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

170 Manual of basic techniques for a health laboratory8. Reconnect the syringe to the filter holder and push the air through the filterover the dish containing disinfectant, to remove excess water from the filter.9. Remove the syringe from the filter holder and draw up approximately 7ml ofair and 3ml of methanol.10. Reconnect the syringe to the filter holder and push the methanol and air throughthe filter over the dish containing disinfectant, to fix the microfilariae and removeexcess methanol from the filter, respectively.11. Remove the syringe from the filter holder. Dismantle the filter holder and removethe membrane filter using forceps.12. Place the membrane filter, top side facing up, on a slide. Allow to air-dry.13. Stain with Giemsa stain as for thick films (see page 175) and examine theentire filter membrane using the ¥ 10 objective.Technique for staining microfilariaeMaterials and reagents● Microscope●Microscope slides● Giemsa stain (reagent no. 29)● Delafield’s haematoxylin stain (reagent no. 19)●Methanol● Buffered water (reagent no. 15).Method1. Prepare a thick blood smear of the deposit as described on page 174. Allow thesmear to air-dry.2. Fix in methanol for 1 minute.3. Stain with Giemsa stain (diluted 1 in 20 with buffered water, pH 6.8) for 30minutes.4. Examine the preparation under the microscope using the ¥ 10 objective. If it isdifficult to distinguish the nuclei of the microfilariae, return the slide to theGiemsa stain solution for another 5–10 seconds.5. Stain with Delafield’s haematoxylin stain (diluted 1 in 10 with buffered water,pH 6.8) for 5 minutes. Wash in buffered water, pH 6.8. (This second stain isrequired because Giemsa stain alone does not stain the sheath of Loa loa verywell.)6. Examine the preparation under the microscope. Use the ¥ 10 objective first tolocate the microfilariae; then identify the filarial species using the ¥ 40 and ¥ 100objectives.ResultsUnder the light microscope microfilariae appear (after appropriate staining) as primitiveorganisms, serpentine in shape, often enclosed in a sheath and filled with thenuclei of many cells (Fig. 4.128).Not all species have a sheath. In those that do, the sheath may extend a short orlong distance beyond either extremity. In some species, depending on the stainused, the sheath displays a unique staining quality which aids in species identification.The nuclei of the cells which fill the body are usually darkly stained and may becrowded together or dispersed (see Fig. 4.128). The anterior extremity is charac-