The Bacteria-Free Culture of a Nematode Parasite

It has been possible for some time to grow the entire life cycle of Neoaplectana glaseri, 1 a nematode parasite of the Japanese beetle, in cultures in which bacterial and fungous growths have been inhibited in various ways but not eliminated. 2 These contaminants undoubtedly introduced a high degree of variability into the results obtained. It therefore seemed advisable to attempt to rear this parasite in cultures free from bacteria. Lapage 3 and Glaser and Stoll 4 developed technics whereby the second ecdysis of strongyloid nematode larvae was easily and consistently obtained in quantity under sterile conditions. It was found necessary to modify one of these technics slightly for work with Neoaplectana. Cultures prepared in the routine manner were permitted to develop for 10 to 15 days, at which time the majority of the parasites were second-stage larval forms in their third or fourth generation.∗ These were removed from the surface of the solid medium and washed with water until free of much debris. To remove the dead larvae they were then filtered through 2 layers of lens paper supported by fine gauze. The larvae were ensheathed by aerating them in 25 cc of water for 3 to 4 days with a change of clean water 3 times daily. They were then washed in 25 cc lots of sterile water 3 times each day for 3 days, after which they were treated for 30 minutes to 1 hour with Labarraque's solution (sodium hypo-chlorite) at a dilution of 1 part to 40 or 50 parts of water. The nemas were again washed 3 times in sterile water, and then placed in water about 5 mm deep for from 15 to 20 hours.† The next day the nemas were again treated with Labarraque's solution and then washed 3 times, followed by another 15 to 20 hours' sojourn in a small amount of water.