A Technique for Determining Live Second-stage Juveniles of Heterodera glycines

We developed a quick and reliable technique to distinguish live and immobile (presumed dead) Heterodera glycines second-stage juveniles (J2) following their treatment with microbial culture filtrates. About 50 J2 in 1 ml of culture filtrate or water were placed in wells of a 24-well tissue-culture plate. After incubation, the nematodes in the wells were observed with the aid of an inverted microscope. The J2 lay straight and their viability could not be determined by direct microscopic observation. With the addition of one or two drops (50 to 100 microl) of 1 N NaOH into the well, the live nematodes changed their body shape from straight to curled or hook-shaped after about 30 seconds. The nematodes that responded to NaOH by changing their body shape within 3 minutes were considered a live, while those nematodes that failed to respond within 3 minutes and were immobile were presumed to be dead. The technique is simple, fast, and useful for the examination of a large number of samples in which one wants to determine the effects of microbial cultural filtrates on nematodes, or in similar tests.