Staining methods for the identification of malaria parasites

Examination of Thick Blood Films for Malaria Parasites

Introduction

The definitive diagnosis of malaria infection is still based on finding malaria parasites in blood films. In thin films the red blood cells are fixed so the morphology of the parasitised cells can be seen. Species identification can be made, based upon the size and shape of the various stages of the parasite and the presence of stippling (ie bright red dots) and fimbriation (ie. ragged ends). However, malaria parasites may be missed on a thin blood film when there is a low parasitaemia. Therefore, examination of a thick blood film is recommended. With a thick blood film, the red cells are approximately 6 - 20 layers thick which results in a larger volume of blood being examined.

Field's stain method for THICK blood films

The method recommended for staining thick blood films by the Hospital for Tropical Diseases is Field's stain which is made from 2 components. Field's A is a buffered solution of azure dye and Field's B is a buffered solution of eosin. Both Field's A and B are supplied ready for use by the manufacturer.

Method

Place a drop of blood on a microscope slide and spread to make an area of approximately 1 cm2 . It should just be possible to read small print through a thick film.

The film is air dried and NOT fixed in methanol.
The slide is dipped into Field's stain A for 3 seconds.
The slide is then dipped into tap water for 3 seconds and gently agitated.
The slide is dipped into Field's stain B for 3 seconds and washed gently in tap water for a few seconds until the excess stain is removed.
The slide is drained vertically and left to dry.
Microscopic examination of the Field's stained thick blood film
The end of the film at the top of the slide when it was draining should be looked at. The edges of the film will also be better than the centre, where the film may be too thick or cracked.
In a well stained film the malaria parasites show deep red chromatin and pale blue cytoplasm.
White cells, platelets and malaria pigment can also be seen on a thick film. The leucocyte nuclei stain purple and the background is pale blue. The red cells are lysed and only background stroma remains. The occasional red cell may fail to lyse.
Schizonts and gametocytes, if present, are also easily recognisable.
A thick film should be examined for at least 10 minutes, which corresponds to approximately 200 oil immersion fields, before declaring the slide negative.

Difficulties in the examination of thick blood films

As a result of haemolysis of the red blood cells due to staining of an unfixed film, the only elements seen are leukocytes and parasites, the appearance of the latter being altered. Consequently:
The young trophozoites appear as incomplete rings or spots of blue cytoplasm with detached chromatin dots.
The stippling of P. vivax and P. ovale may be less obvious although occasionally ghost stippling may be seen.
The cytoplasm of late trophozoites of P. vivax and P. ovale may be fragmented.
Caution should be exercised when examining thick blood films as artefacts and blood platelets may be confused with malaria parasites.
Conclusion

A thick blood film is recommended for routine diagnosis of malaria in addition to the thin film and is particularly valuable in instances of low parasitaemia. However, in order to correctly speciate the parasite, examination of a thin film is required. If the thick film is negative, it is unlikely that parasites will be found in the thin film.



A thick film showing trophozoites and gametocytes of Plasmodium falciparum

Examination of Thin Blood Films

1. Rapid Field's stain for thin films

This is a modification of the original Field's stain to enable rapid staining of fixed thin films. This method is suitable for malaria parasites, Babesia sp., Borrelia sp. and Leishmania sp.

Method.

Air dry the film
Fix in methanol for 1 minute.
Flood the slide with 1 ml of Field's stain B, diluted 1 in 4 with distilled water.
Immediately, add an equal volume of undiluted Field's stain A, mix well and allow to stain for 1 minute.
Rinse well in tap water and drain dry.
Uses.

This is a useful method for rapid presumptive species identification of malarial parasites. It shows adequate staining of all stages including stippling (mainly Maurer's clefts). However, staining with Giemsa is always the method of choice for definitive species differentiation.

2. Giemsa stain for thin films.

Method.

Air dry thin films
Fix in methanol for 1 minute
Wash in tap water and flood the slide with Giemsa diluted 1 in 10 with buffered distilled water pH 7.2. The diluted stain must be freshly prepared each time.
Stain for 25 - 30 minutes.
Run tap water on to the slide to float off the stain and to prevent deposition of precipitate on to the film. Drain dry vertically.
Examine the film using the x100 objective.
Notes on the stained film.

Examine the tail end of the slide where the red cells are separated into a one-cell-layer thick.
An alkaline buffer pH 7.2 is vital for clear differentiation of nuclear and cytoplasmic material and to visualise inclusions such as Schuffner's/James's dots in the red cells.Acidic buffer is unsuitable.
The red cells are fixed in the thin film so the morphology of the parasitised cells and the parasites can be seen.
On a well stained film the chromatin stains red/purple and the cytoplasm blue. Leucocytes have purple nuclei, the red stippling , if present should be clearly visible.