The document outlines the process of tissue processing in histotechnology, detailing key steps such as fixation, dehydration, clearing, impregnation, and embedding. It discusses the principles and reagents involved, various techniques for dehydration and clearing, and the importance of using appropriate methods to ensure the integrity of tissue samples. The conclusion emphasizes the necessity for precise processing to obtain diagnostic-quality sections.

1. + ?
GOOD AFTERNOON

2. TISSUE PROCESSING
. . . . . . . . . . . . . . .
-A
histotechnique.............

3. INTRODUCTION
DEFINITION
AIM
PRINCIPLE
STEPS
CONCLUSION
CONTENTS

4. INTRODUCTION
• History of tissue processing
• Histology
• Histopathology
• Histotechnologist
?
?
?
?
• SECTIONING
•EMBEDING
ALEXANDER BUTLEROV, 1859
MICROTOME

5. GROSS EXAMINATION
SPECIMEN ACCESSIONING
•REQUEST FORM
• NUMBER
•COLOUR
•CONSISTENCY
•TEXTURE

6. SPECIMEN LABELLING
• Should be insoluble
• Should not contaminate
• Should not penetrate
• Should not react
• Clearly identifiable
• Eg:India ink, silver nitrate, alcian blue
SOFT LEAD PENCIL

7. DEFINITION
The tissue must undergo preparatory treatment
before being sectioned, entailing to impregnation of
specimen with an embedding medium to provide support
and suitable consistency for microtome is tissue
processing.
FORMALIN ALCOHOLS XYLENE PARAFIN
10% WAX
PREPARATORY TREATMENT
FIXATION DEHYDRATION CLEARING IMPREGNATION

8. To remove water from tissue and replace with a
medium that solidifies to allow the thin sections of the tissue to
be cut.
AIM
FIXATION DEHYDRATION
4-6mm
HCT-2-3mm
LC-10-20mm

9. PRINCIPLE
“FICK’S LAW”
REAGENTS REAGENTS
TISSUE FLUID TISSUE FLUID
cold heat=
TEMP GRADIENT
STABILIZE POROUS TISSUE
THERMODYNAMIC TENDENCY OF PROCESSING REAGENTS
FICK’S LAW- The rate of diffusion of solutions through tissue is proportional to
the concentration gradient of various tissue processing reagents.

10. FACTORS EFFCTING THE RATE
OF TISSUE PROCESSING(TP)
Vacuum-
Fixation-
Viscosity-
Agitation-
Heat-
(45oc
)
(TP)
TPf Highly flammable
r
V-E-O
Reduced
pressure
15 inches of hg
Removes air trapped,
doesn’t make hard tissueMolten parafin
wax
Temp-hardens, shrinkage
CEDAR WOOD OIL, MOLTEN WAX

11. STEPS
1.FIXATION-
2.DEHYDRATION-
3.CLEARING
4.IMPREGNATION
5.EMBEDDING
Mini-6hrs, max-12hrs to 24hrs,
25times
AIM, washed in running h2o
types
ACTION, ARTIFACTS,FACTORS, TECHNIQUE

12. DEHYDRATION
Removal of unbound water & aqueous fixatives from tissue
components is called Dehydration.
DEHYDRATING AGENTS
Ethanol- fast acting, electron microscopy specimen, clear colourless, flamable liquid
Industrial Methylated Spirit-methonol 1%+IPA, Same physical property as
ethanol
Methanol- subsituted for ethanol,toxic, inflammable
Propan-2-01,Isopropylealcohol- Microwave processing schedule,
Butyl alcohol- plant and animal histology, slow dehydrant
Acetone-Rapid in action with poor penetration, clear colorless & Inflammable
liquid,
Removes lipids & causes brittleness of the tissue
Universal Solvents-Tertiary butanol, Tetra hydro furan, Dioxane,
dehydrate+clear, not for delicate tissues.
No shrinkage &
hardness of the tissue

13. TECHNIQUE
70% 80%
50%
90% 100% 100%
THE GRADUAL INCREASING CONCENTRATIONS OF ALCOHOL
2- 4 hrs
7-8hrs

14. •In celloidin embedding after
dehydration ,tissue is transferred to
a mixture of equal parts of absolute
alcohol & ether.

15. USE OF COPPER SULPHATE IN FINAL ALCOHOLS
.
.....
.....
.....
.....
.....
.....
.....
.....
.....
white white
.....
.....
.....
BLUE BLUE

16. CLEARING
•Intermediate step
•Refractive index
•Boiling point
•Short chain aliphatic hydrocarbons

17. CRITERIA FOR
CHOOSING
CLEARING
AGENTS
Rapid removal of dehydrating agent
Ease of removal
by melting
paraffin
Minimal tissue damageToxicity
Flammability Tim
e Cost
viscocity
Storage
Disposal
CRITERIA FOR CHOOSING CLEARING AGENTS

18. TECHNIQUE OF CLEARING
Volume of clearing agent should be 50-100 times the volume of
tissue.
In chloroform or carbon tetrachloride tissues are left over night for
clearing .
In Xylene,benzene,toluene tissues are given one change after 30 to 60
minutes.
Technique for cedar wood oil is different.

19. CEDAR WOOD OIL
TECHNIQUE:-
Cedar wood oil poured into specimen jar & same quantity of
absolute alcohol superimposed on it .
The specimen is placed into alcohol , it floats at the interface of two
fluids.
As tissue is cleared it sinks into cedar wood oil.
The alcohol is removed & specimen is transferred to fresh cedar
wood oil
Cedar wood oil
Absolute alcohol
Cedar wood oil

20. In processing for electron microscopy clearing is done in
1,2 epoxy propane,2 changes 15 min in each.
15mins 15mins
ELECTRON MICROSCOPY
1,2 epoxy propane
1,2 epoxy propane

21. CLEARING AGENTS
•Chloroform
•Toluene , Xylene , Benzene
•Carbon Tetrachloride
•Cedar Wood oil
•Methyl Benzoate , Methyl Salicylate
•Citrus Fruit Oil

22. CHLOROFORM
•Most common reagent used in processing of CNS
specimens.
•DIS ADV:-
Slower in action, Highly toxic.
Does not effect refractive index of tissue.
End point of clearing can not be determined.
Expensive compared to other clearing agents.
Tissue can be left in this with out any damage for longer
time, non-flammable.
•ADV:-

23. CARBON TETRACHLORIDE
•Similar action as chloroform.
•Much cheaper.
•Non inflamable.
•Is toxic.

24. IMPREGNATION
•Impregnation with paraffin wax is carried out in a oven heated
to 54-56 degree.
•The temperature depends up on the melting point of wax used
for impregnation.
•Types of impregnation oven:
Electric heated oven.
Vacuum embedding oven.
Gas heated oven.
•Permeating the tissue with a supporting medium is called
Impregnation.

25. FACTORS EFFECTING
IMPREGNATION
•Size & type of tissue.
•Clearing agent employed.
•Use of vacuum embedding oven.

26. IMPREGNATION OVEN

27. TECHNIQUE OF
IMPREGNATION
•After clearing the tissue is transferred to oven maintained at 56
degree temperature.
•Volume of wax should be 25-50 times the volume of tissue.
•Wax must be changed at least once during the process.

28. PARAFFIN WAX
•Should be free from dust & foreign particles.
•Should not contain water.
•Melting point should be 54degrees.
•Soft wax(45 dg) for foetal & areolar tissue.
•Hard wax (60 dg) for hard & fibrous tissue

29. PARAPLAST
•Mixture of purified paraffin & plastic polymers.
•Greater elasticity than paraffin .
•Wrinkle free serial sections can be cut.
•Does not need ice application while sectioning

30. ESTER WAX
•From clearing agent transfer to mixture of clearing agent & wax.

31. CELLOIDIN EMBEDDING
2% celloidin:- 5-7 dayS.
4% celloidin:-5-7 days.
8% celloidin:-2-3 days.

32. L.V.N
•5% L.V.N:- 3-5days.
•10%L.V.N:-2-3days.
•20%L.V.N:-1 day.

33. DOUBLE EMBEDDING
Tissue is impregnated in a mixture of celloidin& methyle benzoate.

34. ARTIFACTS
•In adequate impregnation leads to loss of architectural details.

35. AUTOMATICTISSUEPROCESSOR
•All the before mentioned procedures upto the impregnation step can be
done automatically in a single, unmanned instrument , which is the
Automated Tissue processor.

36. AUTOMATIC TISSUE
PROCESSOR
•Transfer tissue both by day & night.
•Reduce time in each fluid by continual agitation.
•The 24hr clock is provided.
•Allows rapid processing.
•Racks to carry 25 cassettes.
•Eliminates human error.

37. •Tissue containers:
Has 24 containers subdivided into 2
Compartments.
•Beakers &Wax baths:
Provided with 10 beakers for reagents & 2
wax baths maintained at 56 degree temp.
.
•Stirring Mechanism: One arm supports stirring mechanism.

38. • Time Mechanism: Consists of 1hr,24hr or 7-day electric
Clock which has a time disc.
•Disc revolves once in 24hrs.

39. MICOROWAVE PROCESSING
•Invented by percy spencer in
1945.
•Based on principle, that heat
peaks up diffusion of liquids in &
out of tissues.
•Steps:-
Dehydration.
Clearing.
Impregnation.

40. PROCESSING SCHEDULES
-Thin sections less than 1mm -- 25 min.
-For medium sections 1-2mm -- 60 min.
-For thick sections 2-5mm --165 min.
•Isopropanol is used as clearing agents in microwave processing
instead of xylene.
PROCESSING SCHEDULE FOR MICROWAVE
PROCESSING
•Dehydration with ethyl alcohol at 67 d-centigrade for 5 min.
•Clearing with isopropyl alcohol at 74 d-centigrade for 3 min.
•Impregnation with paraffin at 65 d-centigrade for 2 min.
•Followed by 80 d-centigrade for 5 min.

43. • Tissue processing is a very much critical step that needs to be
monitored with utmost care.
• Since it takes longer time to process the tissue, any mistakes
alters the tissues requiring repetition.
• The tissues should not be under processed or over processed
that may hamper the tissue details.
• It is essential that the tissues are processed with proper
techniques and rendered them for subsequent steps.
CONCLUSION

44. •To get sections of diagnostic value,
processing of tissue should be done with
appropriate reagents & embedded in suitable
embedding medium depending upon type of
tissue.

45. Thank You!!!
REFERENCES
•Hand book of histopathologic techniques:
C.F.A. CULLING.
•Theory & practice of histological techniques:
JHON.D.BANCROT
•Internet source, W.W.W.histotechniques.com, youtube.com