The Use of Density-Gradient Centrifugation for the Purification of Eggs of Meloidogyne spp.

Investigators studying lipids, enzymes, and soluble proteins of nematodes have used plant tissue (2, 3, 4, 5, 9, 10, 12), fungi (1, 10), yeasts (! , 9), or bacteria (9) for propagating large numbers of nematodes for subsequent b iochemica l analyses . A l t h o u g h most investigators have eliminated viable bacteria from nematodes with surface sterilants, the possibility of macromolecular contamination from nonviable bacteria would remain. McClure et al. (8) recently suggested the use of a screening-centrifugation technique to obtain relatively noncontaminated eggs. In an attempt to obtain eggs of Meloidogyne incognita (Kofoid and White) Chitwood for analyses of structural protein, we found the above method unsatisfactory for eliminating sufficient bacterial and plant contamination. The size and density of nematode eggs are quite different from bacteria and most plant mater ia l . T h e r e f o r e , dens i t y -g r ad i en t centrifugation (DGC) in combination with filtration might be a useful technique for obtaining "clean" eggs for biochemical analysis; this report describes such a technique. Eggs of M. incognita were obtained from roots of tomato (Lycopersicon esculentum Mill. 'Bonny Best'), grown in 30-era clay pots of Lloyd sandy clay-loam:vermiculite mixture (3: i) or builders sand in a greenhouse. Roots were collected 4-8 weeks after planting. Three methods of extracting eggs were compared: (i) the method of McClure et al. (8), (ii) McClure et al. without sodium hypochlorite (SH), and (iii) McClure et al. modified as follows: egg masses and small roots were placed in 500 ml of sterile distilled water instead of I% SH. Eluted eggs and plant debris from a 16 ~m (400-mesh) screen were centrifuged in water instead of sucrose. After centrifuging for 2 min at 1,000 X g in 15-ml conical glass tubes,