F) MICROTOMY

The process by which processed tissue is trimmed and cut into uniformly thin slices or sections. The basic
instrument used is a microtome that is capable of cutting a section at a predetermined thickness by sliding the
block into a cutting tool, usually a steel knife of blade, which is fixed and attached to the machine.
There are five kinds of microtomes, these are:
1) rocking microtome for cutting serial sections of large blocks of paraffin embedded tissues,
2) rotary microtome for cutting paraffin embedded sections which is the most commonly used microtome
that allows the perfect sectioning of the paraffin-embedded tissue specimens,
3) sliding microtome for cutting celloidin embedded sections
4) freezing microtome for cutting the unembedded frozen sections
5) ultrathin microtome for cutting sections fro electron microscope

Whatever the type of microtome is used, the principle remains essentially the same, that is, a spring-
balanced teeth or pawl is brought into contact with, and turns a ratchet feed wheel connected to a micrometer
screw, which is in turn rotated, moving the tissue block at a predetermined distance towards the knife for cutting
sections at uniform thickness.
For the ideal sectioning of the tissue, certain accessories and types of equipment are required apart from
the Microtome and paraffin block which are as follows:
a. A pair of pointed forceps
b. Small squirrel hair brush
c. A sharp scalpel
d. Water bath with thermostatic control
e. Paper towel/Clean cloth
f. Clean microscopic glass slides
g. Section adhesive (Mayer’s Albumin, Gelatin)
h. Blotting paper and ice cubes

PROCEDURE OF SECTION CUTTING/ MICROTOMY

1) Trim the block with the hot knife before beginning the microtomy or section cutting. In a properly
trimmed block face, the top and bottom edges will be parallel; the block face should be in touch with the
knife. All the tissues desired on the slide should be exposed to the face and no scratch marks should be
visible on the surface.
If the scratch marks are visible on the block face, the knife must be moved laterally to use a new portion of the
knife edge and the face is trimmed again. Accordingly, the microtome was prepared for the process of section
cutting:
• The feed mechanism should be screwed back
 • The knife position should be checked
• The indicator for the thickness of the section should be adjusted and kept between 4-6μ.
• If there is enough paraffin in front of the tissue untrimmed, then the block must be cut until the
tissue reaches the knife.
2) The knob attached to the spring
holder was moved quickly, so as to
obtain a ribbon enabling the tissue
to adhere to each other. When the
Serial section/Ribbons are obtained
they may be transferred, with the
help of small squirrel hair brush &
forceps, into the tissue floatation
bath and the temperature should
be maintained below the melting
point of the wax. This will remove
any wrinkles (if present in the
section) and flattens the sections.
3) The ribbons are then cut and
shorten by using a hot pointed forceps into lengths of about two and a half inches and transferred onto
the clean grease free glass slide smeared with the adhesive (e.g. Mayer’s Albumin) for the better adhesion
of the section so that it will not get washed away during the process of staining. Several ribbons may be
placed side by side, and so a large number of sections kept in the order in which they are cut. A mark
should be made on the slide to indicate where the series begins, and each slide should be numbered so
that the exact position of each section in the series can be recognized at once.
4) The slides are kept aside for a few hours to dry and after that, the sections should be stained using
suitable staining technique.

G) CYTOLOGY PREPARATION
Diagnostic cytology is the science of interpretation of cells that are either exfoliated from epithelial
surfaces or removed from various tissues. George N Papanicolou introduced cytology as a tool to detect cancer
and pre-cancer in 1928. It is now a widely accepted method for mass screening in asymptomatic population. The
advantages of diagnostic cytology are that it is a non-invasive, simple procedure, helps in faster reporting, is
relatively inexpensive, has high population acceptance and facilitates cancer screening in the field.

Different methods of diagnostic

cytology includes:
1) Collection and examination of
exfoliated cells such as vaginal
scrapes, sputum, urine, body
fluids etc.
2) Collection of cells by brushing,
scraping or abrasive techniques is
usually employed to confirm or
exclude malignancy
3) Fibreoptic endoscopes
4) Other procedures can be used for collecting samples directly from the internal organs
 Fine-Needle Aspiration Cytology/ Biopsy (FNAC/FNAB) is now a widely accepted diagnostic procedure,
which has largely replaced open biopsy. This method is applicable to lesions that are easily palpable, for example
swellings in Thyroid, Breast, superficial Lymph node etc. Imaging techniques, mainly ultra-sonography and
computed tomography, offer an opportunity for guided FNAC of deeper structures.

H) STAINING
Slides are stained to highlight features. Staining is
the process of applying dyes on the sections to see and study
the architectural pattern of the tissue and physical
characteristics of the cells. This is made possible because
different tissues and cells display vary affinities for most dyes
and stains used during the process, so that they become more
visible, morphologic changes are more easily identified, and
the presence or absence of disease process can be
established. Physical and structural relationships of tissues
and their cells are thereby studied and evaluated.
For the purpose of simplicity, staining of tissues can
be classified into three major groups, namely:
1) Histological staining- process whereby the tissue constituents are demonstrated in sections by direct interaction
with a dye or staining solution, producing coloration of the active tissue component

2) Histochemical staining (Histochemistry)- the process whereby various constituents of tissues are studied thru
chemical reactions that will permit microscopic localization of a specific tissue substance

3) Immunohistochemical staining- this is a combination if immunologic and histochemical technique that allow
phenotype markers to be detected and demonstrated under the microscope, using a wide range of polyclonal or
monoclonal fluorescent labelled or enzyme-labelled antibodies

H&E stain
Routine histology uses the stain combination of hematoxylin and
eosin, commonly referred to as H&E. Hematoxylin is a basic stain with deep
purple or blue color. Structures that are stained by basic stains are described
as basophilic ("base-loving"). Chromatin (i.e., cell nuclei) and ribosomes are
basophilic.
With H&E staining, basophilic structures are stained purple. Eosin is
an acidic stain with a red color. Structures stained by acid stains are described
as acidophilic (or eosinophilic) and include collagen fibers, red blood cells,
muscle filaments, mitochondria. With H&E staining, acidophilic structures are
stained red or pink.

Trichrome stain
Trichrome uses three dyes, including one that is specific for the
extracellular protein collagen. Depending on the particular stain
combination, a trichrome stain may color collagen fibers sky-blue or
bright green.
 The principle use for trichrome is to differentiate collagen from other eosinophilic structures, such as
muscle fibers. Trichrome stains can be especially useful for highlighting an accumulation of scar tissue, as in
glomerulosclerosis of the kidney or cirrhosis of the liver.

For staining, several materials are needed including:

1. Coplin jar – a covered glass vessel that is rectangular in cross section and grooved inside for holding microscope
slides vertical during processing
2. Slotted staining dishes – holding from 5 to 19 slides, over which different solutions are poured. Slides are placed
on end singly or in staggered fashion, in the arm.
3. Metal or glass staining racks or carriers – holding from 10-30 slides upright

I) MOUNTING AND LABELLING

To preserve and support a stained section for light microscopy, it is
mounted on a clear glass slide, and covered with a thin glass coverslip. The slide
and coverslip must be free of optical distortions, to avoid viewing artifacts.
A mounting medium is used to adhere the coverslip to the slide. Aqueous
based mounting media are available, which allow the mounting of tissues directly
from the staining procedure. However, the
water solubility of some stains allows them
to bleed and/or fade in such mountants,
necessitating the use of resinous mounting
media. To use a nonaqueous mountant, the
section must first be dehydrated and
cleared.
Any water carried over to the mounting stage will show up as bubbles
or vacuole-like structures, as the water droplets aggregate and distort the
tissue. It is important to note also that the clearing agent used must be
compatible with the mounting medium, or the sections must be thoroughly dried prior to mounting. Slides for
mounting should be properly labelled with case number to identify the section and avoid any damage or partial
loss of sections caused by wiping the wrong side of the slide and for proper identification.
A good mounting medium must have the following characteristics:
1. To avoid distortion of the image, the refractive index of the mountant should be
as near as possible to that of the glass slide which is 1.518.
2. It should be freely miscible with xylene and toluene.
3. It should not dry quickly.
4. It should not crack or produce artefactual granularity on the slide upon drying.
5. It should not dissolve out or fade tissue sections.
6. It should not cause shrinkage and distortion of tissues.
7. It should not leach out any stain or affect staining.
8. It should not change in color or pH.
9. It should set hard, thereby producing permanent mounting of sections.
J) SLIDE TRAY ASSEMBLY
Slides are gathered and
assembled in slide folders by
case number. Assembled cases are then delivered to pathology offices or to the courier room for delivery to
pathologists located off-site.

K) MICROSCOPIC EXAMINATION
Histopathology benefits greatly from analysis conducted under the microscope, provided the sample is
pure and viable. Analysis under a microscope is necessary for a histopathologist to make an accurate prognosis.
Additionally, the microscope proves to be one of, if not the most, used and relied upon tool by scientists
studying pathogens that cause tissue changes or damage, as it allows them to see the level of tissue degradation
present and therefore, verify the progression of the particular disease.

Microscopy analysis is important in histopathology because it

allows scientists to view tissue changes or signs of infection, even if the
pathogen is not present. Microscopy offers even more benefits in the field
of histopathology not only because it allows for a quicker turn around
time when making a diagnosis but also because it provides scientists with
a means of viewing bacteria that do not cultivate.
Recently, the use and importance of the microscope has increased, as histopathology processes are now
including immunohistochemistry techniques, which basically allows the pathogen to show up as a fluorescent
object, making detection much simpler for the scientist. This technique is helping histopathologists to see
microorganisms that were once undetectable, either because they do not cultivate, cannot be seen through
traditional staining methods, do not stain well, have an unusual morphology, or simply have a small presence. It is
also allowing scientists to view missing DNA sequences on chromosomes, spot intracellular bacteria and learn
more about nucleic acid and RNA.

REFERENCES:
Anderson, H. (2010). Histopathology - Analysis under the Microscope. Retrieved from
https://www.microscopemaster.com/histopathology.html
Mounting Tissue Sections | National Diagnostics. (2011). Retrieved from
https://www.nationaldiagnostics.com/histology/article/mounting-tissue-sections

McLean, C. (2018, March 18). Principles of Staining. Retrieved from

https://www.slideshare.net/CharlotteMcLean4/principles-of-staining

King, D. (2010). SIU SOM Histology INTRO. Retrieved from http://www.siumed.edu/%7Edking2/intro/tissprep.htm

Abraham, Pillai, Mani, jayalal, E. K. K. K. (2005). Manuals in Training for Cancer Control. Manual for Cytology, 1.
Retrieved from https://screening.iarc.fr/doc/Cancer_resource_Manual_3_Cytology_New.pdf

Bahtra, S. (2018, March 2). HISTOPATHOLOGY & CYTOPATHOLOGY NOTES

MICROTOMY – THE ART OF SECTION CUTTING. Retrieved from https://paramedicsworld.com/histopathology-
cytopathology-notes/microtomy-art-section-cutting/medical-paramedical-studynotes

Bahtra, S. (2018, March 2). HISTOPATHOLOGY & CYTOPATHOLOGY NOTES

MICROTOMY – THE ART OF SECTION CUTTING. Retrieved from https://paramedicsworld.com/histopathology-
cytopathology-notes/microtomes/medical-paramedical-studynotes

Gregorios, J. H., & Faldas, M. M. (2016). Histopathologic techniques. Makati City, Philippines: Katha Publishing.