Histopathology Technique - Part 2 (Tissue processor)

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Tissue Processing:
      Nowadays all the process of fixation, dehydration, clearing, and impregnation are carried out in a special equipment which is known as automated tissue processor.

  • It can be a open (hydraulic) system or a closed (Vacuum) type.
  • In the open type, the tissue processor has 12 - 16 glass jars for formalin, ascending grades of alcohol, xylene and thermostatically- controlled two paraffin wax baths to keep paraffin wax in molten state.
  • Tissue moves automatically by hydraulic mechanism from one jar to another after fixed time schedule and the whole process takes 16-22 hours.
  • In closed type of tissue processor, tissue cassettes are placed in a single container while different processing fluids are moved in and out sequentially according to electronically programmed cycle.
  • The closed or vacuum  processor has the advantage that there is no hazard of contamination of the laboratory by toxic fumes unlike in open system.
  • In addition, heat and vacuum can be applied to shorten the processing time.
  • Thus, closed tissue processors can also be applied for short schedules or rapid processing of small biopsies.
Embedding and Blocking:




  • Embedding of tissue is done in molten wax.
  • Wax blocks are conventionally prepared using metallic L (Leuckhart's mould) nowadays plastic moulds of different color for blocking are also available.
  • The moulds are placed over a smooth surfaced glass tile.
  • Molten wax is poured in the cavity in the moulds.
  • The processed tissues pieces are put into wax with number tag and examining surface facing downwards.
  • Wax is allowed to solidify.
  • After solidification, if L-moulds are used they are removed while plastic mould remains with the wax block.
  • In either case, each block contains a tissue piece carrying a identification label.
  • Embedding and blocking can also be performed in a special instrument called embedding centre.
  • It has a wax reservoir, heated area for steel moulds, wax dispenser, and separate hot and cold plates for embedding and blocking.

Section Cutting (Microtomy)

         Microtomy is an equipment for cutting sections. There are 5 types of microtome:
  1. Rotary
  2. Sliding
  3. Freezing
  4. Rocking
  5. Base- sledge
Rotary Microtome:

This is the most commonly used microtome. In this, microtome knife is fixed while the tissue block is movable. The knife in this faces upward and is wedge-shaped. The knife used is of steel but glass knife can also be used.These knives are sharpened by a process known as honing and stropping.

Honing is done manually on a stone or on an electrically operated automatic hone. After honing, a stropping is done which is polishing of its edge over a leather strop. The process of sharpening of microtome knife can also be done by automatic knife sharpener. Nowadays, disposable blades for microtomy are also available.

Sliding Microtome:

In this the tissue is fixed while the knife is movable. These microtomes are used as freezing microtomes.

Rocking Microtome:

This is a simple microtome. The knife is immovable while tissue block is held in a spring - bearing rocking arm. This is more useful when cutting serial sections.

Base- Sledge Microtome:
This type of microtome is used for very hard tissues or large blocks. e.g. pieces of brain and heart.

Procedure for microtome:

  • Put the paraffin block having tissue in it in the rotary microtome.
  • Cut the section by operating the microtome manually after adjusting the thickness at 5-6 mue.m.
  • Sections are picked from the knife with the help of a forceps or camel hair brush.
  • These are made to float in a water-bath which is kept at a temperature of 40-45 degree Celsius.
  • i.e. slightly below the melting point of wax. 
  • This removes folts in the section. 
  • From water-bath sections are picked on a clean glass slide. 
  • The glass slide is placed in an oven maintained at a temperature of 56 degree Celsius for 20-30 minutes for proper drying and better adhesion. 
  • Coating adhesives for sections can be used before picking up sections, these include egg albumin, gelatin, poly-L-lysin etc. 

The section is now ready for staining.

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