IBBS: MBCHB YEAR 2

HISTOLOGY AND ITS METHODS OF STUDY

MRS A/N MWAMBA

 HISTOLOGY
INTRODUCTION
• Histology is the study of the body tissues and
how they are arranged to constitute organs.
• Comes from a Greek root “histo” translated as
either “tissue” or “web,”
 INTRODUCTION;CONT

• Hence tissues are webs of interwoven

filaments and fibers, both cellular and non-
cellular, with membranous linings.

• Have two interacting components: cells and

extracellular matrix (ECM)
• ECM supports cells and cells produce ECM
 PREPARATION OF TISSUES INTO
SECTIONS FOR STUDY
• This is the most common procedure used in
histological research with use of a light
microscope
• The ideal microscopic preparation is first
preserved
• Makes the tissue slide to have the same structure
and molecular composition as it had in the body.
 Tissue fixation and Fixatives
▪ Tissue fixation; Foundation step in the study of
pathology

▪ Exists to prevent the autolysis and degradation

of tissue

▪ Enables observation of tissue microscopically

following sectioning.
 Fixatives
• Compounds used to preserve tissue structure by
cross-linking and denaturing proteins, inactivating
enzymes, and preventing cell autolysis or self-digestion

• Examples of fixatives used in preserving tissues are

chemical substances like formalin, mercuric chloride,
acetic acid, picric acid, ethanol, methanol and
glutaraldehyde and mostly used in combinations.
 COMBINATIONS
1. Bouin’s fluid (formalin, acetic acid and picric
acid)
2. Formal sublimate (formalin and mercuric
chloride)
3. Helly’s fluid (formalin, mercuric chloride and
potassium dichromate)
4. Zenker’s fluid (acetic acid, mercuric chloride
and potassium dichromate)
 Purpose of fixation
• To preserve the morphology and chemical
composition of the tissue
• To prevent autolysis(self-destruction/ tissue
digestion by enzymes present within the cells)
and putrefaction (decay or decomposition)
• To harden the tissue for easy manipulation
• To solidify colloidal material
• To influence staining
NOTE:
• After fixation, Hard tissues like bone and
tooth, which contain large amount of calcium
salts, require an additional step called
decalcification before they are subjected for
dehydration.
 Cont`
• Makes the hard tissues soft, enabling them to
be cut with microtome.
• E.gs decalcifying agents are used:
• 10% nitric acid
• 5 trichloroacetic acid
• Ethylene diamine tetra acetic acid (EDTA).
• Fixation takes 24 hours
 Tissue processing
INVOLVES (3) MAIN STAGES After Fixation
• Dehydration
• Clearing
• embedding
 1. Dehydration
• Water from the tissues is removed in a gradual
manner by immersing the tissues in ascending
grades of alcohol (ethanol) 50%, 70%, 90%
and absolute alcohol
• This is in order to embed it in paraffin wax
which is not miscible in water.
• Tissue remains in each of these grades for 30
– 60 minutes
• This takes a total of about 2 – 4 hours
 2.Clearing
• After dehydration, tissue is treated with
paraffin solvent like xylene, toluene,
chloroform, benzene, or petrol for 2-3 hours.

• These agents penetrate and replace the

alcohol from the tissue and makes it
translucent (clear).
 3.Embedding
• Tissue is infiltrated with embedding medium
• To give a rigid consistency to the tissue.
• Done by placing it in melted paraffin until it becomes
completely infiltrated with this substance.
• various embedding media are paraffin wax,
celloidin, gelatin, plastic resins.
• Paraffin is the routinely used embedding medium for
light microscopy.
 Embedding involves two steps:
a. Impregnation/infiltration

• After clearing, the tissue is impregnated with

molten paraffin wax (at 58°C – 60 °C) in a hot
air oven for 2 hours with three changes.

• The melting point of paraffin wax is 56 °C.

 b. Casting or block making
• After impregnation, the paraffin-infiltrated tissue
is placed in a small ‘L’ molds with melted(molten)
paraffin and allowed to harden.

• The molten wax cube with the tissue is allowed to

cool and the paraffin block is then removed from
the mold.
 Section Cutting (Microtome)
4.Sectioning.
• 5–7 μm-thick sections are cut with a rotary microtome.
• The cut paraffin sections are affixed to albuminised glass
microslides after flattening the sections over warm water.
• The microslides with sections are either air dried or dried
in an incubator overnight at 37 °C and stored for staining at
room temperature.
 5. Staining and mounting Procedure
• Use of a basic and acidic dyes that stain tissue
components selectively is done.
• Tissue components that stain with basic dyes
are termed basophilic and are blue in colour
• Tissue components with an affinity for acid
dyes are termed acidophilic and are
pink/orange in colour.
 Staining and mounting Procedure
cont,
• Most staining solutions are aqueous
• To stain the sections, the wax has to be
dissolved and replaced with
water(rehydration).This is essentially step one
in reverse.
• The sections are passed through xylen,
• Then decreasing strenghths of alcohol(100%
to zero%)
• Finally into water.
 Staining and mounting Procedure
cont,
• Once stained, the section is then dehydrated
once again, and placed in xylen.
• A cover slip is placed on top , to protect the
sample.
• Evaporation of xylen around the edges of
coverslip, dries the mounting medium and
bonds the coverslip firmly to the slide
 MEDICAL APPLICATION
• Biopsies are tissue samples removed during surgery or routine
medical procedures. They are then, fixed in vials of formalin for
later processing and microscopic analysis in a pathology laboratory.
• Purpose: to know whether a growth is malignant, benign or any
other condition by identifying abnormal cells.
• If a condition has already been diagnosed, a biopsy can also be
used to assess its severity (such as the degree of inflammation) and
grade (such as the aggressiveness of a cancer).
 ACTUAL STEPS IN STAINING
A. Deparaffinization
• To remove the paraffin from the section, the
slides are treated with xylen.
 ACTUAL STEPS IN STAINING cont,
B. Hydration
• The slides are passed through the following
series to hydrate the sections:
• Absolute alcohol – 5 min (with 2 changes)
• 90% alcohol – 3 min
• 70% alcohol – 3 min
• 50% alcohol – 3 min
• [Wash in] Distilled water – 3 min
C. Staining
• Starts by Staining with haematoxylin for 5–7
minutes.
• Then washing well in running tap water until the
section becomes blue.
• Differentiation with 1% acid alcohol for 5
seconds.
• Washing in running tap water again, until the
section becomes blue.
• Staining with 1% eosin for 1 minute.
D. Dehydration
• The stained sections are dehydrated in the
following series:
• – 50% alcohol – 10 sec
• – 70% alcohol – 10 sec
• – 90% alcohol – 30 sec
• – Absolute alcohol – 5 min (with 2 changes
E. Clearing and Mounting
• The sections are cleared in xylene and
mounted in DPX (Dibutylphthalate Polystrene
Xylene. A synthetic non-aqueous mounting
medium for Microscopy. It’s a mixture of
Distyrene,plasticizer and Xylene.
• Used to preserve stains and help the sections
to dry quickly.
 BASIC/ACIDIC )AND SPECIAL DYES
• Examples of Basic dyes are : haematoxylin,
toluidine blue, alcian blue and methylene blue.
• Examples of Acidic dyes are : eosin, orange G and
acid fuschin
• Combination of haematoxylin and eosin (H&E) is
most commonly used in histological staining
procedure.
 Acidic Dyes/Colour

• Acid Fuschin - Red

• Eosin - Red

• Orange G - Orange
 Basic Dyes and Colour

• Methyl - Green

• Methylene Blue – Blue

• Pyronin G – Red

• Toluidine Blue – Blue

 Stains
• Chrome alum/Haematoxylin
• Stains nuclei blue and cytoplasm red
• For pancreas, glycogen secreting cells stained
pink
• Insulin secreting cells are stained blue

NOTE: Chrome alum is a mordant used for intensifying stains in cell or tissue
preparations .
 Hematoxylin and Eosin (H&E)
Hematoxylin
• DNA or RNA stain
basophilic (purplish blue)
Eosin
• Cytoplasmic proteins and
collagen stain acidophilic
(pinkish red)
 SPECIAL STAINS
• Special stain” is a term used to refer to
many alternative staining techniques that
are used when the traditional H&E does
not provide all the information the
pathologist or researcher needs from a
tissue slide.
 Periodic acid Schiff reagent (PAS)
• stains complex
carbohydrate containing
glycogen, mucin
granules or glycocalyx
• Stains glycogen red
Osmic Acid (Osmium Tetroxide) Stain
• lipids in general and
lipids in nerve fibers –
stain black
 Aldehyde Fuchsin

• Stains elastic fibers

purple/black
 Mallory Trichrome
• This procedure employs a combination of stains applied in series

• Trichrome means technique uses 3 colors

• Nuclei staining purple; cytoplasm, keratin, and erythrocytes staining

bright red or orange; and collagen bright or light blue.

• Useful in demonstrating cells and small blood vessels of connective

tissue.

• Similar stains, such as Masson trichrome and Gomori trichrome, yield

comparable results except that collagen stains blue-green or green
 Masson’s Trichrome
• Weigert's Hematoxylin,
Biebrich scarlet-acid
fuschin solution, and
Aniline blue
• Nuclei and other
structures are Stained
blue
• Cytoplasm, muscle, rbc &
keratin are stained Bright
red
• Collagen is stained Green
or blue
 Wright-Giemsa Stain
• Two similar combinations of stains that are widely
used on fixed cells of blood or bone marrow smears
• Demonstrate types of blood cells.
• Granules in leukocytes are seen to have differential
affinity for the stain components.
• Nuclei stain purple and erythrocytes stain uniformly
pink or pinkish orange.
Wright-Giemsa Stain
Cajal Stain (Silver and Gold Methods)
• Gold stain
• used to demonstrate
fine structures such as
cell processes in nerves.
• Stains black, brown or
golden stain
Silver stain
• Aid visualization of
intracellular and
extracellular
components such as
DNA, and proteins, such
as type III collagen and
reticulin fibres black.
 Toluidine blue
• Stains chromatin shades
of purple and cytoplasm
and collagen a lighter
violet.
• These stains penetrate
plastic sections more
readily than H&E
• ( acrylic or epoxy
sections)
• Also used for differential
staining of cellular
components, particularly
cytoplasmic granules.
 OTHERS
• Other stains are:
• Alcian blue,
• Van Gieson
• Azan stain

REF:Janqueira
 The end
Thank you