Anesthetization of morphologically similar species with carbon dioxide for separation and culture.

Nematologists frequently encounter congeneric species in sampling for nematodes. No convenient method for the separat ion of morphological ly similar species has been described. For Meloidogyne spp., inoculum consisting of hundreds of eggs can be easily obta ined from a single egg mass and a pure popula t ion reared on a suitable host. T h e separation and culture of other plantparasitic nematode species, such as Paratrichodorus spp., is often more difficult. T h e species may be separated by placing a single female in the rhizosphere of a host plant, but the chances of a single nematode increasing to a high popula t ion in a short t ime are not great. T h e dissecting microscope may also be used for separat ing species which are not very dissimilar, but obta in ing high numbers of a single species is difficult, especially when the dissimilarities between species are not easily discernible. We have also tried separat ing nematodes which had been placed on glass slides and cooled to immobil i ty , bu t their recovery rate was rapid or many specimens were killed. We have developed a method, using vaporphase perfusion of carbon dioxide (CO2), for anesthetization of nematodes. Th is method allowed us to start greenhouse cultures wi th large, pure populat ions of desired species. We were concerned with the separat ion and culture of a mixed popula t ion of Paratrichodorus christiei (Allen) Siddiqi and Paratrichodorus porosus (Allen) Siddiqi. T h e nematodes were extracted from soil by Cobb's method (l) and placed in a drop of water on a glass slide. T h e slide was placed next to a 50-gin piece of dry ice, and bo th were enclosed in an inverted 2-liter beaker. T h e solid CO2 was allowed to sublime and perfuse into the water drop. After 3 min,