This document provides an overview of tissue processing techniques used in histology and histopathology. It discusses the various steps involved, including dehydration, clearing, infiltration, and embedding in paraffin wax or other mediums. It describes the purposes and methods for dehydration, clearing using agents like xylene, infiltration using paraffin wax, and embedding tissues. It also discusses alternative embedding techniques like ester wax, water soluble waxes, gelatin, and celloidin embedding as well as double embedding methods and potential processing artifacts.

2. Dr.Shubham Patel
II year PG Student
Dept. of Oral Pathology and Microbiology

3. CONTENTS
• INTRODUCTION
• DEHYDRATION
• CLEARING
• INFILTRATINGAND EMBEDING REAGENTS
• PARAFFINWAX EMBEDDING
• AUTOMATEDTISSUE PROCSSING
• ESTERWAX EMBEDDING
• WATER SOLUBLEWAXES
• GELATIN EMBEDDING
• CELLOIDIN EMBEDDING
• DOUBLE EMBEDDING

4. Introduction
• Histology
• Histopathology
• Histotechniques

5. }Tissue Processing

6. Tissue Processing
• What is tissue processing
• Principle

7. Dehydration
• Removal of “free “ unbound water and aqueous fixatives from the tissue components.
• Hydrophilic
• Excessive dehydration  hard and brittle
• Incomplete dehydration  impair penetration of clearing reagent
• Use of copper sulphate
• Transfer of tissue directly to higher conc. is risky since it is liable to cause tissue shrinkage
• The concentration of the first fixative depends on the the fixative and size and type of
tissue
• Delicate tissue

8. Dehydrating fluids

9. •Industrial methylated spirit
•Butyl alcohol – slow agent, less hardening and
shrinkage

10. Clearing
• It is an intermediary stage between the dehydration and
infiltration solutions
• It should be miscible with both solution
• Mostly hydrocarbons with refractive index similar to protein
• Boiling point of clearing agents gives an indication of its speed of
replacement by melted paraffin wax

11. • The criteria for choosing a suitable clearing agent are :
Rapid penetration of tissues
Rapid removal of dehydrating agent
Ease of removal by melted paraffin wax
Minimal tissue damage
Low flammability
Low toxicity

12. Clearing agent
• Volume of clearing agent used is optimally 30-40 times the volume of the
specimen
• The smaller pieces of tissues are cleared in 30minutes to 1hour, whereas larger
tissues (>5mm thick) are cleared in 2-4 hours
• The end point of clearing can be noted by transparent appearance of the tissue
against light

13. Xylene
• Flammable , colorless liquid with characteristic petroleum or aromatic odor
• Suitable for less than 5 mm thick (2-4hrs)
• Overexposure- hardening

14. • Toluene  Similar properties to xylene
Less damaging
More flammable and volatile than xylene
• Chloroform  Slower than xylene
Cause less brittleness
Tissue do not become translucent
Non flammable but highly toxic produce phosgene gas when heated
Commonly used in processing of CNS
• Xylene substitute  Aliphatic hydrocarbons that exist in short and long chained forms
Differ in number of C atoms in the carbon chain
Amyl acetate, methyl benzoate and methyl salicylate are chiefly
used as nitrocellulose solvents in double embedding techniques.

15. • Citrus fruit oil limonene reagents
Non toxic and miscible in water
least hardening effect
Disadvantage- can cause sensitization and have strong pungent odor that may cause headache
Mineral deposits such as Cu and Ca may dissolve and leach from tissue
Extremely oily and cant be recycled
• CEDARWOOD OIL Least hardening effects
Used for hard tissue
5-7mm thick (2-5 days)
has a role in forensic histopathology in processing the hardened skin margins of
electrical burns and bullet wounds
Formation of crystalline cedrol in cedarwood oil can be overcome by the
addition of 1 ml xylene or toluene to 80 ml cedarwood oil

16. Infiltrating And Embedding Medium
• It is the process in which the clearing agent is replaced by paraffin or its substitute that
completely fills all tissue cavities
• PARAFFINWAX
Most popular infiltration and embedding medium in histopathology
It is a polycrystalline mixture of solid hydrocarbons produced during refining of coal and
mineral oils
Its properties are varied depending on the melting point used, ranging from 47 to 64°C.
Paraffin wax permeates the tissue in liquid form and solidifies rapidly when cooled.
The tissue is impregnated with the medium, forming a matrix and preventing distortion of
the tissue structure during microtomy.

17. MODIFIED PARAFFIN WAXES:
• Paraplast
• Paraplast plus
• Ester waxes
• Polyester wax
• Water soluble wax

18. Time of impregnation
• The size and type of tissue
• The clearing agent employed
• The use of vacuum embedding oven

19. Paraffin wax embedding
•What is embedding
•Casting/ blocking

20. • 1.‘L’ mould piece
• 2. glass petri dishes
• 3.metal petri dishes
• 4. paper boats
• 5.watch glasses
• 6.test tubes
Moulds

21. • Embedding cassettes (tissue-tek and its modification)

22. Orientation of tissue

24. Automated tissue processing
• The basic principle of tissue processing requires the exchange of fluids using a
series of solutions for a predetermined length of time in a controlled environment

27. WORKINGOFTISSUE PROCESSOR

28. Ester wax embedding
• Steedman (1960)
• Paraffin + celloidin
• Harder
• M.p 46-68
• 1-2 micrometer

29. Water soluble waxes
• Carbowax, solid polyethyl glycol wax
• No need to be dehydrated and
cleared before infiltration
• Less shrinkage
• Miles and linder (1952)
Technique
1. Wash tissue
2. 50 % polyethylene glycol 900 in
distilled water (10-15 mins)
3. Four changes of molten polyethylene
glycol at 28-30 degree (45 mins)
4. Polyethylene + nonex 63B at 39 degree
(30-40 mins)
5. 3 parts of nonex+ 1 part polyethylene
(15mins)
6. 3 changes of nonex at 39 degree (30-45
mins each)

30. Gelatin
• Frozen sections of friable or partially necrotic
tissue
• Immeresed in 10 % formalin
• In phospholipid and enzyme studies tissues
may be infiltrated and embedded in gelatine
Technique
• Tissue is fixed
• Washed for 6-12 hrs
• 10% gelatin in 1% phenol (24 hrs )
• 20% gelatin (12 hrs at 37 degree )
• Embed in 20% gelatin

31. CELLOIDIN EMBEDDING
• Purified form of collodion, or nitro-cellulose
• Used as an embedding medium for tissue requiring special treatment, particularly
exceptionally hard tissues.
• Not require heat at any stage of processing, and it has a rubbery consistency
which gives support to hard tissues in circumstances where paraffin wax would
crumble.
• Possible to cut sections of mixed hard and soft tissue of even thickness, while
preserving the relationship of cell layers.

32. Preparation
• Once dissolved equal amount of ether is added
• Thin solution – 2% celloidin in ethanol/ether
• Medium solution- 4% celloidin
• Thick solution –8% celloidin
Processing
• 70% ethanol 2 changes 24 hrs each
• 95% ethanol 2 changes 48 hrs each
• Absolute alcohol 2 changes over 2-5 days
• Thin 3-5 days
• Medium 5-7 days
• Thick 5-7 days

33. Casting and blocking
• Follwing impregnation in thick solution
• 8% celloidin
• Mould 1 quarter inches in depth
• L pieces cant be used
• Glass petri dishes 2 inches in depth with loose fitting ground glass lids
• Blocks hardedned by evaporation
• Rubbery consistency is enough
• Acceleration by Chloroform vapours
• Block is fixed to vulcanite or hardwood for microtomy

34. Microwave processors
• Now common.
• Shortens processing time from hours to minutes
• Based on principle, that heat peaks up diffusion of liquids in & out of tissues.
• Microwave exposure stimulates the diffusion of the solutions into the tissue by
increasing the internal heat of the specimen, thus accelerating the reaction
• Clearing agents are not necessary because the temperature of the final paraffin
step facilitates evaporation of the alcohols from the tissue
• Disadvantages -process is labor intensive because the solutions are manually
manipulated and temperatures must be maintained

35. DOUBLE EMBEDDINGAND DOUBLE
INFILTRATION METHODS:
• It is the process by which tissues are first embedded or fully infiltrated with a
supporting medium such as agar, celloidin then infiltrated a second time with wax
in which they are also embedded.
• The main use of this method is for cutting sections of delicate tissue and preparing
sections from blocks of tissue of varying consistency eg. eyes where retina is easily
detached.

36. Processing artefacts
• Processing floaters or cutting board metastasis
• Improper dehydration
• Improper clearing
• Improper embedding
• Orientation artifact
• Loss of soluble substances

37. References
• Hand book of histopathologic techniques: C.F.A. CULLING.
• Theory & practice of histological techniques: JHON.D.BANCROT
• Chatterjee S. Artefacts in histopathology. J Oral Maxillofac Pathol 2014;18:111-6.