The document discusses various techniques used in histopathology sample processing including decalcification, fixation, dehydration, clearing, embedding and sectioning. It covers different chemical agents used for each step along with their properties and advantages. Various methods are described such as paraffin, celloidin and vacuum embedding for optimal tissue preservation and section quality. Automatic tissue processors and freeze drying are also mentioned as techniques to reduce processing time.

1. HISTOTECHNIQUES - PART 2
Dr Swati Patil

2. Decalcification
• Tissue : bones & teeth
• Criteria for suitable decalcifying agent:
- Complete removal of Ca salts
- Lack of distortion of cells & connective tissue
- Lack of harmful effect on staining reactions
- Speed for removal of Ca salts

3. Methods of decalcification:
• Chemical method
-Acid reagents
-Nitric acid- 4hrs
-Trichloracetic acid- 48hrs for small
-Formic acid- 12-24hrs
 Chelating agents:
 Ethylene Diamine Tetra acetic acid[EDTA]- 4-40 days
• Ion–exchange resin method
( ammonium form of sulphonated polysterene resin)
• Electrophoretic method

4. Knowing the decalcification end point:
• Chemical test:
after 6-12 hrs of decalcification,
5ml of decalcifying fluid + 5ml ammonium hydroxide +
5ml ammonium oxalate.
-Observation :
Absence of turbidity- complete decalcification
Turbidity appears – specimen is not thoroughly not
decalcified.
Change the decalcifying agent, repeat the test.
• X-ray examination:

5. Washing after fixation
• To remove excess fixative
• Thorough washing in appropriate fluid after fixation is
essential for good staining.
-mercuric chloride, acid fixatives
Is fixation always necessary
 Fixation is 1st step towards preparation of histological
sections.
 Certain structures like chitin, cellulose do not require
fixation
Storage of fixing fluids

6. • Artefacts after fixation:
- Extrinsic:
- Intrinsic:
primary-
secondary-

7. Dehydration
• Definition:
Removal of water & fixative from tissue, & replaced them
with dehydrating fluid.
-For this purpose alcohols of various strengths are used
-As alcohols are hydrophilic in nature, it drags water from
tissues by diffusion.
-So the increasing concentrations starting from 50%, 70%,
90%& two replacements of absolute alcohols are used.
-For delicate tissues like embryos, animal tissue start conc.
with 30% dilution.

8. Steps Of Processing

9. • Time required for dehydration
For tissue thickness <5mm – 2-3 hrs.
Change from one bath to that of another done at hourly
interval.
For absolute alcohol bath - 1-11/2 hr each.
• Volume of dehydrating agent- 10 times that of tissue.

10. • Dehydrating fluids:
-Ethanol: used in final dehydration steps , to ensure total
dehydration.
-Industrial methylated spirit: (denatured alcohol)
ethanol +1% methanol
-Methanol:
-Propanol: isopropyl alcohol: No excised duty. Increasingly
used in histology labs.
especially with methods of microwave.
-Acetone: rapid in action, poor penetration, less cost, only
used for urgent reports.

11. Anhydrous CuSO4:
- Dehydrating agent as well as Indicator of dehydration
It is placed in last alcohol bath in automated tissue
processor.
It changes to blue color in presence of water.
- Indication for presence of water in last bath of
alcohol particularly useful for automatic tissue
processing
-Change of ethanol, from last bath of absolute alcohol.
Discard the first one of 100% ethanol, move down
the others, so that last bath has fresh 100% ethanol

12. Clearing
• Clearing (Dealcoholisation) :
-clearing agent miscible with both alcohol & paraffin wax.
-have similar refractive index with protein
-clearing agents are liquids whose function is to make
tissue transparent by raising refractive index of any
specimen. This makes tissue transparent hence
procedure called as clearing.
• Clearing in Paraffin wax embedding:
• Clearing in mounting:

13. • Criteria for suitable clearing agents:
-Speedy removal of dehydrating agents.
-Ease of removal of molten paraffin wax.
-Minimum tissue damage.
-Flammability
-Toxicity
-Cost
• Factors determining the speed of replacement:
-Boiling point
-Viscosity

14. • Clearing agents suitable for general use:
-Xylene: -used for routine histology schedules < 24 hrs &
tissue size < 5mm
-prolonged use overhardens & shrinks tissue
-inadequate dehydration causes formation of whitish
emulsion
-Toluene & benzene: less damages the tissue even if on
prolonged immersion
-toluene is less toxic than benzene

15. -Chloroform:
-hardening is almost nil as compare to xylene but
-penetration effect is very slow & transparency is less.
-non flammable but releases phosgene gas on heating.
- Cedar-wood oil:
-used to clear both the paraffin & celloidin sections
-penetration is well, improves section cutting
-slow in action & less damaging to tissue.
-traces of oil retained, emersion in xylene is
necessary prior to paraffin impregnation.
-expensive

16. Impregnation
• Definition: Impregnation is process involves placement
of tissues with medium that will fill all natural cavities,
spaces, & interstices of tissues, even inter constituent
space of cell, replaces clearing agent & terminates with
making of block.
- Impregnation (paraffin) supports tissue from all sides
with firmness without producing any injurious effects on
tissues.
-It allows cutting of tissues suitably thin sections without
undue distortion & without alteration of spatial
relationships of tissue & cellular elements.

17. • Methods :
-Paraffin embedding:
-simpler, better for routine use
-allows thin section to be cut
-about 10% shrinkage of section on cooling
-Celloidin embedding:
- suitable for specimens containing large cavities or hollow
spaces and layers which tend to collapse e.g. eyes, larger
embryos
-slow, tedious, does not allow serial sections
-causes much less shrinkage & distortion
-require no heat & heavy microtome
• Volume of impregnating medium: at least 25 times of the

18. Embedding
• After completely impregnation with suitable medium,
solid block of suitable medium containing impregnated
tissue is obtained by embedding.
• Done by
- Filling mould of suitable size with molten paraffin wax.
- Orienting specimen in mould to ensure its being cut in
right plane.
- Cooling mass to promote solidification

19. • Types of embedding medium:
-Ribboning media:
eg. paraffin, soap
-Non-ribboning media:
eg. sugar, gum solution, gelatin
• Methods of embedding:
-Fusion embedding method: eg paraffin embedding
-Evaporation embedding method : eg. Celloidin
embedding

20. Paraffin wax:
Properties :
-saturated hydrocarbons, very stable
-Insoluble in water & alcohol
-Burns with smoky flame
- it’s properties are varied with it’s
melting point ranging between 40oc - 70oc
- higher the melting point, harder the wax.
-strongly hydrophobic
- plastic point of paraffin wax
-low melting point-thick sections
High melting point-thinner sections
• Wax additives -Adding different sub.
increases hardness eg beeswax, rubber,
ceresin etc

21. • Other waxes:
Bees wax, candle wax, ester wax, carbowax (polyethylene
glycol)
• Adv of carbowax:
- It is soluble & miscible in water, hence embedding can be
done directly by eliminating clearing & dehydration.
-because of this lipids & neutral fats can be demonstrated in
thin sections.
-technique is good for enzyme histochemistry.
-reduces processing time.
-reduces shrinkage & distortion.

22. Paraffin Embedding
• Advantages
-Reasonable speed of processing
-Good consistency for serial sectioning
-A wide range of section thickness & durability of block

23. Paraffin embedding
Technique:
- Tissue is transferred to
specimen tube filled with
paraffin wax.
- Keep it in an oven at a temp.
not >5oC above the melting
point of paraffin.
- 2 or 3 changes are done at an
interval at an interval of 2hrs
Recommended melting point
for our set up
58 to 60oC

24. • Storage of Paraffin Block:
-Block should be stored in a cool place
- Cut surface should be protected from dust & vermin

25. Types of Moulds
• Leuckhart’s L pieces:
• Glass petri dishes:
• Metal petri dishes:
• Paper boats:
• Test tubes:
• Plastic embedding rings:
• Tissue Tek 2 embedding rings:

26. Paraffin block making:
-Metal moulds smeared with glycerol
-fill the mould with fresh melted wax.
-orient specimen so that intended cutting surface is pressed
against base
-place identifying label
-after skin of wax has formed completely over surface of
block solidification by immersion in cold water for nearly
15 min.
-When block completely solidify, mould is removed

27. Cellulose Nitrate / Low viscosity
Nitrocellulose
-considerably less shrinkage
-improved cutting qualities of large blocks of dense tissue.
Eg: tooth, embryos
-facilitate production of sections of brain
-preserving relationships of all tissue layers of different
consistency. eg: eye
Disadvantages:
- Sections less than 10 mm are not possible.
- Non ribboning media
- Slow process, taking several weeks

28. Double Embedding
• Combination of celloidin & paraffin embedding
• To get sections with improved cohesion of tissue layers &
plasticity given by celloidin with facility of cutting ribbons of
sections from dry durable block which is characteristic of
paraffin.
• Organs benefited: bone, brain, muscle, large pieces of
dense fibrous tissue

29. Vacuum embedding
-Evaporation of clearing agent
-Impregnation of paraffin wax
-Removal of trapped air in specimen
-Reduce impregnation Time to half.
-Carried out in vacuum embedding oven.
-If conti. Bubbles are coming, indicates inadequate
dehydration & clearing.
Tissues benefited by vacuum impregnation:
Lungs, muscle, spleen, decalcified bone, skin &
nervous tissue.

30. Automatic Tissue Processing
• Principle
Time required for tissue processing may
be considerably reduced when tissue is Suspended in
fluid, Continuously agitated, Moved from one reagent
to another whenever desired, not restricted by working
hours.
• Processors are configured with preset interval for different
schedules of suspension, agitation, &
automatic changeover

31. • Advantages:
- Reduce time taken for
processing
- Superior results are obtained
- Multiple blocks can be
processed together
- Less laborious

32. Freeze Drying
• Theoretically only water is removed from tissue because
of that
- No alteration in arrangement or quantity of any other
tissue components & enzymes.
- routine steps of tissue processing are avoided
• Use: special procedures like histochemical investigations
for different enzymes

33. • Technique:
- Fresh tissue of size 1mm, immediately freeze by
immersion in isopentane cooled by liquid nitrogen (-
1500c)
- Transferred to drying chamber (-30 to -60 0c) under
vacuum,
- Ice is removed by sublimation, & water vapours
absorbed by phosphorus pentoxide (drying agent)
- Impregnate tissue under reduced pressure in paraffin
wax /polyethylene glycol wax
- Block is stored in desiccator in cool place