WET FILM EXAMINATION

      

INDICATION:

A wet blood film is used for the detection of living Trypanosomes, microfilariae of filarial worms. This is easy and rapid method of detection of live Trypanosoma species and microfilariae or first larval stage of blood filarial worms in acute infections.

MATERIALS REQUIRED:

Microslide

Capillary pipette

Coverslip

Low power Microscope

Normal saline solution

10% formalin

 

METHODS:

1.    Direct blood film (Wet film):

Procedure:

·       Place one drop of  blood on a slide, add a droplet of physiological saline, mix and cover with a coverslip.

·       Examine directly under low power (10X) of a microscope for live microfilariae. Larvae can be immobilized by placing a drop of 10% formalin at the edge of the coverslip. This can also be used for detecting trypanosomes.

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2.     Indirect wet film Method:

Drying and fixing of blood smears leads to some alteration of the morphology of intra erythrocytic parasites. The present technique overcomes the problem of biconcavity of erythrocytes so that intra erythrocytic parasites can be visualized because refraction of light induced by the biconcavity of bovine erthrocytes makes it difficult to appreciate the morphology of intracellular, unstained parasites. In a hypotonic environment, an erythrocyte swells to its maximum and becomes a sphere and the parasites and associated structures become visible within the cells. Stained haemoglobin appears to mask intracellular inclusions associated with parasites (eg. Theileria orientalis), which are otherwise visible in the wet blood films.

Procedure:

§  A small drop of blood enough to give a single layer distribution of erythrocytes is taken on a glass slide and mixed with a smaller drop of water using a glass slide.

§  A clean coverslip is gently placed over the preparation avoiding the formation of air bubbles.

§  Excess blood is absorbed using a piece of blotting paper.

§  The smear is then to be examined under oil immersion.

§   Apart from Theileria, Babesia and Anaplasma also can be detected in a live condition using this method.

 

For delay preparation of wet film, the blood should be collected in Alsever solution. Alsever's solution contains Dextrose, Sodium citrate, sodium chloride, citric acid and distilled water.

OBSERVATION:

                                             

                                                           Blood wet mount - under 10x

 

Artefacts can be differentiated as follows:

«  Howell jolly bodies are static and larger than Anaplasma and are seen in immature cells and in less number of RBC’s.

«  Chylomicrons and superimposed platelets appear as bright, translucent objects lodged above or below RBC’s. They tend to get separated on constant observation due to Brownian movement in wet medium and are extra cellular.

«  Erythrocytic pseudopodia appear as bright translucent bodies, lack characteristic movements and the continuity of the erythrocyte membrane can be made out when the RBC’s move. Such pseudopodia or crenations occur when samples are dehydrated.

«  Normoblasts are nucleated and appear as large spherical bodies. These are large and have an entire margin. Static and wavy cytoplasmic movements of Babesia are absent.