Dr. B. Victor presented on the key aspects of histotechnology. He discussed the meaning and branches of histotechnology, the major steps in tissue processing including fixation, dehydration, clearing, embedding and staining. He described different types of fixatives, their characteristics and principles of tissue fixation. The document also covered the paraffin wax technique, types of microtomes used to cut tissue sections, and various staining techniques including double stains and acid/basic stains.
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Histological techniques for life science researchers
1. Presented by
Dr. B.Victor., Ph. D.
Email : bonfiliusvictor@gmail.com
Blog: bonvictor.blogspot.com
2. Presentation outline
Meaning and branches of
histotechnology.
Major steps in tissue
processing
Fixative –definition, kinds,
characteristics.
Micro-anatomical fixatives
Principles of tissue fixation.
Processing of tissues and
tissue sections.
Paraffin wax technique
Types of microtomes
Staining-types, double stains,
acid stains, basic stains.
Clearing tissue sections.
3. Meaning of histotechnology
It is the preparation of tissues for
microscopic examination.
It is an effective diagnostic tool in clinical
pathology.
Histological preparations reveal normal
tissue structure, tissue abnormalities and
cancerous conditions.
4. Branches of histotechnology
Histology- the microscopic study of the
normal tissues.
Histopathology – the microscopic study
of tissues affected by disease.
Histochemistry – the techniques provide
information on the chemical composition
of parts of tissues.
Cytochemistry – the techniques provide
information on the chemical composition
of parts of cells.
5. Steps in the processing of tissues
1. Fixation – preservation of tissues in its
original condition.
2. Dehydration – removal of water from
tissues.
3. Clearing – infiltration of paraffin solvent.
4. Embedding – infiltration of paraffin wax.
5. Microtomy – preparing thin slices of
tissues.
6. Staining – colouring of tissues.
7. Mounting – arranging tissues on slides.
6. What is a fixative ?
A fixative is described as a chemical
substance which will preserve the shape,
structure, relationship and chemical
constituents of tissues and cells after
death.
7. Purpose of fixing agents
1. To kill and preserve living tissues.
2. To stabilize the tissue and cell structure
for subsequent treatments( wax
embedding, sectioning, mounting).
3. To prepare tissue for staining and optical
contrast.
4. To harden the tissue for section cutting
8. Requirements of a good fixative
1. Penetrate the tissue and cells rapidly and
evenly.
2. Prevent autolysis and bacterial decomposition.
3. Preserve tissues in their natural state and fix
all chemical cell components ( proteins,
carbohydrates, fats etc.,)
4. Preserve cell volume.
9. Requirements of a good fixative
(Cont’d)
5.Avoid excessive hardness of fixed tissue.
6.Allow enhanced optical differentiation by
staining.
7.Make the cellular components insoluble
to liquids used in tissue processing.
8.Be nontoxic and non-allergenic
9.Providing iso-osmotic conditions to the
tissues.
10. General principles of fixation
Amount of fixing fluid should be approx.
10 to 20 times more than the volume of
tissue held in a container with a required
fixation time.
Temperature has an important effect.
A lower temperature retard fixation –
reduce autolytic reaction.
A higher temperature will decrease the
required for fixation but will increase
autolysis.
11. Methods of Fixation
Fixation by heat - this denatures and
coagulates proteins resulting in some
distortion but is useful in fixing smears.
Cryostat (freezing) fixing - it does not
denatures proteins and minimizes
distortion. useful in locating particular
chemicals - histochemistry
Fixation by chemicals - chemical fixatives
are used.
12. Kinds of fixatives
Fixatives
Temperature Chemical
fixation fixation
Simple fixatives Compound fixatives
Micro-anatomical Cytological
fixatives fixatives
Nuclear
fixatives
Cytoplasmic
fixatives
13. Common fixatives
Routine Special
Fixatives fixatives
Primary Secondary
fixatives fixatives
Simple Fixation
fixatives mixtures
14. Simple fixatives or primary fixatives
or unmixed fixatives
Formalin Ethyl alcohol
non-coagulant fixative Colourless liquid
Acidic, cheap, Reducing agent
easy to prepare, Osmium tetroxide
relatively stable Strong oxidizing agent
Expensive, poor penetration
Mercuric chloride
Potassium dichromate
Coagulant fixative,
Strong fixative
black precipitate in tissues
Fix lipids
Glacial acetic acid Trichloro acetic acid
Protein precipitant, Protein precipitant
Colourless solution Picric acid
Pungent smell Protein precipitant
Used as saturated solutions
20. Physico-chemical features of
fixatives
Degree of ionization
Oxidation-reduction potential
Reactions with proteins, lipids,
carbohydrates
Rate of penetration
Shrinkage or swelling
Degree of hardening
Methods of washed out
Effect on staining
Compatibility with other fixatives
21. Processing of specimens
Processing
Of tissues
Processing of
specimens
Processing of
Tissue sections
22. Processing of animal tissues
Fix in appropriate fixative
Wash in water / iodine alcohol
Partially dehydrate in 30, 50, % grades of
alcohol – 30 -90 mts each
Store in 70 % or 80% alcohol
23. Processing of plant tissues
Fix in FAA
(formalin-acetic acid-alcohol)
Wash in 50% alcohol
Partially dehydrate in
10, 20, 30, 40, 50 & 60%
Alcohol grades – 20 mts each
Store in 70% alcohol
24. Paraffin wax technique of
tissue blocks
Dehydration-the alcohol method
-the acetone method
- the dioxane (diethylene
dioxide) method
Clearing- de-alcoholisation -clearing
agents- xylene, benzene, toluene,
chloroform.
Embedding- blocking – out in wax-wax
impregnation
25. Paraffin wax technique of
tissue blocks
wax-wax impregnation- It can be cold wax
infiltration and melted wax infiltration.
Complete wax infiltration is essential for the
production of good sections.
Hard tissues requires a higher melting point
wax.
Number wax changes and time in each wax
change, depend upon the density and size of the
tissue.
Embedding media – wax, gelatin, celloidin,
polyester wax.
27. Microtomes
The microtomes cut the tissue at a pre-
determined uniform thickness.
These instruments are designed for the
accurate and serial cutting of thin slices of
tissue.
Several models are available – sliding,
rotary, rocking ultra- thin microtomes.
28. Cambridge rocking microtome
It consists of a heavy base
and two arms ; the lower
arm rests on a column and
supports the upper, both
being pivoted on knife edges
which acts a fulcrum.
The upper arm carries the
block holder.
There is an adjustable cord.
The feed mechanism is
graduated in units of 1or 2
um.
29. The rotary microtome
The section cutting is
effected by the vertical rise
and fall of the object against
an fixed knife edge.
The block holder is
equipped with adjustable
screws.
The block is parallel to the
microtome knife.
The knife holder is movable.
30. The sliding microtome
The block remains stationary, while the
microtome knife moves during the process of
sectioning.
31. The freezing microtome
The optimum cutting
temperature is -20
degree Celsius.
The freezing of the
tissues is done by the
carbon dioxide gas.
32. The cryostat
Sectioning is done on
unfixed tissue.
The microtome is
housed in a deep
freezer cabinet.
The temperature can
be maintained
between -15 to -30
degree Celsius.
34. Processing of paraffin
sections
Hydration
Down-grading –
Drying of sections De-paraffinization
Graded alcohol
series
Staining Dehydration
De-alcoholization
-primary staining Up-grading-
And clearing
-counter-staining Graded alcohol series
Mounting
Observation
In a medium with
In a microscope
Cover glass
35. Staining
Staining is used to obtain contrast
between the constituent parts of a tissue
section.
The depth of colouration is affected by
chemical affinity, density, and permeability.
Certain stains are metachromatic –i.e.
they are capable of imparting one colour
to certain constituents and another to
others.
36. Mordanting
The salts of certain metals are capable of
radically alter the behavior of particular
stains.
These salts are called ‘mordants’.
A mordant is capable entering into
chemical combination with a stain.
The resulting substance is called ‘a lake’.
37. Kinds of staining
Progressive staining Retrogressive staining
The tissue is left in the The tissue is over
stain until the desired stained and decolorized
depth of color is using differentiating
obtained solution
Direct staining Counter staining
The stain combines Two stains are applied
directly with cell one by one with proper
structures destaining in-between.
38. Kinds of staining -2
Acidic Basic Neutral
stains stains stains
A colored organic A colored organic
A colored organic
acid combined base combined
acid is linked
with a metal. with uncolored
chemically to a
acetate, chloride
colored organic
or sulphate
base.
radical.
These are
dissolved in water
or alcohol.
They are dissolved
They tend to stain
in absolute
They tend to stain nucleus.
alcohol.
the cytoplasm.
39. Common double stains
Animal • Borax carmine and eosin Y
• Haematoxylin and eosin Y
• Haematoxylin and van
tissues Geison stain
Plant • Haematoxylin and eosin
• Safranin and light green
tissues • Safranin and haematoxylin
40. Good general stain for
animal tissues
stain solvent effect
Pico carmine water Nucleus-red
Cytoplasm-yellow
Borax carmine alcohol Nucleus-pink
Delafield alcohol Nucleus - blue
haematoxylin
Haemalum water Nucleus - blue
Eosin Y Water or alcohol Cytoplasm - pink
41. Clearing agent
• It makes the processed
Clearing tissue transparent.
agent • Removes alcohol from
tissue sections.
42. Dr.B.Victor is a highly experienced professor,
recently retired from the reputed educational
institution- St. Xavier’ s College, Palayamkottai,
India-627001.
He was the dean of sciences, IQAC coordinator
and assistant controller of examinations.
He has more than 32 years of teaching and
research experience
He has taught a diversity of courses and guided
12 Ph.D scholars.
He is an expert in histological techniques and
photo micrography.
send your comments to :
bonfiliusvictor@gmail.com