BIOLOGICAL TECHNIQUES (BIO 204)

MICROTOMY:

The word “microtomy” originated from the Greek language Mikros meaning small and temnein
meaning to cut. So the word “microtomy” means to cut the tissue in thin sections. Microtomy is
a method for the preparation of thin sections for materials such as bones, minerals and teeth, and
an alternative to electro-polishing and ion milling. Microtome sections can be made thin enough
to section a human hair across its breadth, with section thickness between 50 nm and 100 µm.
For successful microscopic examination, it is necessary to have thin sections of the tissue by
microtomy. Microtomy is the next step after embedding the tissue and preparing the block.

Microtomes
Microtome is a sectioning instrument that allows for the cutting of materials into extremely thin
slices known as sections. Microtomes are used in microscopy, allowing for the preparation of
sample for observation under transmitted light or electrons radiation
Microtomes is the main instrument by which we cut the embedded tissue in the paraffin block
into thin sections.
The different types of microtomes in the traditional histology laboratory are:
(a) Rotary microtome
(b) Rocking microtome
(c) Base sledge microtome
(d) Sliding microtome
(e) Cryo microtome
(f) Ultra microtome
(g) Laser microtome
(h) Vibrating microtome
(i) Saw microtome

(a) Rotary microtome: This is the most commonly used microtome in routine laboratory. The
cutting blade is kept in horizontal position, and the block containing tissue moves up and down
with the help of rotary handle attached with the microtome. A rotary action of the hand wheel
actuate the cutting movement. Here the hard tissues can be cut without vibration. Serial sections
or ribbons of sections can easily be obtained. The block holder or block is mounted on the steel
carriage that moves up and down and is advanced by a micrometer screw. The typical cut
thickness for a rotary microtome is between 1 and 60 µm. For hard materials, such as a sample
embedded in a synthetic resin, this design of microtome can allow for good “semi-thin” sections
with a thickness of as low as 0.5 µm.
From there the tissue can be mounted on a microscope slide, stained with appropriate aqueous
dye(s) after prior removal of the paraffin, and examined using a light microscope.
Advantages:
1. Good-quality 2–3-µm-thin section is possible.
2. Heavy and stable automated rotary microtome reduces health hazard and gives the best-
quality section.
3. Good tissue ribbon production.
4. Easy-to-cut various types of tissue.

1
Disadvantages:
1. Expensive.
2. Unsuitable to cut large block.
3. Knife faces up and so may be dangerous to the technical staff.

(b) Rocking microtome: The rocking microtome is also known as Cambridge rocking
microtome. The word “rocking” is used as there is a rocking action of the microtome like arm
movement. In this type of microtome, the knife is static, and the block of tissue moves in a
rocking motion (arc-like movement of the block).
This is one of the oldest designs of the microtome. The microtome can cut thin section with
ribbons and is ideal for serial section. The sections are slightly curved in this microtome.
Advantages:
1. Thin section
2. Easy to operate
3. Low-cost instrument and reliable
Disadvantages:
1. Tissue is curved and the microtome does not provide flat section.
2. The microtome is of light weight so vibration may occur.

(c) Base sledge microtome: Sledge Microtome is a device where the sample is placed in a fixed
holder (shuttle), the sledge placed upon a linear bearing, a design that allows for the microtome
to readily cut many coarse sections. Applications for this design of microtome are of the
preparation of large samples, such as those embedded in paraffin for biological preparations.
Typical cut thickness achievable on a sledge microtome is between is 10 and 60 microns.
Hard materials like wood, bone and leather require a sledge microtome. These microtomes have heavier
blades and cannot cut as thin as a regular microtome.
In sledge microtome the block is fixed in a static position within a steel carriage. The knife slides to and
fro over the top of the block. This microtome is the best for large tissue sample or the hard tissue.
The tissue sections are usually thick (more than 10 µm) in base sledge microtome.
Advantages:
1. Hard tissue can be cut.
2. Large tissue sample can be cut.
3. The best microtome for ophthalmology and large neuropathology section.
Disadvantages:
1. Difficult to get thin section.
2. Large slides are required.

(d) Sliding microtome: In this microtome the knife is static, and the block moves horizontally
over the knife.
Advantages:
1. Large sections can be cut.
2. Mainly used for colloid in-embedded tissue.
3. Simpler design and easy maintenance.
4. Brain sections can be cut better by this type of microtome.

2
Disadvantages:
1. The knife may glide in case of hard tissue and may jump.
2. Long knives are difficult to sharpen.

(e) Cryo microtome: This type of microtome is used for cutting frozen tissue samples/sections.
The sample is made hard in liquid nitrogen and then cut by the microtome in the chamber that
contains liquid nitrogen. Water-rich tissues are hardened by freezing and cut in the frozen state
with a freezing microtome or cryo-microtome. The reduced temperature allows for the hardness
of the sample to be increased, such as by undergoing a glass transition, which allows for the
preparation of semi thin samples. However the sample temperature and the knife temperature
must be controlled in order to optimize the resultant sample thickness.
Sections are stained and examined with a light microscope. This technique is much faster than
traditional histology (15 minutes vs 16 hours) and is used in conjunction with medical
procedures to achieve a quick diagnosis.
Cryo-sections can also be used in immuno histochemistry as freezing tissue stops degradation of
tissue faster than using a fixative and does not alter or mask its chemical composition as much.

Advantages:
1. To get rapid section for rapid diagnosis
2. To study nerve biopsy
3. To study enzyme histochemistry
Disadvantages:
1. It needs continuous supervision to maintain the temperature.
2. Freezing artefact is often seen.
3. Very expensive instrument.
4. Fixed tissue is very difficult to cut.

(f) Ultra microtome: Ultra microtome is used to cut ultra thin sections. It can allow for the
preparation of extremely thin sections, with the device functioning in the same manner as a
rotational microtome, but with very tight tolerances on the mechanical construction. These
extremely thin cuts are important for use with transmission electron microscope (TEM) and
Serial Block-Face Scanning Electron Microscopy (SBFSEM), and are sometimes also important
for light-optical microscopy. The typical thickness of these cuts is between 40 and 100 nm for
transmission electron microscopy and often between 30 and 50 nm for SBFSEM.
Thicker sections up to 500 nm thick are also taken for specialized TEM applications or for light
microscopy survey sections to select an area for the final thin sections.
Diamond knives (preferably) and glass knives are used with ultra microtomes.
After embedding the tissues in epoxy resin, sections are cut between 40 and 100 nano micron
thickness with the help of glass knife or diamond knife. The tissue is at first trimmed to make
small block of 1 × 1 mm size, and then the section is cut by this ultra microtome with the help of
optical microscope. The cut section is allowed to float on the water hold by a boat and then
finally picked up on a metal grid. These sections are of 0.5 to 1 µm thickness and are mounted on
a glass slide and stained to locate areas of interest under a light microscope prior to thin
sectioning for the Transmission Electron Microscope (TEM). Thin sectioning for the TEM is
often done with a gem quality diamond knife.

3
 (g) Laser microtome: The laser microtome is an instrument for contact free slicing. Prior preparation
of the sample through embedding, freezing or chemical fixation is not required, thereby minimizing
the artifacts from preparation methods. Alternately this design of microtome can also be used for very
hard materials, such as bones or teeth as well as some ceramics. Dependent upon the properties of the
sample material, the thickness achievable is between 10 and 100 µm.
The device operates using a cutting action of an infra-red laser. As the laser emits a radiation in the near
infra-red, in this wavelength regime the laser can interact with biological materials. Through sharp
focusing of the probe within the sample, a focal point of very high intensity, up to TW/cm 2 , can be
achieved. Through the non-linear interaction of the optical penetration in the focal region a material
separation in a process known as photo-disruption is introduced. By limiting the laser pulse durations to
the fem to seconds range, the energy expended at the target region is precisely controlled, thereby
limiting the interaction zone of the cut to under a micro metre. External to this zone the ultra short beam
application time introduces minimal to no thermal damage to the remainder of the sample.

(h) Vibrating microtome:

The vibrating microtome operates by cutting using a vibrating blade, allowing the resultant cut to be
made with less pressure than would be required for a stationary blade. The vibrating microtome is
usually used for difficult biological samples. The cut thickness is usually around 30-500 µm for live
tissue and 10 - 500 µm for fixed tissue.

(i) Saw microtome

The saw microtome is especially for hard materials such as teeth or bones. The microtome of this
type has a recessed rotating saw, which slices through the sample. The minimal cut thickness is
approximately 30 µm, and can be made for comparatively large samples.

Microtome Knife
The microtome knife is important to cut uniform and thin section of tissue. These are made of
steel. Various types of knife profiles are available for different types of microtomes. The most
commonly used knife profile is the wedge profile.
The various types of microtome knife include :
1. Plano concave: One side of the knife is plain and the other side is concave.
Originally these knives were used for cutting colloid in-embedded tissue. This is a very sharp
knife and is used for cutting soft tissues. However, presently these knives are less frequently
used.
2. Biconcave : The knife is concave on both sides. The knife was used for rocking microtome.
The concavity of the knife is often difficult to identify. This is a less rigid type of knife and often
vibrates during cutting.
3. Wedge : This knife is plain on both sides. This is the most widely used knife for routine
microtomy and it is compatible with the different type of microtome. This type of knife is also
easy to sharpen.
4. Tool edge : The knife resembles chisel used in wood working. Both sides of the knife are
plain; however the cutting edge is steep. It is mainly used for cutting hard tissues such as
decalcified bone.

4
Factors Involved in Cutting
1. Temperature: Lowering the temperature facilitates section cutting.
2. Angle of rake: Higher rake angle helps in smooth flow of ribbons. Lower rake angle is used
for hard tissue.
3. Consistency of tissue: Soft tissue is cut at a slow rate than the hard tissue.

STEPS FOR PROCESSING TISSUES:

1. After fixing in Bouin’s fluid for 12-24 hours, wash well in tap water and then in distilled
water. Remove as much of picric acid as possible by repeated washing.
2. Dehydrate the tissues in 30%, 50%, 70%, 90% and absolute alcohol, 4-6 hours in each
grade. You can leave the tissue in 70% alcohol for any length of time and process a few pieces
at a time.
3. Clear in xylene for 15-30 minutes. Small sized tissues are better as they become transparent
quickly.
Embedding in wax
Pour molten wax (50-60 C range) in the bottle containing the tissues. Put in the incubator for
0

12-18 hours. Incubator should be set at 58-60 C temperature, preferably at 60C. Keep filtered
0

molten wax in beakers at the same melting range in the incubator. After infiltration of wax into
the tissues for 12-18 hours make the tissue blocks. Two L-shaped brass metallic pieces are put on
the glass plate smeared with glycerin. Pour the molten wax into the cavity formed by connecting
together the two metallic pieces and transfer the material in the middle of the wax-containing
cavity. Remove the wax block after setting and drying. Trim the paraffin blocks to proper size
and keep in paper covers to be used later.

Sectioning the Paraffin Block

The following instruments are essential for section cutting:
1. Microtome with blade
2. Water bath
3. Paraffin block with embedded tissue to cut
4. Ice tray
5. A blunt forceps
6. Camel brush
7. Slide rack with slides
Water Bath (Floatation Chamber): Water bath is used to float the tissue after cutting .The
temperature of the water bath is usually controlled automatically by a thermostat. The
temperature of water in the water bath should be 10 °C below the melting point of the embedded
paraffin wax and is usually kept in 40–50 °C. It is necessary to prevent formation of any air
bubbles within the water bath. For adequate floating of the tissue, one can add a few drops of
alcohol or little amount of detergent. This reduces the surface tension of the water and tissue
floats smoothly.
Blunt Forceps: Blunt forceps helps to manipulate the floating tissue section.
Camel Hair Brush: Camel hair brush is used to clean the blade.

5
Slide Rack with Clean Glass Slides: The clean slides are kept in the slide rack. The slides can
be already labelled by diamond pencil or on the frosted side by lead pencil. Alternatively this can
be marked after lifting the tissue section.

Adhesive: In case of routine section and staining, no adhesive is required. However, in certain
situations we use cell adhesive material
The most commonly used adhesives include:
• Mayer’s egg albumin and glycerol:
• Poly-l-lysine:
• 3-Aminopropyltriethoxysilane (ACEP):

Steps of Tissue Sectioning

1. Trimming the tissue: Trimming of the tissue is needed to expose the tissue piece within the
paraffin wax for cutting. The block is fixed in the chuck of the microtome, and the paraffin is cut
till the tissue is fully exposed. Adequate caution should be taken not to overcut tissue as this may
produce various artefactual changes.
2. Cooling the block: After the initial trimming, the blocks are kept for cooling for 15–20 min.
This will help to maintain the same consistency of the paraffin and tissue. This helps in easy
cutting.
3. Cutting proper: The block is fixed in the chuck of the microtome. The cutting surface of the
block should be parallel to the knife. The angle of clearance should be only 2–5° to have good
section . The tissue in the block is cut by gentle, smooth and slow stroke. The ribbon-like tissue
sections are produced. The tip of the ribbon is held by forceps, and the end part of the ribbon is
removed from the knife edge by brush. In case of any difficulty to get the flat section, the cutting
surface should be gently warmed by warm water.
4. Floating the ribbon: The ribbon of the tissue is floated in the water bath, and this makes the
tissue flat and removes any wrinkling of the tissue. With the help of the forceps, the individual
sections are separated from each other.
As mentioned before, the temperature of the water bath should be constantly maintained below
the melting point of the paraffin wax. Note: In case of temperature variation in the bath, air
bubbles may be formed that may rupture the tissue.
5. Picking up the tissue: The slide is placed vertically within the water bath in front of the tissue,
and when the tissue is touched, the slide is withdrawn vertically from the water. The tissue
pickup process must be gentle and smooth. To prevent any mix-up, the water bath should be
cleaned immediately after cutting each block.
6. Drying the section: The slide containing the picked-up section is kept in slide rack. The slides
are now kept in hot oven to get dry. The temperature of the oven should be slightly more than the
melting point of the paraffin.

Some hints while cutting sections

Cut at 7m thickness.
Prepare water bath at 58 or 60 C.
0

Smear albumen (Mayer’s albumen:-White albumen, 50 ml; glycerin, 50 ml; sodium salicylate,
1.0 gram) on the slide. It will fix sections on the slide.
Put a few drops of 30% alcohol on the albumen smeared slide.
Cut the paraffin ribbon into bits containing 5 or 6 sections and put ribbon-bits on the slide with alcohol.

6
Put the ribbon into the water bath. The ribbon will completely stretch in the water bath without
melting. Lift it with the same slide coated with Mayer’s albumen.
Rest the slide at an angle of 45 C against a solid surface. Water under the material will get
0

drained out in 6-8 minutes.

Put the slides in the incubator overnight (24 hours) at temperature of 56-58 C.
0

Remove the slides next day and store them in a slide box as long as required.
Prepare the staining set in staining jars that contain grooves for holding slides.

Problems In Tissue Sectioning

(i) Multiple air bubbles which may rupture the tissue; The air bubbles are produced due
to uncontrolled temperature in the water bath.
(ii) Freezing artefact of the tissue due to immediate putting the tissue from the
refrigerator to formalin.
(iii) Uneven staining pattern due to poor de-paraffinization of the tissue