BOT205/3

Microscopy and
Histology Technique

SCHOOL OF BIOLOGICAL SCIENCES

Course coordinator: Dr. Azlinda Abu Bakar

azlindaab@usm.my
Co-lecturers : Dr. Siti Khayriyyah Mohd Hanafiah
kye@usm.my
Introduction to Histology
Lecture 1
Dr. Azlinda Abu Bakar
Email: azlindaab@usm.my; Ext: 3509
4 April 2022
What is histology and why is it important?

1. The study of the microscopic structure of biological material and the ways in which
individual components are structurally and functionally related.

2. Critical to biological and medical science:

o Bridge between biochemistry, molecular biology, physiology, disease processes and

the effects of diseases.

o Knowledge of normal histologic appearance is essential if abnormal diseases

structures are to be recognized.

3. Challenges:

o Most fresh tissue specimens are colorless and squishy.

o They provide little useful information.

o For scientific or diagnostic purposes, tissue specimens must undergo substantial

alteration in preparation for viewing under a microscope.
Terms in histology

o Histotechnology: technique of processing the tissues submitted for

histopathological study until the preparation of the stained section on a glass
microscopic slide ready for study

o Histology is the study of tissue sectioned as a thin slice using a microtome and
it can be described as microscopic anatomy.

o Histography: the photographing of stained cells

o Histopathology: microscopic study of diseased tissue

o Histotechnician/histotechnologist: trained scientists who perform the

preparation of histological sections
HISTOTECHNICIAN

HISTOTECHNOLOGY

HISTOGRAPHY HISTOPATHOLOGY
FIG 1 Observation of the highly efficient lytic activity of
LysGH15: effects on the strain USA300 after treatment with
LysGH15 for 0 (A), 1 (B), and 2 (C) min.
Types Of Surgical Biopsies:

o Incisional biopsy when a piece of the tumor is taken

for the diagnosis.

o An excisional biopsy removes the whole tumor and Incisional

submitted for surgical pathology report for diagnosis excisional
and resected margins.

o Punch biopsy is done to remove a small growth by

removing a core of tissue with a skin punch. The
biopsy area is then covered with a bandage.
Punch
o A needle biopsy may be used to take tissue or fluid
samples from muscles, bones, and other organs, such
as the liver or lungs. Fine needle when the bore of the
needle is 23 to 25 gauge.
needle
o Curretage biopsy when the endometrium biopsy is
taken.

Surgical Pathology May Be Carried As:

1. Paraffin section (paraffin-embedded method).

2. Frozen section (the tissue is frozen and sectioned).

This is a rapid method and available during surgical
operation. Curretage
Steps in the Processing of a Biopsy tissue
Step 4ii: Embedding:
Tissue(s) are placed into
Step 2: Dehydration: Tissue(s) is placed into melted paraffin (58°C)
alcohol or acetone in various grades or and kept there at least for
concentrations. This step will remove water and 2 to 4 hours.
replaced by alcohol. This step takes at least 4 to
6 hours.

Step 3: Clearing:
Step 1: Fixation: A biopsy tissue is Step 4i: Infiltration: Add
Tissue(s) is placed
kept in formalin for at least 12 to 24 the crystals of paraffin wax
into xylene which
hours. This step is very important. If to the tissue gradually and
will replace
the tissue(s) is not fixed properly reduce the clearing
alcohol.
then you will not get good slides for solution. This to prepare the
interpretation. tissue for the embedding
step. This process will
require approximately 24-
48 hrs. Now the tissue is fit
for embedding. This
process will require 1 to 2
hours.
 Step 8: Imaging

Step 7: Staining: Stain slide(s)

with haematoxylin and eosin.
Mounting: Stained slide(s) is
mounted with cover glass.

Step 6: Sectioning: Block(s) is cut

by a microtome with a thickness of
4 to 7 microns. The cut sections
are placed on slides.

Step 5: Block making: Tissue(s) is removed from

paraffin and paraffin block(s) is made.
Purpose of Microscopic Examination
o Understanding the anatomy of the structure.

o Studying the distribution of the constituent cell types in relation to each other.

o Studying the cell contents and cell division process (cytological investigations).

o Studying the development of organs.

o Studying the chromosomes, their structure and number, behavior during cell
division etc.

o Studying the chemistry of different cell constituents (histochemistry) and

cytochemical localization of materials
Four Typesof major microscopicslides
1. Wet mount - Whole frame 2. Prepared slide – sectioning and staining

3. Smearing 4. Dry mount

Wet mount - Whole frame

o The whole specimen is preserved to study its structures.

o Specimens have to be killed first, then fixed; in order to keep or preserve them.

o Normally, specimens are fixed in 70% alcohol so that it become transparent.

o To study the inner structures; specimens need to be stained properly.

Prepared slide – Sectioning and staining

o Individual cells in any organ can be observed if it is properly sectioned or

dissected.

o Sectioning can be conducted on both animal and plant tissues.

o However, animal and plant tissues are too soft to be sectioned into thin layers.

o Therefore, tissues need to be hardened first before they are fixed.

o The harden tissues are washed and preserved in 70% alcohol before sectioning
into thin layers.
Smearing
o Smearing is done for samples/specimens such as blood, urine or bacterial cells.

o A thin layer of sample is smeared on a clean microscopic slide.

o The smear needs to be stained with proper dyes/stains in order to observe cell
structures easily.

o The stained slides can be kept for future reference.