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Staining methods for microfilariae. (i) Supravital staining. Reagent. 0.75% cresyl blue in saline or 1.0% methylene blue in saline. These reagents can be used to stain live microfilariae by allowing the stain to flow
under the coverslip on to a polycarbonate membrane preparation or a centrifuged preparation. The dye will stain the nuclei of the microfilariae and also provide a contrasting background to look for a sheath. It may take
several minutes for the dye to penetrate the organisms and the slide should be kept in a moist chamber to prevent the preparation from drying out. (ii) Permanent staining.
Permanent stains should show up the nuclei, including the pattern of nuclei in the tail region and stain the sheath if necessary. The stains of choice are;
1. Haematoxylin 1. Giemsa 2. Rapid Field's
1. Haematoxylin. Delafield's haematoxylin will stain the nuclei and the sheath well and unlike Ehrlich's haematoxylin does not require heating Reagents.
Delafield's haematoxylin (BDH) 1% acid alcohol Methanol
Method.
a. Make thin films, allow to air dry then fix in methanol for 5 minutes b. Stain with Delafield's haematoxylin for 20 minutes c. "Blue " the nuclei by placing the
slide in a coplin jar and allow a stream of running water to flow into the jar for 20 minutes. d. Decolourise with 1% acid alcohol for 5-10 seconds before "blueing" in tapwater again. Control
this process by examination under the microscope until the nuclei are clear and distinct. e. Allow the slide to dry before mounting in DPX f. The nuclei should stain blue and the sheath grey
2. Giemsa. See method for staining thin blood films. Use Giemsa at a dilution of 1 in 6 in pH 6.8 buffered distilled water. Rinse with buffered water.
3. Rapid Field's See method for staining thin blood films.
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| [UKNEQAS Parasitology] [Teaching Information] [Intended results] [UKNEQAS Study Day] |