Use perfectly de-oiled slides: they should be washed with scouring powder, rinsed in water, then with an extemporaneous alcohol-ether mixture.

Blood collection 

• by venous puncture using a hemogram tube (EDTA or heparine), which means that hematological tests may be carried out on the same sample.
• by pricking the finger tip with a micro-lance lancet. 

Blood film formation

•  ideally by using a pre-prepared slide, which should be carefully cleaned after each use, with alcohol and a compress.
• alternatively, by using a cover-slip (to be destroyed after use). 

The smear should be thin, regular and contain a head, a body, a tail and two margins. The film should be spread with a regular movement, neither too slowly (the smear will be to thin and spill over the end of the slide), nor too quickly (the smear will be too short and thick).  

Once it has been prepared, the smear should be dried immediately, either manually by agitating, or by means of an electric fan. It should be precisely identified (name, date) with a lead pencil, by writing on the head of the smear once it is completely dry.  

It should be stored in a place protected from flies. If it is planned to store it for longer than 6 hours, the smear should be fixed with methanol (a 3 minute bath or evaporation of a film covering the smear) The smear should NEVER be fixed using a bacteriological type sterilization. 

Four staining techniques may be used:

• May-Grunwald-Giemsa staining: gives the best results. The colors are bright with good contrast. 
• Giemsa staining: the technique is simpler, but less discriminatory. 
• Panoptic staining: this very rapid technique has the inconvenience of giving "pastel" shades which require a certain experience to read them. 
• RAL 555 rapid smear staining: provides a good quality/speed compromise.  

This identification can only be carried out on a good quality smear. A mediocre smear is the cause of most false positive results. 

Beware of poly-parasitism

• By venous puncture using a hemogram tube (EDTA or heparine), which means that hematological tests may be carried out on the same sample.
• by pricking the finger tip with a micro-lance lancet.  

Bio-ecology of the Plasmodium

Alongside the biological elements, knowledge of the cycle of the different plasmodium species and their geographical distribution provides interesting information:

• it is very rare to be able to diagnose Plasmodium vivax in a melanoderm subject for there is a resistance due to the absence of the antigenic determinants Fy a and Fy b (Duffy blood group system). 
• Account should be taken of the existence of hypnozoites for Plasmodium vivax and Plasmodium ovale, which explain the possibility of a relapse after schizontocidal treatment. 
• Geographically speaking, Plasmodium falciparum is distributed throughout the inter-tropical zone limited by the isotherm 18°C, Plasmodium vivax is to be found in the warm temperate inter-tropical zone. It is rare in Africa, except in North Africa and on the neighboring islands (Madagascar and the Comoro Islands). Plasmodium malariae  is to be found erratically throughout Africa. Plasmodium ovale is limited to Central Africa and the Comoro Islands.  

Use of a score

A diagnostic orientation may be given by the use of a score, if the parasitic density allows: each parasite seen is given the name of the most probable morphological species. The count is made on at least 50 parasites and the species retained is that which obtained the highest score. 

ATTENTION

It is important not to confuse diagnostic uncertainty and therapeutic attitude 

When it is possible and well implemented, the microscopic examination is currently the key player in the diagnosis. The individual diagnosis of malaria may therefore be based on optical microscopy on the condition, however, that the result may be communicated quickly to the therapist.