Processing of Tissue for Microscopic Examination
Prabhakar Kumar
Department of Anatomy
College of Veterinary Sciences & Animal Husbandry
SVPUAT, Meerut
Tissue Processing in Histology
Tissue processing describes the steps required to take animal or human tissue from
fixation to the state where it is completely infiltrated with a suitable histological wax and can be
embedded ready for section cutting on the microtome. Tissues from animal/ human body taken
for microscopic study (diagnosis of disease) must be processed in the histology laboratory to
produce microscopic slides that are viewed under the microscope. The techniques for processing
the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy, are
described below.
This purpose of this course is to discuss the appropriate application of the most common
tissue processing steps utilized in today's histology laboratory and provide troubleshooting
guidelines for tissue that has been improperly processed.
Objectives
• To describe the steps involved in processing tissue and the various reagents used in the
process.
• To identify the common reagents used in conventional tissue processing and their effect
on tissue.
• To get hands on training on handling of instruments used for tissue processing.
• Identify the various factors affecting processing and compare how different tissue types
require different processing cycles.
Steps for Tissue Processing:
1. Collection of Tissue
2. Fixation
3. Overnight Washing – to remove fixative from tissue (in running tap water)
4. Dehydration – ascending grades of alcohol/ acetone
5. Clearing – three changes of clearing agent (tissue become clear)
6. Infilteration – Paraffin Wax
7. Block Making
Collection of Tissue: Fresh representative tissue specimens of proper size are collected come
from various sources. It is important that they are handled carefully and appropriately fixed as
soon as possible after dissection. Ideally fixation should take place at the site of removal, perhaps
in the operating theatre, or, if this is not possible, immediately following transport to the
laboratory.
Following points must be taken into account while collecting tissue:
• The tissue should be collected from animals as early as possible after death of animal.
• The size of tissue should not be more than 5 mm and should be collected directly in the
fixatives. The small size facilitates homogenous and smooth fixation.
• The tissue from hollow organs like intestine, oviducts etc should be cut transversely and
placed on hard paper, then it should be cut longitudinally in such a way that serosal layer
sticks to paper, After this it should be placed in fixative alongwith paper.
• The tissue from encapsulated organs should be collected with the capsule.
Fixation: The purpose of fixation is to preserve tissues permanently in as life-like a state as
possible. Fixation should be carried out as soon as possible after removal of the tissues (in the
case of surgical pathology) or soon after death (with autopsy) to prevent autolysis. There is no
perfect fixative. The choice of fixative depends on the the type of investigation required.
Therefore, a variety of fixatives is available for use, depending on the type of tissue present and
features to be demonstrated.
There are five major groups of fixatives, classified according to mechanism of action:
a. Aldehydes
b. Mercurials
c. Alcohols
d. Oxidizing agents
e. Picrates
Aldehydes:
Include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by cross-
linkages formed in the proteins, particularly between lysine residues. This cross-linkage
does not harm the structure of proteins greatly, so that antigenicity is not lost. Therefore,
formaldehyde is good for immunohistochemical techniques. Formalin penetrates tissue
well, but is relatively slow. The standard solution is 10% neutral buffered formalin
(NBF). A buffer prevents acidity that would promote autolysis and cause precipitation of
formol-heme pigment in the tissues.
Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not
good for immunohistochemical staining. However, it fixes very quickly so is good for
electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and
nuclear detail. The standard solution is a 2% buffered glutaraldehyde.
Mercurials fix tissue by an unknown mechanism. They contain mercuric chloride
and include such well-known fixatives as Zenker's fluid. These fixatives penetrate
relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear
detail.
Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are
protein denaturants and are not used routinely for tissues because they cause too much
brittleness and hardness. However, they are very good for cytologic smears because they
act quickly and give good nuclear detail. Spray cans of alcohol fixatives are marketed to
physicians doing PAP smears, but cheap hairsprays do just as well.
Fixation - factors affecting fixation
a. Buffering –
Fixation is best carried out close to neutral pH, in the range of 6-8. Hypoxia of
tissues lowers the pH, so there must be buffering capacity in the fixative to
prevent excessive acidity. Acidity favors formation of formalin-heme pigment
that appears as black, polarizable deposits in tissue. Common buffers include
phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered
with phosphate at a pH of 7.
b. Penetration –
Penetration of tissues depends upon the diffusability of each individual fixative,
which is a constant. Formalin and alcohol penetrate the best, and glutaraldehyde
the worst. Mercurials and others are somewhere in between. One way to get
around this problem is sectioning the tissues thinly (2 to 3 mm). Penetration into a
thin section will occur more rapidly than for a thick section.
c. Volume - There should be a 10:1 ratio of fixative to tissue.
d. Temperature
e. Concentration
f. Time interval
Washing: After proper fixation, the tissues are cut into small blocks and kept in tissue capsules
along with identification mark written with pencil on small paper. The tissue capsules are then
kept in running tap water overnight.
Dehydration
Because melted paraffin wax is hydrophobic (immiscible with water), most of the water
in a specimen must be removed before it can be infiltrated with wax. This process is commonly
carried out by immersing specimens in ascending grades of ethanol (alcohol). Ethanol is miscible
with water in all proportions so that the water in the specimen is progressively replaced by the
alcohol. A series of increasing concentrations is used to avoid excessive distortion of the tissue.
A typical dehydration sequence for specimens not more than 4mm thick would be:
1. 70% ethanol 1 Hr
2. 80% ethanol 1 Hr
3. 90% ethanol 1 Hr
4. 95% ethanol 1 Hr
5. 100 % ethanol 1 Hr
6. 100% ethanol 1 Hr
7. 100% ethanol 1 Hr
Clearing - Clearing agents impart an optical clarity or transparency to the tissue due to their
relatively high refractive index. Another important role of the clearing agent is to remove a
substantial amount of fat from the tissue which otherwise presents a barrier to wax infiltration.
Eg. Xylene, toluene, cedar wood oil etc
A typical clearing sequence for specimens not more than 4mm thick would be:
1. xylene 20 min
2. xylene 20 min
3. xylene 20 min
Impregnation/ Infiltration:
The tissue can now be infiltrated with three changes of paraffin wax. A typical wax is
liquid at 60°C and can be infiltrated into tissue at this temperature then allowed to cool to 20°C
where it solidifies to a consistency that allows sections to be consistently cut. In this process the
empty spaces in tissues and cells, after removal of clearing agent, are taken by molten wax
Hardens the tissue – helps in section cutting.
A typical infiltration sequence for specimens not more than 4mm thick would be:
1. wax 2 Hr
2. wax 2 Hr
3. wax 2 Hr
Block Making:
Now that the specimen is thoroughly infiltrated with wax it must be formed into a
“block” which can be clamped into a microtome for section cutting. For this an small amount of
molten wax is poured into the rectangular space made by combining two L-moulds, immediately
the tissue is placed with proper orientation then finally the space in the mould is filled with
molten wax and allowed to get cool and hardened.
Sectioning: 5-6 µ thick section
Once the tissues have been embedded, they must be cut into sections that can be placed
on a slide. This is done with a microtome. The microtome is nothing more than a knife with a
mechanism for advancing a paraffin block standard distances across it. Sectioning tissues is a
real art and takes much skill and practice. Histotechnologists are the artists of the laboratory.
A microtome is used to cut extremely thin slices or sections of tissue for light microscopy
studies. The most commonly used microtomes in the histology laboratory are the rotary
microtome. Microtomes use steel, glass, or diamond blades depending upon the specimen and
thickness of the section required. Nowadays, disposable steel blades are generally used to
prepare paraffin sections of tissues for light microscopy histology.
Before actual tissue sectioning the the tissue block is trimmed to remove extra wax from
sides and to expose the tissue then the block is fixed to microtome with block holder and the 15-
20 µ thick sections are cut to reach the tissue properly. Then with the held of new blade 5-6 µ
thick sections are made.
The ribbon of properly cut sections are taken to water bath from where it is taken on glass
slides made sticky (or adding gelatin to water bath).
Procedure for H&E (haematoxyling & eosin) staining: Most common and routine stain
1. Deparaffinize the section: flame the slide on burner and place in the xylene.
2. Hydration : Hydrate the tissue section by passing through decreasing concentration of
alcohol baths and water. (100%, 90%, 80%, 70%)
3. Stain in hematoxylin for 3-5 minutes
4. Wash in running tap water until sections “blue” for 5 minutes or less.
5. Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 5 minutes.
6. Wash in running tap water until the sections are again blue by dipping in an alkaline
solution (eg. ammonia water) followed by tap water wash.
7. Stain in 1% Eosin Y for 2-5 minutes
8. Wash in tap water for 1-5 minutes
9. Dehydrate in increasing concentration of alcohols and clear in xylene
10. Mount in mounting media
11. Observe under microscope
Equipments/ Instruments:
Rotary Microtome, Knife, L-mould, Hot air woven, Water bath, Block Holder, Tissue
capsule, Forceps, Brush etc.
Glasswares:
Glass slides, coverslips, Couplin jars, reagent bottles, beakers etc.
Reagents:
Formalin, alcohol, acetone, xylene, Paraffin wax, DPX, Gelatin, glycerine,
Haematoxylin, Eosin, Ammonia, Acid etc.
Xylene-I Xylene-II Xylene-III Xyl+ Alc Abs Alc I Abs Alc II 95% Alc
2-4 min 2-4 min 2-4 min 2-4 min 2-4 min 2-4 min 2-4 min
90% Alc
2-4 min
Tap Wat. Haematoxylin Dist. Wat. 70% Alc 70% Alc 80% Alc
4-6 min 5-10 min 2-4 min 2-4 min 2-4 min 2-4 min
Acid Alc
2-5 Sec
Dist. Wat. Amm. Wat. Dist. Wat. Eosin 70% Alc 80% Alc 90% Alc
Dip 1-2 dip Dip 2-5 min 1-2 min 2-4 min 2-4 min
95% Alc
2-4 min
Examine Mount Xylene-II Xylene-I Xyl+ Alc Abs Alc II Abs Alc I
Micorscope with DPX 10 min 10 min 5 min 2-4 min 2-4 min