Histological Techniques for LM

Question 1

Fixation

Answer 1

1st Step

A small piece of fresh tissue is immersed in a fixative that stops enzyme actions and prevents post mortem deterioration.

Fixatives include: Formalin (10%), Alcohol, picric acid, mercury dichloride, potassium dichromate

*Alcoholic fixatives are useful in preventing water-soluble substances from dissolving out of the tissue

Question 2

Dehydration

Answer 2

2nd Step

Block of tissue must eventually be embedded in some solid material for cutting. The embedding materials are not water-soluble so the unbound water must be removed.

Accomplished by passing the tissue through alcohol solutions of increasing concentration (ascending alcohols), starting with 50% alcohol and ending with absolute alcohol.

Question 3

Clearing

Answer 3

3rd Step

Standard embedding medium, paraffin, is not soluble in alcohol. The tissue block must be saturated with some chemical which will dissolve paraffin.

Xylol is usually used. The term “clearing” became applied to this step, since xylol and similar chemicals render the tissue transparent.

Question 4

Embedding

Answer 4

4th Step

Cleared block is transferred to melted paraffin, which infiltrates the xylol-saturated tissue.

On hardening, the paraffin block containing the specimen is trimmed to a convenient size and shape.

Question 5

Sectioning

Answer 5

5th Step

The tissue is now sectioned into thin slices on a microtome.

The standard section thickness is 5-10 μm.

Question 6

Placing section on slide

Answer 6

Section (still in paraffin) is flattened on a warm water surface and transferred to a glass slide.

A section will adhere to a slide, which has been made sticky by treating it with a weak solution of egg albumin.

Question 7

Removal of Paraffin and Hydration

Answer 7

7th Step

These steps are necessary since most dyes are water-soluble.

Paraffin is removed from the section by immersing the slide in xylol. The slide is then passed through descending alcohols (absolute to 50%) and into distilled water.

Question 8

Staining

Answer 8

8th Step

Although hundreds of staining methods are available for various purposes, staining with haematoxylin and eosin remains the standard procedure.

Haematoxylin stains the cell nuclei and eosin the cytoplasm.

Question 9

Mounting

Answer 9

9th Step

The stained section is dehydrated by passing the slide through ascending alcohols and into xylol.

For permanent mounting, a drop of Canada Balsam is placed on the section and a cover slip is applied.