Tissue Processing : Factors, Steps Of Tissue Processing, Types -part 1

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Principles of Tissue Processing

  • The technique of getting fixed tissue into paraffin is called tissue processing
  • Tissue processing is designed to remove all extractable water from the tissue, replacing it with a support medium that provides sufficient rigidity to enable sectioning of the tissue without damage or distortion

Fick’s Law

  • To equalize concentrations inside and outside blocks of tissue this depends on Fick’s Law:
  • the rate of solution diffusion through tissues is proportional to the concentration gradient (the difference between the concentrations of the fluids inside and outside the tissue) as a multiple of temperature dependant constants for specific substances.

Factors influencing tissue processing

  • Agitation
  • Heat
  • viscosity
  • Vacuum


  • Agitation increases the flow of fresh solutions around the tissue.
  • Automated processors incorporate vertical or rotary oscillation.
  • Efficient agitation may reduce the processing time up to 30%.


  • Heat increases the rate of penetration and fluid exchange.
  • Temperatures limited to 45°C can be used
  • Temperatures higher than that can cause tissue brittleness.


  • Viscosity is the property of resistance to the flow of a fluid.
  • The smaller the size of the molecules in the solution, the faster the rate of fluid penetration (low viscosity) and vice versa
  • Most of the processing solutions dehydration and clearing have similar viscosities with the exception of cedar wood oil.
  • Embedding mediums have varying viscosities.
  • Paraffin has a lower viscosity in the fluid (melted) state, enhancing the rapidity of the impregnation.


  • Using pressure to increase the rate of infiltration decreases the processing time.
  • Vacuum will remove reagents from the tissue but only if they are more volatile than the reagent being replaced.
  • Vacuum can also aid in the removal of trapped air in porous tissue. Impregnation time for dense fatty tissue can be greatly reduced with the addition of vacuum during processing






  • The first stage of processing is the removal of ‘free’ unbound water and aqueous fixatives from the tissue components.
  • Many dehydrating reagents are hydrophilic and interact with the water molecules in the tissue by hydrogen bonding.
  • Other reagents affect dehydration by repeated dilution of the aqueous tissue fluids.
  • Dehydration should be accomplished slowly.
  • If the concentration gradient is excessive diffusion currents across the cell membranes may increase the possibility of cell distortion.
  • For this reason specimens are processed through a graded series of reagents of increasing concentration.
  • Excessive dehydration may cause the tissue to become hard, brittle and shrunken.
  • Incomplete dehydration will impair the penetration of the clearing reagents into the tissue leaving the specimen soft and non-receptive to infiltration.

Dilution dehydration

  • Most commonly used method.
  • Specimens are transferred through increasing concentrations of hydrophilic or water miscible fluids which dilute and eventually replace free water in the tissues.

Chemical dehydration

  • Where the dehydrant, acidified dimethoxypropane or diethoxypropane, is hydrolyzed by free water present in tissues to form acetone and methanol in an endothermic reaction
  • In the paraffin wax method dehydration usually initiated in 60%-70% ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage.
  • Dehydration is necessary except where tissues are externally supported by an aqueous embedding medium.
  • Choice of a dehydrant is determined by the nature of the task, the embedding medium, processing method, and economic factors.
  • Dehydrant differ in their capacity to cause tissue shrinkage
  • The dehydrant concentration at which processing is initiated depends largely upon the fixative employed.
  • Following fixation in alcoholic fixatives such as Carnoy’s fluid dehydration can be initiated in 100% ethanol
  • Duration of dehydration should be kept to the minimum consistent with the tissues being processed.
  • Tissue blocks 1 µm thick should receive up to 30 minutes in each alcohol,
  • Blocks of 5 µm thick require up to 90 minutes or longer in each change.
  • Tissues may be held and stored indefinitely in 70% ethanol without harm.

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Dehydrating agents

  • Ethanol
  • Methanol
  • Methylated spirit
  • Isopropanol
  • Normal and tertiary butanols
  • Dioxane
  • Polyethylene glycols
  • Acetone
  • 2,2 dimethoxypropane (DMP)
  • 2,2 diethoxypropane (DEP)
  • Phenol
  • Anhydrous Cupper sulfate
  • HCL
  • Tetrahydrofuran

Additives to dehydrating agents

  • Phenol can be added to dehydrating agents as a softening agent for hard tissues such as tendon, nail, dense fibrous tissue and keratin masses.
  • Phenol (4%) should be added to each of the 95% ethanol stations.
  • Hard tissue can be immersed in a glycerol/alcohol mixture.
  • Anhydrous CuSOcan act both as dehydrating agent as well as indicator of water
  • content of last bath of 100% alcohol. Copper sulfate is layered in the final dehydrating bath and covered with a filter paper if there is any water present copper sulfate will turn blue

Universal solvents

  • Universal solvents both dehydrate and clear tissues during tissue processing.
  • Examples include Dioxane, tertiary butanol and tetrahydrofuran.
  • They are no longer used for routine processing due to their hazardous properties and due to their hardening properties for delicate tissues

2.  Clearing

  • Clearing is the transition/intermediate step between dehydration and embedding.
  • Consists of removal of the dehydrant with a substance that will be miscible with both the embedding medium (paraffin) and dehydrating agent.
  •   Most clearing agents  are hydrocarbons with high refractive indices (approaching that of dehydrated fixed tissue protein) and, on immersion, anhydrous tissues are rendered transparent or clear similar to protein so they are termed as  ‘clearing agent’.

Criterion for selection of clearing agents

  • rapid penetration of tissues
  • rapid removal of dehydrating agent
  • ease of removal by melted paraffin wax
  • minimal tissue damage
  • low flammability
  • low toxicity
  • low cost

Criterion for selection of clearing agents

  • Type of tissues processed
  • Type of processing and the processor system to be used
  • Processing conditions such as temperature, vacuum and pressure

Factors of clearing

Clearing of the tissues depends on
  • Boiling point of clearing agent
  • Viscosity
  • The boiling point of the clearing agent gives an indication of its speed of replacement by melted paraffin wax. Fluids with a low boiling point are generally more readily replaced
  • Viscosity influences the speed of penetration of the clearing agent

Transition solvents

  • Toluene
  • xylene
  • Chloroform
  • Carbon tetrachloride
  • Trichloroethane
  • Esters
  • n-Butyl acetate
  • Amyl acetate, methyl benzoate and methyl salicylate


  • Flammable and colorless liquid with a characteristic petroleum odour
  • Miscible with organic solvents and paraffin wax.
  • Over exposure may cause tissue hardness.
  • Most commonly used clearing agent in routine histology and is also recyclable.


  • Same properties as Xylene
  • it is less damaging with prolonged immersion of tissue.
  • It is more flammable and volatile than xylene.


  • Slower in action
  • Little brittleness
  • Thicker blocks may be up to 1 mm in thickness can be processed
  • Tissues placed in chloroform do not become translucent
  • non-flammable but highly toxic and releases the toxic gas phosgene

Citrus fruit oils – limonene reagents

  • Limonene reagents are extracts from orange and lemon rinds
  • they are non-toxic and miscible with water.
  • The main disadvantages are that they can cause sensitization and have a strong pungent odor that may cause headaches.
  • small mineral deposits such as copper or calcium may dissolve and leach from tissues.
  • They are extremely oily and cannot be recycled.

Automated tissue processors

  • Tissue processing can be done manually or on automated tissue processers
  • Two types of machines can be used

  1.  Carousel type               2.  Enclosed pump fluid type         
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