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Cytology · Year 1 · Medical University of Sofia

03

Main stages in the preparation of a permanent histological preparation. Hematoxylin-eosin staining

Free notes for topic 03 of the Cytology syllabus, open without an account. Written by a senior student against the syllabus question and checked line by line by a second student before publishing. How content is made

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Summary

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A permanent histological preparation is made by the paraffin technique, which has eight steps: removal of tissue, fixation, dehydration, clearing, infiltration, embedding, sectioning and staining.

The logic running through it is that a soft, wet, colourless tissue has to be turned into a hard, dry, coloured one. Everything up to sectioning exists to make the tissue hard enough to cut into slices thin enough for light to pass through. Staining exists because those slices are then colourless.

Hematoxylin and eosin, H&E, is the routine stain. Hematoxylin is a basic dye and stains DNA in the nucleus dark blue or purple; eosin is an acidic dye and stains cytoplasmic structures and collagen pink.

1. The paraffin technique

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The paraffin technique is the standard route to a permanent histological preparation. Its eight steps are described one at a time below.

Staining is listed as the last step and is only necessary for light microscopy. Paraffin sections are colourless, so the tissues have to be stained with dyes before there is anything to see.

Step 1. Removal of tissue

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The tissue is obtained by biopsy from a living patient, or by necropsy after death.

Step 2. Fixation

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Small pieces of tissue are placed in solutions of chemicals to prevent enzyme digestion. This preserves the cell and tissue structure.

There are two main groups of fixative:

  • Simple fixatives, containing one chemical: aldehydes such as formaldehyde, alcohols such as methanol, and oxidizing agents such as chromic acid.
  • Compound fixatives, which are mixed solutions of simple fixatives. Carnoy's and Flemming's fixatives are the named examples.

Step 3. Dehydration

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The specimen is washed and dehydrated in concentrated alcohol, in order to remove the water.

Water and paraffin do not mix, so every trace of water has to leave the tissue before paraffin can enter it.

Step 4. Clearing

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Organic solvents are used to remove the alcohol.

The alcohol that replaced the water now has to be replaced in its turn, because paraffin does not mix with alcohol either. The solvent used is one that mixes with both alcohol and molten paraffin, which is what makes it the bridge between the two.

Step 5. Infiltration

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The tissue is placed in melted paraffin until it becomes completely infiltrated with this substance.

Step 6. Embedding

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The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden.

Step 7. Sectioning

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The hardened paraffin block is trimmed and mounted for sectioning in an instrument called a microtome, which cuts it into sections.

Step 8. Staining

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Staining is only necessary for light microscopy.

Paraffin sections are colourless, so the tissues have to be stained with dyes.

2. Basic and acidic dyes

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Dyes fall into two groups, and which group a dye belongs to decides what it will colour.

Basic dyesColour
Methyl greenGreen
Methylene blueBlue
Pyronin GRed
Toluidine blueBlue
Acidic dyesColour
Acid fuchsinRed
Aniline blueBlue
EosinRed
Orange GOrange

Notice that the name of the group has nothing to do with the colour. Methylene blue and aniline blue are both blue and belong to opposite groups. What the group tells you is the charge the dye carries, and therefore what it will bind to.

3. Basophilia and acidophilia

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This is the rule that makes every stained section readable.

Basophilic compounds are anionic, they carry a negative charge, and they react with basic dyes such as toluidine blue and methylene blue. The basophilic components of the cell are DNA, RNA and glycosaminoglycans.

Acidophilic compounds are cationic, they carry a positive charge, and they react with acidic dyes such as eosin and acid fuchsin. The acidophilic components of the cell are mitochondria, secretory granules and collagen.

Nucleic acids carry a negative charge, so they are basophilic. That single fact is why the nucleus takes up hematoxylin and the cytoplasm does not.

4. Hematoxylin-eosin staining

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Hematoxylin and eosin is the most commonly used staining combination.

Hematoxylin stains DNA in the cell nucleus. The DNA-rich and RNA-rich portions of the cell therefore come out dark blue or purple.

Eosin stains other cytoplasmic structures, and collagen, pink.

A section stained this way is read as a two-colour map: blue or purple wherever nucleic acid is concentrated, pink wherever it is not.

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