Tissue processing

3. Provide sufficient rigidity
to the tissue.
• If the tissue had enough
rigidity then the tissue can
be cut into thin section.
• Then used for microscopic
examination.

4. Principle of processing:
1. To remove water within the
tissue.
2. To impregnate paraffin wax in
the tissue.
Paraffin wax provides support to the
tissue.

5. Dehydration
Clearing
Infiltration & impregnation

6. •Water must be removed
from the tissue.
•Why ??
1.Dehydration:

7. • Water doesn't mix with wax.
• Wet fixed tissues (in aqueous
solutions) cannot be directly
infiltrated with paraffin.

8. • To clear/ remove the dehydrating
agent
• The clearing agents is miscible
to:
— Dehydrating agent.
— Impregnating medium.
2.Clearing:

9.  The tissue is infiltrated with a
supporting medium.
 This supporting medium will
provide adequate rigidity of
the tissue.
• To make thin section for
next step.
3. Infiltration & impregnation:

10. Dehydration
“ Every tissue contains some amount of
free or unbound water molecule “

11. Things you should
do in dehydration
process

12. 1. Dehydration should be done
gradually from low to high
concentration of dehydration
fluid.

13. 2. The tissue should be kept in the
dehydration fluid for optimal
time.
• Too much time  cause the tissue
hard and brittle.
• Too little time insufficient for
removal of free water molecule

14. 3. Smaller size need less time.
• Thin 2–3 mm tissue needs less
time in dehydration fluid than
• Thick 5 mm tissue need more
time.

16. Dehydrating Agents
Alcohol
Ethanol
Methylated spirit
Methanol
Butanol
Isopropanol
Other agents
Dioxane
Ethylene glycol
Acetone

17. Characteristics:
• The most commonly used.
• Clear and colorless fluid.
• flammable liquid.
It needs license from the government to
purchase ethyl alcohol for laboratory use.

18. Concentration:
1. Use graduate ethanol (50, 70, 90 and 100%)
• For delicate/ soft tissue  started from 30%
concentration.
2. Use anhydrous cupric sulphate in final
container
Time:
• 2 h each
 Don’t immerse the tissue in the ethanol for
long time.
• Causes hard and brittle tissue.
Laboratory use:

19. Anhydrous cupric sulphate (CUSO4)
• White powder that draws water from the
alcohol.
• Helps in dehydration.

20. How to use anhydrous cupric
sulphate?
1. About 1 cm layer of this powder is kept in the
bottom of the container.
2. Then covered with 2-3 layers of filter paper.
• to prevent any colouring of the tissue.
3. When the CuSO4 becomes hydrated, the
colour of the powder changes to blue.
• This gives warning signal to change the alcohol
and the CuSO4 powder.

21. Filter paper

22. Advantages:
• Increases the life span of alcohol
• Better dehydration
• Good indicator to change alcohol

23. Methylated spirit
 known as denatured alcohol.
 Components:
• 99% ethanol
• 1% methanol / isopropyl alcohol).
 Uses:
• Used in the same manner as ethanol.

24. Methanol
 Characteristics:
• Clear, colourless
• Volatile.
• Inflammable liquid.
• Rarely used in laboratory.
— its volatility and high cost.

25. Butyl alcohol
• n-Butanol
• isobutanol
• tertiary butanol
• Uses:
• Dehydrating agents in:
• Animal tissue processing.
• Plant histology processing.

26. Advantage:
• Tissue shrinkage is less than other
alcohols.

27. Disadvantage:
• Slowly acting dehydrating agent.
• Takes longer time than ethanol.

28. Isopropyl alcohol
• Isopropyl alcohol is available as isopropanol
(99.8%).
• This is miscible with water and liquid paraffin.

29. Advantages:
• Rapid acting dehydrating agent
• Non-toxic.
• Less tissue shrinkage.
• Good solvent.

30. Other Dehydrating agents

31. Dioxane
• 1,4-diethylene dioxide.
• Characterestics:
• Miscible with both water and molten paraffin
wax.

32. • Rapid dehydrating.
• Causes less tissue shrinkage.
• Tissue can be kept in dioxane
without any harm.
Advantages:

33. • Liberates highly toxic gas
 Solution:
• Use proper ventilation.
Disadvantages:

34. Ethylene glycol
 Other names:
• Ethylene glycol monoethyl ether
• Cellosolve.
 Characteristics:
• It is a colourless and odourless fluid.

35. Advantages:
• Rapidly acting dehydrating
• Tissue can be kept in Ethylene glycol
for long duration.

36. Acetone
 Characteristics:
 Colorless
 Inflammable liquid
 Sharp ketonic smell.
 Miscible with:
— Water
— Alcohol

37. Advantages:
• Best used for fatty tissue
processing.

38. Disadvantages:
• Causes tissue shrinkage
• Prolonged use may cause
brittleness of tissue.

40. Aims of clearing:
1. Removal of dehydrating agent (e.g.
alcohol)
• to facilitate impregnation of paraffin
wax
2.To make the tissue clear and
improve the microscopic
examination

41. • Clearing is the process which leaves
the tissues clear and transparent.
• Transparent tissue = complete
dehydration.
• Any opacity of tissue = Incomplete
dehydration.
• The amount of clearing agent should
be 40 times of the volume of tissue
for clearing.

42. Clearing agents

43.  Characteristics:
• Most commonly used clearing agent in the
laboratory.
• Clear
• Inflammable liquid.

44. • The small pieces of tissue are
cleared rapidly by xylene
 Time:
• 30–60 min
• Note:
• Prolonged exposure to xylene may
make the tissue hard and brittle.

45.  Characteristics:
• Almost similar properties as that of
xylene.
• Flammable.
• Action is slightly slower than xylene.
• Toxic.
• Prolonged exposure does not make the
tissue hard.

46.  Characteristics:
• Highly volatile
• Non-inflammable
• Expensive.
• Toxic.
• Rarely used in laboratory.

47. Uses:
• Used in dense tissue.
• Example: uterus, muscle.
Penetrating power:
• Chloroform is slower than xylene.
 Advantage:
• Does not cause any tissue shrinkage.

48.  Different esters:
— Amyl nitrate
— Methyl salicylate
— Methyl benzoate

49. • Less toxic.
• Used in manual processing.
• Do not cause tissue hardening
even under prolonged exposure.

50. • Expensive
• Rapid clearing agent
• Used in clearing dense tissue.

51. • Clear liquid.
 Advantage:
• Does not cause any tissue
hardening.
 Disadvantage:
• Its removal from the tissue by
paraffin wax is difficult.

52. How to select an
appropriate clearing
agent?

53. Type of tissue:
• large tissue takes more time.
Type of processor:
• manual versus automatic.
Processing condition:
• Temperature and vacuum

54. Speed of removal of
dehydrating agent
Ease of replacement by molten
wax
Safety factors:
• flammability and toxicity
Cost

55. BOOKS:
"Basic and Advanced Laboratory Techniques in
Histopathology and Cytology" by Pranab Dey