Previous work from this laboratory has shown that sterile culture of Colpoda duodenaria is possible in a medium containing heat-labile plasmoptyzate prepared from marine Pseudomonas (Taylor and van Wagtendonk, '41 ; van Wagtendonk and Taylor, '42) supplemented by three vitamins, nicotinic acid, thiamin, and pyridoxin (Tatum, Qarnjobst and Taylor, '42). Successful serial transfer of Colpoda in this medium indicated that all of the essential growth-factors and nutrients were present, although growth was not as rapid as in medium containing brewers' yeast-extract. Under these conditions no need for or effects of additional vitamins could be demonstrated.
Since the plasmoptyzate was the only undefined constituent of the essential medium it must have contained all the unknown factors required by Colpoda. Further analysis of the nutritive requirements of Colpoda was possible only through a more thorough investigation into the nature of the materials supplied by the plasmoptyzate. The results of this work are reported in this communication.
EXPERIMENTAL
Methods.
Essentially the same general methods as described previously (Tatum, Garnjobst and Taylor, '42) were used to culture, inoculate, count, and test Colpoda cultures for sterility, and to culture the marine Pseudomonas and prepare the plasmoptyzate. The basal medium contained per ml.: 20 y each of t,hiamin, pyridoxin and nicotinamide; 0.2 ml. bacterial plasmoptyzate ; and 0.8 ml. Osterhout's salt solution. Trace elements were added to the Osterhout's salt solution in order to prevent any possible mineral deficiencies with further purification of the bacterial plasmoptyzate. Exceptions and additional techniques are noted in connection with particular experiments.
Purification o f p l w m o p t y z a t e . Previous work (van Wagtendonk and Taylor, '42) had indicated that the plasmoptyzate, although relatively 'Supported by a grant from the Rockefeller Foundation.