Thin blood smear

This shows the presence of undistorted parasites. The thin blood smear permits identification and also calculation of the parasitaemia (% of parasitised red blood cells). This is necessary to start appropriate therapy (P. vivax is treated differently from P. falciparum). If the parasite cannot be identified it is regarded as a P. falciparum as a safety precaution. Mixed infections do occur. For staining, Giemsa is used with a slightly alkaline pH. It is a good habit to prepare the buffer solutions each day in the morning (interaction with CO₂ from the atmosphere changes the pH of older solutions). Phosphate buffers are mostly used.

1. First a stock solution is made of KH₂PO₄. This is made by placing 9.078 g KH₂PO₄ in one litre of purified water. This stock solution can be used for weeks if correctly stored (in a closed bottle).

2. A stock solution of Na₂HPO₄.2H₂0 is also made by mixing 11.877 g with one litre of purified water. If one uses the anhydrate (Na₂HPO₄) in place of Na₂HPO₄.2H₂0, only 9.474 g per litre is used. This stock solution can also be used for weeks if correctly stored (in a closed bottle).

3. To obtain a buffer with a pH of 8, add 5.5 ml of KH₂PO₄ solution to 94.5 ml of Na₂HPO₄.2H₂0 solution and dilute with 900 ml of distilled water. One then has 1 litre of buffered water with a pH of 8. This can then be used for the malaria blood smears for the rest of the day.

4. For the leukocytic formula it is best to stain with a slightly acid pH of 6.4. For this a different phosphate buffer is used. This requires different ratios. Now 26 ml of the Na₂HPO₄.2H₂0 solution is mixed with 74 ml of the KH₂PO₄ solution and then 900 ml water is added.

An alternative to Giemsa is acridine orange, but the day-to-day use of this technique is quite unpleasant and it is necessary to have a special microscope (ocular filter, halogen light, interference filter above the condenser). [This latter filter restricts transmission to some narrow spectral bands].

Category: Medicine Notes