Page 133 - LECTURE NOTES

5. When dry, fix the smear by covering it with a few drops of absolute

methanol – Fix for 2-3 minutes and stain the smear using the Giemsa
technique.

5.7.2. Giemsa staining techniques

Reagents required: -

• Giemsa stain
• Buffered water, PH 7-1 – 7.2 or

• Buffered saline, PH 7.1- 7.2

Method

A. Immediately before use, dilute the Giemsa stain to make 10% solution. This

solution requires minutes staining time;
Preparation of 10% solution: Measure 45 ml of buffered water

Ph 7.1 - 7.2 in a 50ml cylinder- Add 5 ml of Giemsa stain (to 50 ml mark) and mix
gently

B. Place the slides in a shallow tray, supported on two rods, in a coplin jar, or in a

staining rack for immersion in a staining trough
C. C Pour the diluted stain into the shallow tray, Coplin jar, or stain thoroughly and

stain for 10 minutes
D. Wash the stain from the staining container using clean water

B. Wipe back of the slide clean and place it in a draining rack to air –dry.

6. When the smear is dry, spread a drop of immersion oil on it and examine first with

the 10 x and 40 x objectives to detect macrophages which may contain amastigotes
(the parasites can also be found outside macrophages) use the 100-X oil immersion

objective to identify the amastigotes.

Morphological characteristic of amastigotes

- Amastigotes are – small round to oval bodies measuring 2-4 μm

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