• History of tissue processing
ALEXANDER BUTLEROV, 1859
• Should be insoluble
• Should not contaminate
• Should not penetrate
• Should not react
• Clearly identifiable
• Eg:India ink, silver nitrate, alcian blue
SOFT LEAD PENCIL
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
FORMALIN ALCOHOLS XYLENE PARAFIN
FIXATION DEHYDRATION CLEARING IMPREGNATION
To remove water from tissue and replace with a
medium that solidifies to allow the thin sections of the tissue to
TISSUE FLUID TISSUE FLUID
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.
FACTORS EFFCTING THE RATE
OF TISSUE PROCESSING(TP)
TPf Highly flammable
15 inches of hg
Removes air trapped,
doesn’t make hard tissueMolten parafin
CEDAR WOOD OIL, MOLTEN WAX
Removal of unbound water & aqueous fixatives from tissue
components is called Dehydration.
Ethanol- fast acting, electron microscopy specimen, clear colourless, flamable liquid
Industrial Methylated Spirit-methonol 1%+IPA, Same physical property as
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
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
90% 100% 100%
THE GRADUAL INCREASING CONCENTRATIONS OF ALCOHOL
2- 4 hrs
•In celloidin embedding after
dehydration ,tissue is transferred to
a mixture of equal parts of absolute
alcohol & ether.
USE OF COPPER SULPHATE IN FINAL ALCOHOLS
•Short chain aliphatic hydrocarbons
Rapid removal of dehydrating agent
Ease of removal
Minimal tissue damageToxicity
CRITERIA FOR CHOOSING CLEARING AGENTS
TECHNIQUE OF CLEARING
Volume of clearing agent should be 50-100 times the volume of
In chloroform or carbon tetrachloride tissues are left over night for
In Xylene,benzene,toluene tissues are given one change after 30 to 60
Technique for cedar wood oil is different.
CEDAR WOOD OIL
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
As tissue is cleared it sinks into cedar wood oil.
The alcohol is removed & specimen is transferred to fresh cedar
Cedar wood oil
Cedar wood oil
In processing for electron microscopy clearing is done in
1,2 epoxy propane,2 changes 15 min in each.
1,2 epoxy propane
1,2 epoxy propane
•Most common reagent used in processing of CNS
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
•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
•Types of impregnation oven:
Electric heated oven.
Vacuum embedding oven.
Gas heated oven.
•Permeating the tissue with a supporting medium is called
•Size & type of tissue.
•Clearing agent employed.
•Use of vacuum embedding oven.
•After clearing the tissue is transferred to oven maintained at 56
•Volume of wax should be 25-50 times the volume of tissue.
•Wax must be changed at least once during the process.
•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
•Mixture of purified paraffin & plastic polymers.
•Greater elasticity than paraffin .
•Wrinkle free serial sections can be cut.
•Does not need ice application while sectioning
•From clearing agent transfer to mixture of clearing agent & wax.
Tissue is impregnated in a mixture of celloidin& methyle benzoate.
•In adequate impregnation leads to loss of architectural details.
•All the before mentioned procedures upto the impregnation step can be
done automatically in a single, unmanned instrument , which is the
Automated 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.
Has 24 containers subdivided into 2
•Beakers &Wax baths:
Provided with 10 beakers for reagents & 2
wax baths maintained at 56 degree temp.
•Stirring Mechanism: One arm supports stirring mechanism.
• Time Mechanism: Consists of 1hr,24hr or 7-day electric
Clock which has a time disc.
•Disc revolves once in 24hrs.
•Invented by percy spencer in
•Based on principle, that heat
peaks up diffusion of liquids in &
out of tissues.
-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
•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.
• 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.
•To get sections of diagnostic value,
processing of tissue should be done with
appropriate reagents & embedded in suitable
embedding medium depending upon type of
•Hand book of histopathologic techniques:
•Theory & practice of histological techniques:
•Internet source, W.W.W.histotechniques.com, youtube.com