Species	Geographic location	Periodicity	Collection time
Wuchereria bancrofti	Tropics/Subtropics	Nocturnal	12.midnight.
Wuchereria bancrofti	Pacific	Diurnal subperiodic	16.00 hours
Brugia malayi	SE Asia and SW India	Nocturnal	12.midnight.
Brugia malayi	Indonesia	Nocturnal subperiodic	21.00 hours
Brugia timori	Indonesia	Nocturnal	12.midnight
Loa loa	West/Central Africa	Diurnal	13.00 hours
Mansonella perstans	Africa/S. America	Non periodic	Any time
Mansonella ozzardi	Central&S America	Non periodic	Any time

The specimen collection times should be selected in accordance with the patient's clinical symptoms and travel history.

(i) Polycarbonate membrane filtration.

This technique is very sensitive, enabling very low parasitaemias to be detected. It is now the most widely used technique for separating microfilariae from blood.

Nucleopore polycarbonate membranes, 25 mm diameter, 5 µm pore size, are held in a Millipore Swinnex filter holder, using a rubber gasket to secure the membrane.

Method.

a. Place the membrane on the holder with a drop of water.

b. Draw up 10-20 ml of 1:1 saline diluted blood into a 20 ml syringe

c. Connect the syringe to the filter and gently push the blood through the filter membrane.

d. Repeat until all of the blood has been filtered.

e. Draw up 20 ml of saline into the syringe, flush through the filter, repeat using air.

f. Unscrew the top of the filter and discard the gasket into chloros; use forceps to transfer the membrane to a slide.

g. Add a drop of saline to the membrane and cover with a coverslip.

h. Examine the membrane under the microscope, using a x10 objective. Examine any microfilariae found using a x40 objective to note the presence of a sheath.

 Confirmation of species can be made by using appropriate staining methods to demonstrate nuclear morphology.

 (ii) Saline/saponin method.

 Reagent.

1% saponin in normal saline.

 Method.

a. Deliver 2 ml of blood (fresh or anticoagulated) into a centrifuge tube and add 8 ml of  1% saponin in saline.

b. Mix the blood by inversion, then allow to stand at room temperature for 15 minutes to allow the blood to haemolyse.

c. Centrifuge at 2,000 rpm for 15 minutes to deposit the microfilariae.

d. Discard the supernatant and use the deposit to make a wet preparation.

e. Examine the slide using the x10 objective. Active microfilariae can be seen and produce a snake-like movement as they disturb the cell suspension.