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.

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

15. Compound fixatives or fixation
mixtures
Micro-anatomical
fixatives
Compound Nuclear
fixatives fixatives
Cytological
fixatives
Cytoplasmic
fixatives

16. Micro-anatomical fixatives
 10% formal saline
 10% neutral buffered formalin
 Heidenhain’s Susa
 Formal-sublimate
 Formal-saline sublimate
 Zenker’s fluid
 Helly’s fluid
 Bouin’s fluid
 Gendre’s fluid

17. Nuclear fixatives
Flemming’s • Chromic acid (1%)-15 ml
• Aqueous osmium tetroxide(2%)-4 ml
fluid • Glacial acetic acid- 1 ml
Carnoy’s • Absolute alcohol- 60 ml
• Chloroform – 30 ml
fluid • Glacial acetic acid – 10ml

18. Cytoplasmic fixatives
Flemming’s
• Chromic acid (1%) – 15 ml
Fluid without • Aqueous osmium tetroxide (2%)-4 ml
Acetic acid
• Mercuric chloride – 5 gm
• Potassium dichromate -2.5 gm
Helly’s fluid • Sodium sulphate – 1.0 gm
• Distilled water -100 ml
• 5 ml of 40% formalin before use.
Formal • formalin
Saline 10% • Sodium chloride

19. Category of fixatives
Mercury
• Zenker’s fluid
fixative
Chromate • Helly’s
fixative fluid
Picric acid • Bouin’s
fixative fluid
Alcohol • Carnoy’s fluid
Fixative

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.

26. Cutting of tissue sections or
microtomy
Rotary
microtome
Ordinary Rocking
microtome microtome
Freezing Sliding
microtome Microtome/ microtome
cryostat
Ultra - microtome

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.

33. Paraffin processing of tissue
Post-fixation Initial
fixation
treatment dehydration
Wax De- Complete
infiltration alcoholization dehydration
Wax Trimming the
block microtomy
embedding

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

43. Thank you for watching