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Giemsa Staining Protocol for Tissue Sections

Principle

Giemsa's stain is a member of the Romanowski group of stains, which are defined as being the black precipitate formed from the addition of aqueous solutions of methylene blue and eosin, dissolved in methanol. The variants of the Romanowski group differ in the degree of oxidation (polychroming) of the methylene blue stain prior to the precipitation.
The stain class was originally designed to incorporate cytoplasmic (pink) staining with nuclear (blue) staining and fixation as a single step for smears and thin films of tissue (spread preparations of omentum). Minor modifications of working stain concentration and staining time have been made over the years for fixed tissue sections.
The Romanowski stains are extremely tedious to prepare, and are best purchased as the commercially available pre-made stock stain.


Technical Points

1. Usually the staining is performed at room temperature overnight, however, increasing the stain temperature shortens staining time. Sections stained at 37°C for several hours, (staining time assessed by microscopical examination), produce better results than sections stained at 60°C for a shorter period. The higher the staining temperature, the greater the intensity of blue staining, but without the equivalently increased red staining - see technical point 2 below.
2. Differentiation with acetic acid will vary according to the staining time and temperature, but it is generally achieved within 30 secs. The differentiating agent removes only the blue dye component, thus increasing the apparent intensity of the red component.


Method

1. Bring sections to distilled water
2. Stain with diluted Giemsa's stain made up fresh (see technical point 1)
3. Rinse in distilled water
4. Differentiate with 0.5% aqueous acetic acid (see technical point 2)
5. Dehydrate rapidly
6. Clear and mount

 

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