Axenic Cultivation of the Parasitic Nematode, Neoaplectana glaseri, in a Fluid Medium Containing Raw Liver Extract

Abstract

Neoaplectana glaseri Steiner, 1929 originally discovered grubs of the Japanese beetle, Popillia japonica Newm. by Glaser and Fox (1930), is now known to parasitize several species of insects, especially coleopterous larvae inhabiting the soil (Christie, 1941; Swain, 1943; Dumbleton, 1945). In a series of experiments which demonstrated the value of the nematode as a biological control agent against the Japanese beetle, Glaser (1931, 1932, 1940a), McCoy and Glaser (1936), and Girth, McCoy and Glaser (1940) worked out methods of growing the oxyurid non-sterilely on veal infusion agar and on ground raw potatoes seeded with live yeast, and on veal pulp (McCoy and Girth, 1938). With a technique devised by Glaser and Stoll (1940) for sterilizing Haemonchus contortus larvae, Glaser (1940b) later reported success ridding N. glaseri of contaminants and culturing it bacteria-free. Successive life cycles would ensue on pieces of fresh, sterile rabbit kidney on dextrose agar slants, and the organism could be serially transplanted, apparently indefinitely. This exceptional result of generation following generation axenically vitro, without return to the host, was the first recorded for a worm parasite. It has been reported for only one other species, the closely related Neoaplectana chresimla, by Glaser, McCoy and Girth (1942), who were able to carry it sterilely for 2 years through 28 transplants, although it failed to survive when bacterially contaminated. Culture in liquid media containing kidney extracts devoid of particulate matter was also briefly noted for N. glaseri (Glaser, 1940b), but without details. Several months after Dr. Glaser's death, the present author, impressed by the potential value for experimentation of a parasitic nematode that could be carried the laboratory on stock cultures, began a study of it fluid media. In contrast to the ease of culturing the organisms on kidney tissue, growth and cyclical development kidney extracts or other liquid media was not initially obtained. This difficulty was later resolved by adding to veal infusion broth an aqueous extract of raw liver (RLE), prepared without heat, acidified, and sterilized by filtration (Seitz). The present report deals with its use, and concomitant conditions found favorable for culturing. Results obtained at the Princeton laboratory prior to July 1950, have been confirmed and extended at New York.1