semi-permanent mount of the organism and usually
with fruiting structures intact. Some worken have cautioned that in many microcells aeration is
inadequate and abnormal development may occur.
A p w e l l technique (20)
1. Pour o petri dish with desired culture medium
2. Using a flamed a d p e l cut the agar into 4 quarters.
3. Lift up each section and place a sterile cover slip under each.
4. Turn plate over and outline cover slip with a grease pencil.
5. Cut and remove a small square of agar'above each cover slip exposing most of the cover dip.
6, Inoculate near the excavated agar.
7. A f t a suitable growth cut and remove agar to free cover dip.
8. Remove cover slip and place a drop of lactophenol, warm slightly, invert on a microscope slide and
osll if desired.
W techdque (4)
TbJa method i s a modification of methods of Riddell (77) and Taschdjian (89). It lessens
condenstion by the use of glycerine agar instead of water and eliminates the need of cutting and
handling of mall squares of agar.
'~dwudGum Ltd., 42 Up- Richmond Rod West, London, S. W. 14.

1. Pour 50 ml of agar containing 5% (w/v) glycerine and 1%(wlv) agar at 50 C into a square plastic
pctd dish (100 mm2). Other size dishes can be used.
2. Place 3 sterile microscope slides on surface of agar after it has set.
3. Remove a cover slip (12 x 12 mm) from 95% ethanol, drain and flame lightly.
4. Dip the cover slip into the desired melted agar medium near 100 C and transfer to the center of one
of the slides in the petri dish.
5. Inoculate the 4 edges of the agar coated cover dip.
6. Place a large cover slip that has been removed from the ethanol (drain off alcohol and flame)
squarely on top of smaller cover slip. Replace cover of petri dish.
7. After incubation remove the larger cover slip and place with fungus growth upward.
8. Remove smaller cover slip and discard.
9. Place a drop of 95%ethanol on fungus growth on the large cover slip and the slide.
10. Just before the alcohol is evaporated add a drop of liquid mountant.
11. Lower clean slide on to cover slip preparation and a clean cover slip on to slide preparation.
Channeled slide technique (85)

Shoemaker (85) suggests a simple microculture slide. An unpolished channel %" wide and slightly
less than 1 rnm deep is cut across a standard 1" x 3" microscope slide. A cover slip and the slide is
sterilized separately. After placing the cover slip across the channel, melted seeded agar is added via a
Pasteur pipette until the space is filled. The slide is placed in a moist chamber.
Van Tieghem cell

A popular microculture cell consisting of a glass ring about 1 cm in diameter and 1 cm high,
cemented by paraffm, zut, epoxy or other water proof cement to a glass slide. The cover slip on top is
held in place by vaseline, and a loopful or two of agar is placed on its lower surface. Water is added to
the cell to maintain high humidity. Cells may also be placed in a petri dish, located by matching holes
punched in moistened filter paper (Fig. 16).
Tlun culture ceII(15)

This cell was devised to permit observation and photography at high magnification. The chamber
shown in Figure 17. The procedure is as follows:
, Seal a sterile 60 x 24 rnrn no. 1 coverslip to one side of the glass slide with nail varnish covering the
hole and the slot.

2. If desired, assembly can be irradiated with a germicidal W lamp for 15 min.
3. Place a sterile thin card, the width of the hole x 5 rnm, across the circular chamber parallel t o the
short axis of the slide.
Fb 16. Van Tieghem odlr in r petri dish. 17. Thin culture con.
218 Microscopic Techniques
4. Add agar to the semicirculat chamber formed by the card on the side of the card away from the slot.
5. Inoculate agar.
6. Seal second 60 x 24 mm cover slip over the hole and slot.
7. Support chamber in petri dish by a V.shaped glass rod laid horizontally on top of filter paper
moistened with 5m glycerol.
DOUBLE COVER GLASS TECHNIQUE

This method is an attempt t o make semi-permanent microscopic mounts "permanent." Chupp
(14) modified the technique of Diehl (28). It is given below:
1. Mount material in Patterson's solution (A) on a No. two 12 mm2 cover glass.
2. Heat gently over a microburner until most of the Iiquid is evaporated.
3. Add a drop of pure glycerine and heat slightly.
4, lnvert the 12 mrn2 coverslip on a no. one 22 or 18 1nm2 coverslip and press firmly down. Wipe all
excess glycerine from margin of the smaller cover slip.
5. Place a generous drop of medium heavy balsam on a microscope slide.
6. Heat balsam gently, then place the pair of coverslips with the smaller one underneath onto the drop
of balsam.
7. After the balsam has spread far enough to seal the glycerine, the whole mount can be pressed down
until the balsam exudes slightly from the edge of the larger cover slip.
Zuck (97) substituted Clearcol, a watermiscible substance, for balsam, thus avoiding the need ti
drive off the water by heating. His procedures are:
1, Add a drop or two of Patterson's solution (B) to a round 22 mm cover slip.
2. Place the specimen in the drop.
3. Place a round 18 mm cover slip over this and add 2 or 3 drops of clearcol on top of the smaller cover
glass.
4, Lower a clean microscope slide over the clearcol until touching. The whole is brought upright and
the 2 cover glasses allowed to settle by their own weight.
CELLOPHANE MOUNT (32)

This technique is a refinement of the method of Butler and Mann (10). It gives a mount of intact
fungal structures with moderate optical qualities.
1. Place a clean square (1 8 mm2)cover glass on a microscope slide.
2. Place a dust-free segment of "Scotch double-stick' tape," 12 mm width, across and even with the
upper edge of the cover glass. Apply tape tautly and evenly.
3, Remove excess tape by cutting tape even with cover glass with a razor blade. The tape should adhere
firmly t o the cover glass t o eliminate air bubbles and to avoid seepage of the mounting medium
between the tape and the cover glass.
4. Place the cover glass with the sticky surface down on the surface bearing the fungus, attempting to
use areas of light sporulation. (Dried plant disease specimens.as well as agar cultures are amendable.)
5. After light pressure and no lateral movement, pick up cover glass.
6, Place the cover glass over a drop of water or other suitable liquid mounting media. Alcohol or
surfactants added to mounting media should decrease air bubbles. Endo (32) says Hoycr's mounting
medium appears very promising for making permanent mounts.
Mlcroscoplc Techniques 219
DFUED REFERENCE CULTURE

This technique (72) is a modification of the one used at the Commonwealth Mycological Institute
(31). It gives a permanent dried culture.
1. Grow fungus culture on any suitable agar substrate in plastic petri dishes.
2. When culture is sufficiently mature, expose to formalin vapors for several days.
3. Allow culture to dry partially (dry enough if the entire agar culture can be removed from the plate).
4, Immediately pour 15 ml of 2.5% glycerol agar into lid of petri dish.
5. Float the culture on the hot agar and gently smooth the edges.
6. Allow the culture t o dry completely, peel out the aiar culture,
7. Clue the upper surface of culture to a snap out cardboard ring 114" wide. The ring and culture is
then inserted into a cardboard holder having a 3-113" diam opening. A holder for a culture and
-
microscope slide is manufactured by Thrifty Paper, Inc., 2508 24th St. N.E.,Washington, D.C.
CELLOPHANE METHOD
There are several methods (1 2, 50, 54) using a cellophane as a vehicle for reference cultures and
also for cytological work (52, 78). The major problems with this material is to get the cellophane in
complete and smooth contact with the agar medium, and after suitable fungus growth, to'dry the
cultureceIlophane without excessive distortion. Uniform contact is usually accomplished by soaking
the sterile cellophane (sterilized dry between sheets of filter paper) in water or alcohol before applying
t o the surface of the medium Less distortion is accomplished either by drying in a specially designed
press (12) or by drying on glass plates (discarded 9 x 12 cm Kodak M plates (54)). The cultures are
afflxed to the @ass plates by a mixture of glycerine 3.0 rnl, gelatin 3.0 g, phenol 0.2 g, and 100 ml
water.
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64. Morton, D. J. 1960. A quick method of preparing barley embryos for loore smut examination.
Phytopathology 50:270.272.
65. Morton, D. J. 1961. Trypan blue and boiling lactophenol for staining and clearing barley tissue
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66. Munford, D: L. 1966. Factors associated with resistance in barley t o spot blotch. Phytopathology
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67. Noble, Mary. 1966. Handbook on seed health testing. Series 3. Int. Seed Test. Assoc.,
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68. Pady, S. M. 1935. Aecioopore infection in Gynvrowniu hfmtlra&s by penetration of cuticle.
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Microscopic Techniques
Parris, G. K. 194-4. A simple nuclear stain and staining technique for Helntintltosporia.
Phytopathology 34:700.702.
Peterson, C. W. 1967. Dothistrorna needle blight of Austrian and Ponderosa pines: epidetniology
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Petersen, L. J. 1956. A method for observing stotnatal penetration by uredospore germ tubes of
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Pollack, Flora C. 1967. A simple method for preparing dried reference culturcs. hlycologia
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Popp, W. 1958. An improved method of detecting loose-smut myceliurn in whole embryos of
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Writing for Publication
by George B. Cun~mins
Introduction
Manuscript Preparation
1. Prior to Preparation of the hfanuscript
2. The Plan of the Manuscript
3. The First Draft of the Manuscript
4. The Revivd >fanuscript
5. The Galley Proof
Research is done to advance the state of knowledge and add new truths, theories, and hypotheses
t o man's store of information. This goal has not been reached until the results of research are made
available. Research is not done merely to satisfy the curiosity of the individual. Hence, the publication
of significant results is an obligation that the scientist owes to society. It is his further obligation to
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rHANUSCRIPT PREPARATION
1. Prior to Preparation of the Manuscript. Several matters should be considered before a
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Writing for Publication 225
one column or or both.

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. ."
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226 Writing for Publication
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Appendix
Humidity Control
Tabk I. The rcktionship ofdensity o f sulfuric acid and relative humidity
Tabk 2. The relationship of specific gravity or refractive index of aqueous glycerine solutions md rebtivc
humidjty
Table 3. The relative humidities of various saturated salt solutions at various temperatures
Refercnas
HUMIDITY CONTROL
It is frequently useful to be able to maintain a constant relative humidity (RH) for the purpose of
determining the effects of moisture on spore germination (5,6,7), spore longevity (15) or a multitude
of other processes. The effect of RH on the fungicidal activity of fumigants (I 2) emphasizes the nccd
for control of RH in fumigant tests and suggests its importance in other biological tests. It should be
recognized as Anderson points out (2) that moisture stress may be of more significance than relative
humidity. Vapor pressure deficit (VPD), a measure of moisture stress, is defined as the difference
between the actual amount of water vapor present and the amount present at the saturation point at
the same temperature (2). Thus, the higher the VPD, measured in units of pressure - inches or mm of
-
Hg the greater the rate of evaporation regardless of relative humidity. If the temperature and relative
humidity are known, WD can be determined. For example, according to the tables of Hodgman (9) the
vapor pressure at 100% RH at 20 C is 17.535 mm of Hg, 70% RH is 12.274 mm of Hg. The vapor
pressure deficit is therefore 5.261.
Schein (13) reminds us that we must exert careful temperature control with relative humidities of
90% and above as only a I C drop will result in dew formation.
Humidity can be maintained by basically two methods:
Air may be humidified t o the degree desired by the physical introduction of water (7, 10) or by
the az of aqueous chemical solutions. The fust method requires a considerable amount of equipment
and careful instnrmentation. It is the method of choice in large chambers. An excellent example is
deocnied by Delp (7). The second method using chemical solutions is of use in small cllambers where
control a n be achieved without elaborate equipment. Two groups of chemicals have been used in this
btter method
ChmumucaI solutions: Different humidities may be achieved by varying concentrations of the
chemical in water. Examples are: sulfuric acid (table I), glycerine (table 2), CaCI, arld NaOIi solutians.
Of the^ sulfuric acid and glycerins are recommended by the American Society for Testing klaterials
(1). These solutions will maintain approximately the same RH at different temperatures. However, as
humidity is related to concentration of the chemical in solution, any change such as that caused by
evaporation or by absorption by material piaced in the containcr will change the RH.This can be partly
overcome by using large quantities of the solution ~JI the container or by previously conditioning the air
to the desired RH by passing it fust through a saturated salt solution that gives the approximate desired
RH (I 1).
227
228 Appendix
Humidity may also be controlled by its use of saturated salt solutions (table 3) each of which
maintains a different RH. For additional solutions see (20). Saturated salt solutions arc valuable
because the concentration does not have t o be precisely maintained; the requirements being that the
solution remains saturated, and the chemical is pure. A substantial excess of the chemical is desirable,
some recommending a "slushy" solution. Unfortunately, most falt solutions do not effect the same
relative humidity at different temperatures.
Some recommendatlonr about chemical solutions used to maintain humidity:
I. Containers should be essentially mnisiure tight as to prevent creeping of salts or evaporation of
water from glycerine or sulfuric acid solutions. If using continuous flow, humidify the air by
first passing through water and a conditioning solution.
2, Sudden temperature fluctuations should be prevented. If they are unavoidable, insulation with
1" glass wool will negate temperature fluctuations of 3C (1). As changes in humidity come
about from differences between temperature of the solution and the air in the container, a
shaking mechanism is also of value. It can be specially constructed (S), or a Warburg apparatus
can LC utilized (6, 7). The smaller the container, the less chance of a temperature differential.
3. Over-all surface of the material ideally should be less than the surface area of the solution.
For considerable detail on the measurement and control of moisture in gases and in solids see
(16).
Table 1. The relationship of density of sulfuric acid and relative humidity.' (ASTM (1).
Percent Relative Hurnidity
Or@nlc matter must not come into contact with add. as toxic fumes will be liberated.
Daennined by hydrometer with o minimum scale division ,001, solution should be at 25 C. A temperature
conversion frctor may be needed to change specific gavity to density for very precise work Formula: denslty of
water at 2s C. (0.99707) x specific gravity.
Table 2. The relationship of specific gravity or refractive index of
aqueous glycerine soIutions and relative humidity.
Aqueous Glycerine Solution ASTM (1) Aqueous glycerine solutions (Braun and Braun (3))
Refractive Percent Relative Percent
Index at Humidity at Specific Temp. Relative
25 C.' 25 C, Gravity . -
C. Humidity
13463 98.0' 1 .0oO3m20) 23 100
13560 96.0 1.049 23 95
13602 95.O 1.082 24 90
13773 90.0 1.107 24 86
13905 85.O 1.130 23 81
1.4015 80.0 1.135 (1.137)4 24 80
1.4109 75.O 1 .I 56 24 74
1.4191 70.0 1.165 24 71
1.4264 65.O 1.168 (I -170) 23 70
1.4329 60.0 1.186 24 62
1.4387 55.0 1.192 (1.193) 24 60
1.a0 60.0 1.207 (1.209) 24 51
1 A486 45.O 1.216 25 47
1.227 24 39
1.243 20 27
1.255 20 17
1.260 24 3.7
1.261 e r e glycerine) -- 0
Determined by a rcfnctornetet using a rodicrn light with an accuracy of 0.0003.

Them figures haw m accuracy of .t 0.2%;for intermedirtc rh a formula may k u ~ s d
''
Determined by a Wertphd balanw.
Me-d by Hydrometer.
Instructions:
A good industrW grade of glycerine should be used with dittilIed water. Copper sulfate,O.l by wcight.9 added
to prevent mold growth. Four drops of a srturrted solution per 100 ml of glycerins solution will equal this
concentration.
Appendix
Appendix 23 1
REFERENCES
Amer. Society for Testing Materids. 1958. Maintaining constant relative humidity by means of
aqueous solutions. ASTM standards part 9: 1947-195 1.
Anderson, D. B. 1936. Relative humidity or vapor presswe deficit. Ecology 17:277-282.
Braun, f. V. and J. D. Braun. 1958. The measurement and control of humidity for preparing
solutions of glycerol and water for humidity control. Corrosion 14: 17-19.
Corr, D. S. and B. L. Ilarris. 1949. Solutions for maintaining constant relative humidity. Ind. and
Eng. Chem. 4 1:20 14-2015 ,
Clayton, C. N. 1942. The germination of fungous spores in relation t o controlled humidity.
Cochran, V. W. 1945. The common leaf rust of cultivated roses caused by PhragmIdium
mucmnatunr (Fr.) Schlecht. Cornell Univ. Agr. Exp. Sta. 3femoir 268.
Delp, C. J. 1954. Effects of temperature and humidity on the grape powdery muucw fungus.
Hart, M. P. and D. hi. MacLeod. 1955. An apparatus for determining the effects of temperature
and humidity on germination of fungous spores. Can. J. Bot. 33:289-297.
Hodgman, C. D. 1954. Handbook of Chemistry and Physics. Chemical Rubber Pub. Co.,
Cleveland, Ohio.
Hopp, H. 1936. Control of atmospheric humidity in culture studies. Bot. Gaz. 98:25-44.
Hunter, J. 1968. Personal communication.
Munnecke, D. E., R. A. Ludwig and R E. Sampson. 1959. The fungicidal activity of methyl
bromide. Can. J. Res. 37:s 1-58,
Schein, R. D. 1964. Comments on the moisture requirements of fungus germination. Phyto-
pathology 54: 1427.
Stokes, R H. and R. A. Robinson. 1949. Standard solutions for humidity control at 25 C. Ind.
Eng. Chem 41:2013.
TeiteU, L. 1958. Effects of relative humidity on viability of conidia of Asperplh. Amer. J. Bot.
45:478-753.
Wexier, A. (Ed.) 1962. Humidity and moisture (4 volumes). Reinhold Pub.Co., Sew York
Wexler, A. and S. Hosegawa. 1954. Relative humidity-temperature relationships of some
sturated salt solutions in the temperature range of 0' to 50' C. f. Rcs. Sat. Bur. Stand.
53: 19-26.
Wilson,R E. 1921. Humidity control by means of suIfilric acid solutions with active compilation
of vapor pressure data. J. lnd. Eng. Chem. 13:326.
Wink, W. A. and G. R. Sears. 1950. Instrumentation Studies LVlI. Equilibrium relative humidities
above saturated selt solutions at various temperatures. TAPPI. 33:96A-9YA.
Winston, P. W. and D. H. Bates. 1960. Saturated solutions for the control of humidity in
biological research. Ecology 4 1 :232-237.
List of Organisms
Actinomyces, 35,40,42,56,64,75,98 C. cunesccns. 150
Aflobucruium. 28.126 C alpsici, 1S0
A. nzdiobacter. 12, 126 C ementa. 150
A. fumcfuciens, 12, 99. 126, 178 C. do visil, 1 50
Albugo occidcntalis, 195 C. dubh. 150
AIIomyces. 30,43 C. fesruwe, 150
Aftemarh, 119 C. kikuchii, 142, 150
A. brassicue, 1 3. 144 C.niwthnue, 150
A. bmssicola. 144 C.penniseti, 150
A. chrysonthemi. 144 C.personata. 150
A. mphanf, 144 C. physalidis. 15 0
A. soloni, 43.52, 146,148 C. pueram'cob, 1 50
A. tenuis, 144 C.sesami, 150
A. zlnniae, 144,146 C. sorghi, 1 5 0
Aphanomyces. 64.80.97, 1 do, 101, 1 19. C. stizlobii, 150
A. tuteiches. 15.34.45.99 C. rebnna, 144, 150
A. raphani. 59 Chnetomium, 57, 80, 1 19
Arthrinum, 1 19 23
ChaImopsiis thielrr~~bfdes,
Ascobolus imnrems, 144 Chwnephora, 194
Ascochytrr, 200 C. confuncra. 144
A. caulicolo, 1 26, 1 5 0 C. cucurbltonrm, 143, 144
A. imperfects, 154 Clborinia, 160
A. melilota, 150 Ckrhrospow diplcspora. 142
A. phuseolonrm, 149 Ctxcodioidcs immitfs,4 8
A. pinnodel&, 144 Cochliobolus, 1 19
A. p W 144 C. homorphus. 144
Aspergifus. 1 19 C. kusanoi, 144
A. fhvus, 20 C xativus. 153
A. ~ ~ 27,45,93
s . Cdlctorrichum. 1 19,200
A. ni&lons, 5 6 C. atramcnrurium. 15 1
A. nigu, 67, 197 C. coccqdes. 62. 151
Bkkeslea mkporu. 144 C. bgemrlum. 35
B&~toclndiellP, 2 2 C Ilndcmuth&num, 18.49, 15 1
B o fiyotinia. 160 C. phomoides. 1 5 1
Botrysphaeria ribis, 144 Coprinus &gopus. 3 2, 34, 15 1
Botrytis, 5 2. 194.206 Cwynebacterium, 126
5. ulli. 2 0 C. betoe, 126
B. cinerea, 20. 143. 144, 180 C facbns, 126
B. fake, 180,182 C.ficcum faden, 1 26
Brrmh &ctucue. 206 ' C ilicis. 126
Cephalosporium, 1 19 . C. Insidiosum. 125, 126, 195
C#tocystis, 1 19 C michageneirse, 126
C faguccc~urn,50,150 C poinsettke, 126
C flmbrkta, 23 C. rathuyi, 1 26
C. ulmi, 29.30.150 C. xcpedonicum, 2 1.6 1. 126
Cefospd*o, 99.1 19,206 Cbrynesporu. 1 19
C. ubelioe. 150 Coryneum. 144
C apfi, 150 Ckonurtium ribicoh. 1 23
C kfhh. 100.144,l50,180 arvularh, 1 19
C brochlrcr. 1SO C #enhbta, 153
List of Organisms
C Intermedia. 154 M. pinodes, 144

Cumluta lunata, 153 Myxomycetrs, 39
Cylindrocarpon, 1 1 9 Necrria plligrna, 144
C. mdicicoh, 143 N. glichdioides, 144
Cylindrrrcladium, 1 19 N. peziza, 144
Dkrporthe. 1 19 Neurospora, 49.50,75
Diplocarpon earliano. 20 N.crassa, 68
Diplodia maydis (D. zeae). 15 1 Ophiobolus graminis, 144
Drtchslera, 68 Papuhrta anr~~ditlisa, 143
Entomophthora, 1 19 Pectobocteriurn, 127
Epicaccum nigtum, 144 P. carotovorum, 124
Eremascur, 35 Penicilliurn, 97,254
Envinia, 126 Peronorporo, 1 22
E. atnylovora, 30.80, 126 P. effusa, 195
E. carotovora, 69 Pestalotia, 19 1
E. nigrifluens, 30 P. rheae, 144
Erysiphe ciclioracearum, 1 02 Phoma nerbatum, 144,154, 180
Erysiphe gmrninis, 179,181, 195,200,206 P. trifolii, 144
Fuzottum, 16,19,33,44,48,97, 119, 147 Phomopsis ju~~iperovora, 144
F.grarninearum (F.rosnrm), 15 1 Phyllosricta, 144
F. nivale, 144 Phymatotrichum om~~lvotum, 19
F. oxysporum. 24,33,52,69,144, 152, 180,184 Physonrrrt nrosirm, 144
F.roseum, 144,151 Physoderma, 1 19
F, solani, 144,152 Phytopltthora, 25,34,59,74,78,97,99, 100,101,
Gibberella zeae (G,rosettm), 25, 152, 178 119,142,146,154,155,156,157,158,194,
Gliuchdum, 144 195,196,211
Gloeosporiirm, 1 19 P. boehmeriae, 158
C. musarum, 34.179 P. cactonrnl, 18, 144,157,158
Glomerella cingrrlata, 5 2 , 148 P, capstci, 36, 144, 157, 158
Guiwrdia bid,vellii. 144 P.cinnamo11i,18,51,157,158 .
Helminthosporium. 62,68,147 P. citrlcola, 158, 159
H. avenue, 144 P. citrophthoro, 157, 159
H. carbonrrm, 40,153 P. colocasiae, 157
H. cynodontis. 154 P. cryptogea, 159
H.dictyoides, 146 P. drechsleri, 144, 157, 159
H. gramincum, 180 P.erythroseptica, 54, 144. 157,159
H. holmii, 153 P. fragariae, 39, 157, 159
H. homotnorphus, 154 P. heveae, 144, 157,159
H. kusanoi. 154, P. hibernalis, 159
H. mydis, 153 P. ilicls, 54, 157, 159
H. nodulosum. 153 P. Infestans, 24,41,54,61,66,73, 154,178, 186
H. oryzae, 60, 144 P. lateralis, 157
H.sativum, 146,182 P. megasperma, 41,74,lS6,157,159
H. sorokinianum. 153,180 P. nicotlnae, 144, 157, 158, 159
H.spicifemrn, !33 P. plmfvora, 73, 144,158, 159
H.turcicum, 40, 153 P. pnrasitica, 36,65,73, 144, 158,159
H. wgans, 43,146 P.phaseoli, 150, 159, 195
Hetcrodera glycincs. 1 2 3 P. primulae, 158, 159
Hererosporium, 1 1 9 P. quinlnea. 159
Hypomyces rolani, 144,152 Pllobolus, 29
Hypoxykn, 1 1 9 Pirtcularia oryzae, 60, 143, 144,182
Kabatiella. 99 Pleospora. 1 19
Leptodiscus terrestris. 144 P. herbamm, 144
Leptosphaerulina arachldicola, 144 P. phaeocomes, 142,
Leptosphaenrlina brioslna, 96, 144, 180 P. trichostoma, 142
Leptosph~erulitiatrifolii, 144 Po& ambigua, 144
Macrophominia phaseoli, 144 Pmdomonas, 31,58,124,125,126,127,141
Mehmspora lini. 1 97,200 P. atrofaciens, 125
Melanconium fuligineum, 46 P. fiycinea, 1 95
Monilim~jkticola.148 P. Iachtymnns, 74
Monachaetia, 1 19 P. mmprunorum, 58
Mucar, 68 P. phascolicola, 69,124
M. hiemalis, 143 P, solanaceamm, 69, 159,200,124
MycaspharrcUa lethal&. 150 Anrdopeztb medicaginis, 96. 180
M mu&o&, 180 , h c c i n h coranata, 193, 197
List of Organisms
P. mmlnk, 123,179,181,182,196,204 Septoria, 99

P,rccondita, 10 1 S glycines, 100
P. sMlfomis (P.glumarum), 180, 197, 198 S. nodomm, 144
Pyrenocharto, 76 S. psserinl, 180
P. teestis, 44, 144 S. rritici, 144, 160, 180. 186
q7enophora, 68 Sordoria destrens, 16
P. bromi, 142,144 Sphaerobolu~stellatus, 144
Pythium, 2 3 , 2 5 , 3 4 , 4 5 , 5 6 , 5 9 , 7 4 , 9 3 , 9 7 , 100, 101, Stemph~lium,1 19, 206
119,142,146,154,155,156, 158, 181,194, S. bolicki, 56
195,196,197,200 S. botryosum, 144
P.aphanidermotum, 158,159 S. floridnnum, 144
P. arrhenomones, 159 S,trifolii, 144
P. butleri, 159 Sneptotnyces, 17,19,23,35,38,51.56,57.70,72,
P. complecrens, 159 122
P. debaryanum, 159,178 S. scabies, 72
P. marsipium, 159 smmp.12 3
P. myriotylum, 159 Synchyntum, 1 19
P. oldochilum, 158,15 9 TaphfiM, 1 19
P. oiigandrum, 158, 159 Thcrnuf ephorus cucumcris ( P e l l i c u l a ~filornentow,
P. palingenes, 15 8, 15 9 Cerorobosidiumjilomenrosum, Cortirium
P. periplocum, 159, w p m ) . 159
P. tomlosum, 159 ThiclavL, 1 19
P. ultimum, 158,159 ntielmtiopsis bosicoh, 23,46, 74,99
P. unudlatum, 158 TUlerio controverw, 70,71
Rhizoctonia, 57,65,97,99 Wchodermrr, 93
R,hiemlis, 15 9 T.lignorum, 144
R. praticolu, 159 Mchometasphoerio, 15 3
R. solani, 159 Ummyces phoseoli, 196
Rhhopus, 53,93 Ustilago nuda, 207
R. nigriccms, 52 U. m'tici, 18 1, 200
R. stolonifer, 20, 3 1 Venturta inaequnlis, 15, 16, 142, 143, 161
Rhynchosporiurn orthosponrm, 4 1 Verticilkum, 13
R. secolis, 4 1, 93 V. a l b a m t m ; 68, 144,161, 184
Saccaromyces postorionus, 184 V. dohlioe, 66.68
Schlroph.vllirrn cotnn~une,62, 143, 160 V. laren'turn, 144
Scierophthora rnacrospora, 2 11 Wojirowicio gmminis, 144
Sclcrospora, 1 19 Xanfhomonus, 36,67,125,127,141
Sclerotinia frucficola, 5 2 , 1 60 X. phaseoli, 125, 128,179, 195, 196
S. trlfoliorum, 144. 147 X. rteuwrti, 38, 127
Scolecotrichum graminis, 13,52, 144 X. tmnslucens. 99
Stlenophoma, 119 X. uredovorus, 127
X. vesifotorh, 79, 124, 125, 179.184
Index
Enclosures, 3
Epiderrnd impression, 205
Ethylene Oxide, 84
A p r : purification. 10-1 1; silica gel as a substitute. Filter paper sporulation techniques, 146
214.215 Flagella staining, 214
A g r : a-ell technique, 215 Free hand sections, 21 8
Andrade's indicator, 15 Galley proof, 225
Antibiotics. 95.96 General nsistance. 177
Autoclave: recommended time for sterilimtian, 83; Glass ware cleaning. 9, 10
nr.8 3 Cram stain method. 21 3
Bactdophage: isolation, 105; purification. 106; Hemacytometer, 183
guntitation. 106; u r in identification of High frequency spark coil tester, 198
bacteria. 125 High-vacuum drying. 200
Baiting, 99 Host susceptibility, 178
Bbcuit cutter, 101, 105 Humidity control: uses, 227; sulfuric acid, 228;
c8rnoy's fluid. 213 glycerine solutions, 229; saturated salt solutions,
k i n hydrolysate: pmcation, 3; amino acid 230
~ b s t i t u t c .14; see also media containing Hydration of spores, 200
Cellophane: use in culture technique. 146; malting Hyphal tip isolation, 100
microrape mounts aith, 218: for reference Identification of plant pathogens: the host, 118;
cultures. 2 19 Fungi: fruiting structures, 119; monographs. 119;
Cellulou: stain for. 21 1 ;see also m d i a containing host index. 119; difficult or a new fungus, 120;
Channelad slide technique. 2 17 culture collections, 121
Counting cells. 183 Bacteria: culture collections, I21 ;pathogenicity,
Chitin: tmt for. 209; see ofso media eon hlning 124; symptomology, 124; procedure for
Colloidin technique. 205 identification, 125; diagnostic features of
C~yopnicnomge, 196 genera, 126,127
Culture conectionr, 121 Nematodes, 128
Cyeocel. 148 lnmasc of inoculum: factors involved. 142-144;
Mey isohtor. 103,105 special techniques, 146, 147; obligate parasites,
Detwhed leaf culturn. 186 148
Dfagmsk of p b t disease: r e f e m on hosts. 116, Incubation: definition, 177
118; diagnosis check list. 117; characters aaoci- Infection: definition. 176
4bd with actinomyates. 113; air pollution. 1 13; Infestation of soil, 181
betair, 113; fun& 113: genetic abemtion. 113; Inhibitory compounds, 93.95.96
gutation, 113; herbicide dmuge, 113; higher plant Inoculation: definition. 176; objectives, 177; require-
prrrritrr. 114; h t s and mites, 114; m i n d de- ments for, 177-180; general techniques. 180.182;
f i c h e k , 114; mineral exce*~.1 14; nematodes, 114; quantitative methods, 182-183; measuring
plant hormones. 114; saline soils, 114; spray dam- hoculum, 183-184; moist chambers, 185-186; use
age. 114; toxic plmts. I 15: to& plant residues, of detached leaves, 186-187; see also increrae of
115;- 115; m-eothcr-frott injury, minter inoculum
injury, excess u l t n , 115 Isolation of microorlpmisrns: specialized media. 3.4,
D n u t k a plate techniques, 9 7 4 9 5; factors involved. 93; standard isolation media,
Mwrr: criteria. 177; major requirements for. 177 93; methods, 96; from soil, 98-99; slow growing
Doublc cover @ass technique. 2 1 8 otpnlsms, 99; tools used. 105
~ r i c rdaerrr
d d r w e , 219 obligate parasites, 99; sin@e sporing, 101-102
bacterl, 98; sinple cell, 103.104 7.8, 157; phytone, 147, 148, 183; poly-
fungi: fmm retail pknt parts, 96; woody tissue placturonic acid, 66; proline, 230; salicin, 38;
and h i t s , 97; fine roots and d a m p d o f f dka p l . 214.215; soytone. 146; sorbose, 31;
eallinp. 97; single cells. IOU-103; freekg n u c h , 29.45.69. 105,224; sterol, 87; sucrose,
from bacteria, 100 14.29,33,36,49,56,57,58,71,75,95, 152,
bacteriophage. 105-106 153.154,199.202,229,230,240,24~6,
Kq-worih h l a t o r , 103.105 253,255,256,257,261,262,264,268,269;
Koch's postulates, 177 hnnic acid, 79; trypticaw, 70; tryptone, 203,
l a c t d u c h d n . 210 226,243,244; tyrosine, 245,246, 247
Lctophenol, 209 Microculture techniques, 216-2 18
h b e r t isoh tor. 103 Millipore filter, 87
Loose smut of u k a t : inoculation of. 181;detection Mist dcriccans, 197
of mycelium, 207 Mice control, 195
Lugol's iodine, 66 Mounting and staining media for fungi, 204-21 1
Lyophilization: pneral considerations, 197; prefreezing, bloist chambers: design, 184, 185; maintenance of
197-198; centrifugation, 198.200; h i g h m u m s~turptedair in, 184, 185, 186
drying, 200 Morton filter, 85.87
Ma~Iurriptpreparation, 224 New fungal species, 12 1
McLeod gauge, 198,199 N u c k v staining, 2 12
Media: prcparation, 2; units commonly u r d , 3; Partial racuum inoculation, 181
purif~ationof chemical constituents, 3; litmature Pattewn's mounting fluid, 21 0
coacuning, 8,9; purification of agar, 10-1 1 Peracetic acid, 85
u ~ isolate,
: culture and/or identify artinomycete, Photographs for publication: requirements, 225;
3; mlate and/or culture bacteria. 4; isolate and arrangement, 225
culture dermatophytic or other pathogenic fungi, Plant nutrient solutions, 7
4; isolate and culture fund from soil andlor Plant herbarium specimens, 123
water, 4; isc~latefungi from plant materid, 4: Plant press, 123
induct sporuhtion in fling&4,5,6.144; culture Predisposition, 178
andlor for the study of the nutrition of fungi Prescnation of microorganisms: method^ for specific
and bacteria, 6; aid in the identification of fungi, 194; periodic transfer, 194; oil overlay, 195;
fungi, 7; from plants, 7; aid in the identification frming, 195; lyophilization, 197; soil cultures,
of bacteria. 7; culture plant tissue or intact plants, 200; dried host tissue, 200; dried media cultures,
7 200; silica gel, 200; water preservation, 200
arntpining these compounds, beginning p. 12 Ropykne oxide, 84,153
(NOTE:Compounds indexed by medh no.) P W ~ a t i o n :of media ingredients, 3; of glassware, 9,
rlnnine, 7, 80: adenine, 198; arginine, 7. 8. 186; 10; of agar, 10. 11;of funpl cultures, 100
rsparginate, 91; asparagine, 10, 35,46,67, 81, 82. Ouvlntine requirements, 122
92, 112,199, 209,231,234; cawrmino acids. 83.
156; casein,44,45, 107, 247; casein hydrolysate, Radiation: effect on sporulation, 143; effect on spore
28.31.84. 113,214,233,246;ceUul~.5 , 6 , 4 6 , gemination, 179; effect on disease development,
SO, 72, 198: chitin, 120, 200; dulcitol, 38; ethyl 180
itrohol, 2.74; fumaric acid, 84; gallic acid. 79: Sealants for liquid mounts, 215
p l t i n , 145;$uwse, 3,IO. 13, 14. IS. 28.29,30. Seitz filter, 87
35,37,38,47.54,60,62,66.67,70,76,77,78, Semiprrmrnent mounts of fungi, 208,209
80,81,82,83,84,85,86,87,88,89,90,107, Settling tower, 182
112,115,121,123,125.1271129t137,138,139, Shiffs reagent, 213
142.146.147,148,150,151,152.157,161,162, Shipping of fungi and bacteria, 122
IH, 166,167,168,169,170,171,183,185,190, Single cctl isolation: value and limitations, 100: of rust
191,203,206,209,210,211,212,214.219,220, rpor.cs, 101; of fungi, 103,104; of bacteria, 103,
W.231,233.243.2s 1,252.270.272; Clutamic 104; tools uscd, 104
rcid.7.253; Jycerol, 31,44,73,91,92,106, S i n t u d glass filter, 85,86
186,195,267;glycine, 7, 123,261,262; lactic Skim milk, 197
rid, 74; bctose, 59,71,113,332; maltose, 19, Slide technique, 216217
114,130,135, 136,156,235,267;manitol 1, Soil plate method, 99
38.234; methionint, 7.10.125.269; peain, 176, Soil sterilization, 88
177,178,217,225,263; peptone, 18,26,27,35, Speciru resistance. 177
37.38,59,60.70,71,73.74,88,89,97,103, Spore lerminotion: tests to determine plcncy. 178;
10(, 114.121,129,130,133,135,137,138,139, affected by light, 179,
142,14S,149,150,151,I65,179,180,195,204, Sporuhtion of fung: media use to induce, 4 4 ; spec*
106,212,233,235,264,261,269; phenylilulim, techniques, 146,147,148;check list, 147;see atw,
ndhrion
Staining fungi: in host tissue, 205-208; with liquid Synthetic media, List of, 6 ; support oporulation of
mounting media, 209-2 12 fungi. 145,146
Staining nuclei, 2 12 Staining solutions, see mounting and staining media
Sterilization: autoclave, 82; steam without pressure, Thin culture cell, 217,218
83; dry air, 84; p s . 84; disinfectants and preserva- Ultraviolet sterilization, 88
tives, 85,86; filtration, liquid, 85; filtration, air, V~porpressure deficit, 227
88; radiation, 88 Van T i e e m cell, 217
Swinny filter adapter, 87 Water holding capacity of soil, 200
Symptom expression: influenced by light, 180; tem- Yeast extract, amino acid substitute, 14
perature, 179; humidity, 119 Zut, 216
J I C R I S A T
L I B R A R Y
DATE DUE
b
lCRlSAT L I B R A R Y
Ga?
Acc No.0 1848
Title Plant pathological methods

Plant Pathological Methods Fungi & Bacteria

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T h i s i s an a u t h o r i z e d f a c s i m i l e o f t h e o r T g i n a l

book, and was produced i n 1974 by m i c r o f i l m -

My Devoted Wife Camile

SUGGESTIONS ON PREPARATION OF h1EDlA AND RELATED SUBJECTS

TABLE 1. LISTING OF MEDIA BY USE

B. h h t e andlor culture bactaia Glucose nitrate agar (modified)

see alro Glucose-peptone medium Kldn y bean agar ~ h y w p h t h o r a f r r p p ~ e )

H. Medm t o aid in the identification of fungi

TABLE 2. LITERATURE ON CULTURE 3IEDI.4

Aaotha formula for acid dichromate:

1. Soak glassware in a mixture of 95 ml. of concentrated H2S04 plus 3-5 ml of concentrated

1. 1 Ib of agar b placed in a 6 1 flask.

1. Phcc agar in a muslin bag.

MEDIA AND THEIR INCREDLESTS

1. Agrobacterium I.wlation Medium

3. Alfalfa Decotion Agar

4. Alfalfa Seed Decotion

6. Alphaal Medium (modified)

7. Amino Acid Substitute for Casein Hydrolysate

8. Amino Add Substitute for 1% Yeast Extract (Difco)

h r g i n i n e HCI For purines and pyrimidines

10. Aphanomyces Synthetic (Haglund & King)

11, Aphanomyces Washing Solution

12. Apple Infusion Agar

13. Apple Infufioa Agar

Sucrose or glucose 2% Uwd to increase wilt Fusmia. 72 hr

15. Asthana and Hawker Medium A

16. A-ZSolution (Hiss and Reed)

19. Barnett Maltose Casamim, Medium

20. Bas11 b l i n d Sllltr Agar

22. Bean Juice Agar

23. Bean Meal Agar

24. Bean Pod Agar

26. Beef Peptone Agar

27. Beef Peptone Agar

28. Bennett Agar

29. Bilai Medium (modification by Joffe)

30. Bonner-Addicott Medium

31. Botrytis Separation Agar

32. Botran Isolation Medium

Ekan pod ='g Make a distilled water extract of

34. h a d Crumb Agar

35. Burkholda Medium

36. Burkholder and Nickell Tumor Medium

37. Cantino PYC Agdr

38. Carbohydrate Fermentation Medium (Fdwards and Ewing)

39. Carbon Utilization Medium

42. Carrot Decotion Agar

43. Carrot Discs

44. Casein Glycerol Medium

45. Casein Starch Medium (modified no. 44)

47. C a e b s e Ammonium Nitrate Medium

48. Charcoal Water

49. Chick-pea Sucrose Agar

50. CMC Medium

51. Corn Meal Agar (Shear)

52. Another Version of Corn hIeal Agar

53. Corn Meal PPP hledium

54. Corn Steep Dextrose

55. Crone Solution (modified by Bond)