<P> 2 Add twice the volume of pH 6.8 buffered water (i.e. if e.g. 7 drops of stain was used, then use 14 drops water) to dilute the stain, taking caution that the stain should not overflow (which will make the dilution inaccurate). However, the standard operating procedure published by WHO suggests adding equal (instead of twice) volume of water . Mix the water with the stain underneath by gently blowing with a straw or using a plastic bulb pipette . Allow to stain for 10--12 minutes (time may require adjusting). In this method, better ionization during the dilution by the aqueous buffer in this step is necessary to complete the staining . The WHO protocol also clarifies that during this incubation </P> <Dl> <Dd> "the appearance of a polychromatic' scum' on the surface of the slide is merely a result of oxidation of the dye components and can be ignored ." </Dd> </Dl> <Dd> "the appearance of a polychromatic' scum' on the surface of the slide is merely a result of oxidation of the dye components and can be ignored ." </Dd> <P> 3 . Wash off the stain with clean (or filtered) tap water . If the stain is tiped off instead of washing, this will leave a fine deposit covering the film . Wipe the back of the slide clean and stand it in a draining rack to dry . The stained smear should grossly appear neither too pink nor too blue (verify final results microscopically). If the tap water is highly acidic, resulting in smear turning grossly pink too fast or highly alkaline, resulting in the smear remaining too blue, try using boiled cooled water or filtered rain water or pH 6.8 buffered water which can be used as an additional flooding step after washing in running water . </P>

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