Manual of basic techniques for a health laboratory - libdoc.who.int

9. Haematology 303Drying the filmAdequate drying is essential to preserve the quality of the film, especially inhumid climates. The film can be left to air-dry in dry climates.In the wet season (in the tropics)Dry the film by waving it rapidly about 5cm away from the flame of a spiritlamp or Bunsen burner: hold the slide to the side and slightly above (butnever directly over) the flame (Fig. 9.71). If necessary, protect the blood filmfrom flies.Mark the dry film with the patient’s name or number. Write with a leadpencil on the thick part of the film not used for examination.Fig. 9.71 Drying the blood film overa spirit lampFixation of the filmIf the film is intended for determining leukocyte type number fractions, it shouldbe fixed with methanol before staining with May–Grünwald stain (see below).If the film is intended for detection of parasites, it should be fixed with methanolbefore staining with Giemsa or Field stain (see below).PrecautionsCare is required to avoid the formation of deposits of stain, which appear on thefilm as masses of little black spots. A number of precautions are also required toavoid staining the films too blue, too pink or too dark; these are briefly describedbelow.● Use perfectly clean glassware. Wash it every day. Do not use acid. Remove staindeposits with methanol.● Use neutral water (buffered if possible, except for Field stain). The preparationtechnique is described in section 2.4.4. Acid water produces a film that is toored; alkaline water one that is too blue. Neutral water must be freshly preparedas it becomes acid when exposed to air.Staining of the filmMethod for Leishman stain1. Fix the thin blood film with methanol for 2–3 minutes.2. Prepare a 1 in 3 dilution of Leishman stain using one volume of stain and twovolumes of buffered water. Mix.Example: Use 10ml of stain and 20ml of buffered water.Prepare sufficient stain for 1 day’s use only, as the diluted stain does not keepwell.3. Cover the slide with the diluted stain for 7–10 minutes.Important: The staining time may need to be adjusted, especially when a newbatch of stain is received or the stain has been stored for a long time.4. Wash the stain off in a stream of buffered water. Do not tip the stain off as thiswill leave a deposit of stain on the film.5. Leave clean water on the slide for 2–3 minutes to differentiate the film. (Thetime taken for differentiation will depend on the stain and the pH of the waterused.)The pH of the water is of vital importance in differentiating the different types ofleukocyte with Leishman stain. It should be between 6.8 and 7.2, and preferablybetween 7.0 and 7.2.6. Tip the water off and place the slide in a draining rack to dry.