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4.2.2.2. Giemsa stain

Giemsa is a mixture of eosin and methylene blue. Stock solutions of Giemsa may be purchased commercially. Some brands are better than others. The stock solution of Giemsa stain is easily prepared from commercially available Giemsa powder.

Dilutions of Giemsa stain

Stock solutions of Giemsa stain must always be diluted by mixing an appropriate amount of stain with distilled neutral or slightly alkaline water; buffered saline is preferred because it provides a cleaner background and better preservation of parasite morphology. Always pour the stock Giemsa into the buffer, not the buffer into the stock, to avoid precipitation.

In the case of Giemsa, the staining solution is often poured below the slide (rather than on the slide) to avoid stain precipitates

Credits: Marcel Hommel

In the case of Giemsa, the staining solution is often poured below the slide (rather than on the slide) to avoid stain precipitates

A buffer solution which gives a pH of 7.2 is prepared as follows:

  • Potassium dihydrogen phosphate KH2PO4 0.7g
  • Disodium hydrogen phosphate Na2HPO4 1.0g
  • Distilled water: 1 litre

Tablets of phosphate buffer salts can be obtained commercially for 100 ml or 1,000 ml of water.

Technique of staining of films using Giemsa stain

  • 1. The thin film should be fixed in absolute methyl alcohol for 30 seconds. This can be done simply by immersing the film in methyl alcohol or by putting a few drops of methyl alcohol on it using a pipette. Thick films should be dried, but not by heating the slide (It is important to note that thick films are not fixed prior to staining).
  • 2. The staining solution must be freshly prepared by mixing 5 ml of stock solution with 100 ml of buffered water. For one or two slides, less staining solution is adequate providing that it contains 5% of stock stain.
  • 3. Transfer slide to staining solution in a Coplin jar or place the slide face downwards in a shallow tray on two glass rods. Stain for 20-30 minutes.
  • 4. Flush slide with tap water, wipe the back of the slide clean and stand upright to dry.

Note:
In emergencies, the staining time can be reduced to 5-10 minutes using a 10 % Giemsa solution.

Glass ‘Laveran dish’, for Giemsa staining

Credits: drawing from the Précis de Microscopie de M. Langeron, 1942

Glass ‘Laveran dish’, for Giemsa staining

Slide holder used to dry the stained slides

Credits: Marcel Hommel

Slide holder used to dry the stained slides

May-Grünwald-Giemsa (or panoptic method)

The May-Grünwald liquid (a methanol eosinate of methylene blue) is preferred by some microscopists as a fixative, instead of methanol. In this technique, 10 drops of May-Grünwald liquid are poured onto the slide and left for 3 minutes. Ten drops of distilled water are then added to the slide and gently mixed : the slide is then left to stand for a further minute. The fixative is then poured off and, without any washing or drying, the slide is stained with Giemsa (as above).
The advantage of the panoptic method is to result in better differential staining of all granules.