1. Cover the blood film (preferably methanol pre- fixed, ) with undiluted stain but do not flood the slide. If using a dropper bottle count the number of drops required to cover the film.
The undiluted stain not only acts as a fixative but also partially stains the smear. This stage is required to obtain the best possible staining results.
2. Add twice the volume of pH 6.8 buffered water (i.e. twice the number of drops as stain). The stain should not overflow. Ensure the water is well mixed with the stain by blowing on the diluted stain or mixing the stain and water using a plastic bulb pipette. Allow to stain for 10 minutes (time may require adjusting).
Diluting the stain in buffered water brings about full staining of the blood cells. The exact staining time to use should be decided when a new batch of stain is prepared.
3. Wash off the stain with tap water (filtered if not clean). Do not tip off the stain, because this will leave a fine deposit covering the film. Wipe the back of the slide clean and stand it in a draining rack for the smear to dry. The blood film should
appear neither too pink nor too blue (check
results microscopically).
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