Microfilaria Detection: Prior to the development of CFA assays, detection of microfilariae in blood was the standard approach to diagnosing lymphatic filarial infection (or loiasis), and it is the one still required today for both brugian filariasis and those situations where the antigen detection test is not available. For such assessments one must take into account the parasites' possible nocturnal periodicity in selecting the optimal blood drawing time (10pm-2am for most brugian filariasis and bancroftian infections). The simplest technique for examining blood or other fluids (including hydrocele fluid, articular effusions and urine) is to spread 20 microliters evenly over a clean slide that is dried and then stained with Giemsa or a similar stain. A wet smear may also be made by diluting 20-40 microliters of anti-coagulated blood with water or 2% saponin, which will lyse the red blood cells but still allow the microfilariae to remain motile and thus more readily identifiable. The larger the blood volumes examined, the greater will be the likelihood of detecting low parasitaemias. Knott's concentration technique has been used to examine 1 ml volumes of anti-coagulated blood by mixing the blood with 10 ml of 2% formalin, centrifuging the preparation and examining the sediment either unstained or fixed and stained; the microfilariae are non-motile and generally straight, and they can be easily missed if the viscous sediment is not searched diligently. More recently, membrane filtration has been advanced as the most sensitive technique for detecting and quantitating microfilariae in blood, urine or other body fluids. Polycarbonate (Nuclepore®) filters with a 3µm pore size have proved most satisfactory. A known volume of anti-coagulated blood or other fluid is passed through a Swinnex holder containing the filter, followed by a large volume (about 35 ml) of pre-filtered water that lyses the red blood cells. A volume of air then follows the water, and the filter is removed, placed on a slide and stained. The morphology of parasites present on filters is much more difficult to assess than those present in the more standard stained blood slides, but detection and quantitation is more straightforward.