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

168 Manual of basic techniques for a health laboratoryAlternative method using saponin lysing solution1. Add 10ml of citrated blood (see above) to 10ml of saponin lysing solution.2. Mix the blood gently and leave for 15 minutes to allow the erythrocytes to lyse.3. Centrifuge at 2000g for 15 minutes.4. Remove the supernatant with a pipette and discard it into a dish containingdisinfectant.5. Transfer the deposit to a slide and cover with a coverslip.6. Examine the entire deposit for motile microfilariae using the ¥ 10 objective.(Microfilariae will still be motile in a “night blood” sample examined thefollowing morning.)7. Count the number of microfilariae in the preparation and divide by 10 to givethe number of microfilariae per ml of blood.Considerable experience is required to identify unstained microfilariae. It isrecommended that identification be performed on stained preparations (see page170).Microscopic examination of venous blood concentrated by filtrationMaterials and reagents● Microscope●●●●Microscope slidesCoverslipsSyringe, 15mlSwinnex-type filter holder● Polycarbonate membrane filter (25mm diameter, 5mm pore size) 1●●●Filter-paper pad (25mm diameter)Shallow dish, 15ml, with lidBlunt forceps● Sodium chloride, 0.85% solution (reagent no. 53)●●Absolute methanolDistilled water.Method1. Draw up 10ml of distilled water into a syringe.2. Draw 1ml of fresh blood or citrated blood into the syringe (Fig. 4.125). Rotategently to mix the contents. Wait for 2–3 minutes, for the erythrocytes to lyse.3. Moisten the filter-paper pad with a few drops of distilled water and cover withthe membrane filter. Place the filter on the filter holder.4. Connect the syringe to the filter holder. Gently push the blood through thefilter into a dish containing disinfectant solution (Fig. 4.126).5. Remove the syringe from the filter holder (taking care to avoid disturbing thefilter) and draw up 10ml of distilled water.6. Reconnect the syringe to the filter holder and gently push the water throughthe filter into the dish containing disinfectant solution, to remove the debrisfrom the filter (Fig. 4.127).1In areas endemic for Mansonella perstans, a membrane filter with a pore size of 3mm should beused.