1. HISTOTECHNIQUES - PART-2
Dr Swati Patil

2. INTRODUCTION
• Histological technique deals with preparation of
tissue for microscopic examination.
Total or a selected part undergoes series of
processes, namely,
- Fixation
- Dehydration
- Clearing
- Embedding
- Cutting
- Staining

3. DECALCIFICATION
• Tissue : bones & teeth
• Criteria for
decalcifying agent:
- Complete removal of Ca salts
- No damage to tissue
- Lack of harmful effect on
staining reactions
- Speed for removal of Ca salts

4. METHODS OF DECALCIFICATION
• Chemical method
-Acid reagents
-Nitric acid
-Trichloracetic acid
-Formic acid
-Chelating agents:
- Ethylene Diamine Tetra acetic acid[EDTA]
• Ion–exchange resin method
( ammonium form of sulphonated polysterene resin)
• Electrophoretic method

5. DEHYDRATION
Definition:
Alcohols of different
strengths
Mechanism of action:
Time required for
dehydration:
Volume of dehydrating
agent:

6. STEPS OF PROCESSING

7. DEHYDRATING FLUIDS
• Ethanol:
• Industrial methylated
spirit: (denatured alcohol)
ethanol +1% methanol
• Methanol:
• Propanol:
• Acetone:

8. ANHYDROUS CuSO4:
Indicator of dehydration
Changes into blue
colour

9. CLEARING
Dealcoholisation:
Definition:
Clearing agents:

10. Criteria for suitable clearing agents:
-Speedy removal of dehydrating agents.
-Ease of removal of molten paraffin wax.
-Minimum tissue damage.
-Flammability
-Toxicity
-Cost

11. Clearing agents suitable for general
use:
-Xylene: -widely used
-rapid in action
-cheap, inflammable
-shrinkage & overhardening
- block 3-5mm : 15 – 30 mins
- Benzene: -cheap, inflammable
-vapors show toxicity
-no shrinkage & overhardening
-Toluene:

12. -Chloroform: - expensive
-high specific gravity, non inflammable – phosgene gas
-slower in action, less transparency
- little hardening
- Cedar-wood oil: -thin fluid, expensive
-used to clear both the paraffin & celloidin sections
-penetration is well, improves section cutting
-slow in action & less damaging to tissue
-emersion in xylene necessary prior to paraffin impregnation

13. IMPREGNATION
Definition: -placement
of tissues with medium
- that fill natural
cavities, spaces, replaces
clearing agent
-supports tissue
-firmness to tissue
-without any injurious
effects

14. 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

15. Types of embedding medium:
-Ribboning media:
eg. paraffin, soap
-Non-ribboning media:
eg. sugar, gum solution, gelatin
 Embedding media:
-Water soluble media:-
eg. gelatin, gum
-Water insoluble media:-
eg. Paraffin wax, nitrocellulose

16. METHODS OF EMBEDDING
• Fusion embedding method:- eg. Paraffin embedding:
-simpler, better for routine use
-allows thin section to be cut
-about 10% shrinkage of section on cooling
• Evaporation embedding method : eg. Celloidin
embedding
- suitable for specimens containing large cavities
-slow, tedious, no serial sections
-less shrinkage & distortion
-no heat & heavy microtome
Volume of impregnating medium: at least 25 times of the
volume of tissue.

17. PARAFFIN WAX
• Saturated hydrocarbons, very stable
• Water insoluble, burns with smoky
flame
• Melting pt- 35-65 C
• Strongly hydrophobic
• Plastic point of paraffin wax
• Low melting pt – thick sections
• High melting pt – thin sections
Wax additives – hardness
eg beeswax, rubber, ceresin etc

18. Other waxes:
-Bees wax
-Candle wax
-Ester wax
-Carbowax
Advantages of carbowax
-soluble & miscible with water
-lipids & natural fats
-enzyme histochemistry
- thinner sections

19. PARAFFIN EMBEDDING
Advantages
-Reasonable speed of processing
-Good consistency for serial sectioning
-A wide range of section thickness & durability of block

20. STEPS IN PARAFFIN EMBEDDING
Selection of proper size of
L shaped moulds smeared
with glycerol
Filling with paraffin wax
Selection of tissue
Orientation of tissue
Insertion of label
Cooling of block

21. L MOULDS

24. Automatic Tissue Processing
AUTOMATED 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

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

26. MICROTOMES
To cut small
Cutting engines
3 main srtuctures
-block holder
-knife carrier
-rachet device

27. TYPES OF MICROTOME
Cambridge rocking
microtome Rotary microtome
Rotary microtome
Sliding & base sledge
microtome
Freezing microtome
Sledge
microtome

28. TYPES OF MICROTOME
Freezing microtome
Ultramicrotome
Freezing microtome
Cryomicrotome

29. MICROTOME KNIVES
Made up of steel
Parts – blade
-knife carrier
-handle if knife
-bevel

30. Bevel angle:
Breadth of knife:
- Bevel angle less, breadth
will be large
- Bevel angle is more, narrow
bevel

31. MICROTOME KNIVES
Obliquity of knife: angle
of knife edge & edge of
tissue block
Angle of tilt: angle, plane
passing through back edge
of knife with plane of
section

32. MICROTOME KNIVES
Profile
• Profile A: biconcave
• Profile B: planoconcave
• Profile C: wedge shaped
- Wide application
• Profile D: chisel shaped

33. CARE OF MICROTOMES
Cover properly
Avoid rust
Application of light oil
Removing of wax
Cleaning of metallic
parts

34. CARE OF MICROTOMES KNIVES
Sharpening of
microtome: manual
sharpening
-Removal of gross nicks
-Honing – removal of fine HONING
nicks
-motion always be from
heel to toe
-Stropping – sharpen knife
which is free of nicks
-on leather strop
-reverse motion of honing
STROPPING

35. SECTIONING
Insert block into
microtome chuck
Blade should be angled
Discard paraffin ribbon
Well ribboning block, pick
with fine brush, float them
on surface of water bath
Float sections onto glass
slides

37. SECTIONING
No ribboning – place block
on ice cubes
Place slides with parffin
sections in 65 c oven for 20
mins
Slides stored overnight at
RT

38. Ready to stain

39. THANK YOU