Tissue preparation for microscopic study

Paraffin sections involve steps in the following sequence.

Collection of specimen

  • A small chunk of tissue collected during life or as early as possible after death.
  • A piece is removed with a sharp clean knife in a particular orientation.
  • The size of the piece should have an optimum thickness of about 1 cm, so as to achieve better penetration of fixative.
  • Wash the specimen with normal saline to achieve maximum penetration of fixative.

Fixation

    The objective of fixation is to preserve protoplasm with the least alterations from the living state and to protect cells from distortion and shrinkage. Fixation facilitates the proper staining of tissues. The volume of fixation should be 20-50 times the volume of the specimen.

Characteristics of a good fixative

  • It should penetrate quickly.
  • It should coagulate protoplasm into insoluble material.
  • It should prevent the tissue against shrinkage and distortion.

Commonly used fixative

                                                                              Formal saline
40% formaldehyde                                                           100 ml
Sodium chloride                                                                    9 gm
Tap water                                                                           900 ml
                                                                 Neutral Buffered Formaldehyde
40% formaldehyde                                                           100 ml
Distilled water                                                                   900 ml
Sodium Phosphate monobasic                                              4 gm
Sodium phosphate dibasic (anhydrous)                                6 gm

 Best overall fixative. Tissue can be stored indefinitely in this solution.

                                                                             Alcohol Formaldehyde
37-40% formaldehyde                                                       100 ml
80 % alcohol                                                                      900 gm

 Up to 5 g calcium acetate may be added to the mixture to ensure neutrality. Excellent for glycogen preservation.

                                                                               Zenker's Fluid
Distilled water                                                                   1000 ml
Mercuric chloride                                                                  50 gm
Potassium                                                                              25 gm
Sodium sulfate 10 gm

 Add 5 ml of glacial acetic acid to 95 ml of Zenker's fluid before use. It is used to fix the bone marrow aspiration specimens.

                                                                                Bousin's Fluid
Picric acid, saturated aqueous solution                                 750 ml
35-40 % formaldehyde                                                          250 ml
Glacial acetic acid                                                                   50 ml

 Fix blocks for 4-12 hours depending on size. Wash in several changes of 50 % alcohol for 5-6 hours. Store in 70 % alcohol.

Washing

Wash the tissue in running tap water for 6-8 hours or overnight to remove fixative.

Dehydration

The specimen is put into ascending grades of ethanol. The following schedule is practiced.
  1. 30 % alcohol for 2 hours
  2. 50 % alcohol for 2 hours
  3. 70 % alcohol for 2 hours
  4. 80 % alcohol for 1 hour
  5. 95 % alcohol for 1 hour
  6. 95 % alcohol for 2 hours
  7. 100 % alcohol for half an hour
  8. 100 % alcohol for half an hour
The tissue can be held in 70 % alcohol for an indefinite period. Another agent used for dehydration is acetone.

Clearing

  • It involves the removal of the dehydrating agents and replacement with some fluid miscible both with the dehydrating agents and the embedding medium. Xylene is most commonly used.
  • Put the specimen in Xylene for 1 hour and then in fresh xylene for another 1 hour. Other clearing agents are toluene, cedarwood oil, chloroform, and benzene.
Toluene is expensive and slower in action.
Chloroform is a more expensive, and good clearing agent.
Benzene is cheap, excellent clearing agent, vapors are toxic.
Cedarwood oil is expensive, yet a good clearing agent.

Infiltration

The tissue is impregnated with melted wax. The schedule is given below:
  • Saturate xylene with paraffin. Leave the tissue for 1 hour.
  • Leave the tissue in melted paraffin for 1 hour.
  • Transfer tissue in a fresh dish of melted paraffin and put in a vacuum oven for another 1 hour.

Embedding

The tissue in the melted wax is allowed to solidify in the form of cubes.

Sectioning

Thin (5-7 um) sections are cut with the help of microtome. The knife should be properly sharpened and free from nicks. Hone, strap and automatic knife sharpeners can be used. The motion of the wheel of the microtome should be uniform.

Mounting of section

  1. Smear an extremely thin layer of Meyer's egg albumin onto the clean slide.
  2. Sections are floated on warm water (50-55oC) in a water bath.
  3. Dip the smeared slides under the section and lift.
  4. Drain off water.
  5. Dry the slide in an incubator (45-50oC) for 30 minutes.
  6. Label the slide properly with a waterproof pencil.

Staining

The sections are, in routine, stained with Hematoxylin and Eosin (H&E) stain. Specific stains may also be used.

Mounting of coverslip

Put a drop of mounting medium (Canada balsam) onto the section. Place clean dry coverslip. Avoid air bubbles. Allow drying in an incubator at 37oC for 2 hours.

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