32 AQUATIC PHYCOMYCETES 



The piesent isolation of Rhizophlyctis rosea was greatly facilitated by the 

 fact that it first appeared in a highly selective enrichment culture. The en- 

 richment medium — prepared in an attempt to isolate cellulose-decomposing 

 myxobacteria — consisted of a mineral agar plate covered with a round of 

 filter paper, which was inoculated with compost and incubated at 28° C. 

 After four days the filter paper had become entirely covered with a bright 

 pink growth. Microscopic examination showed the presence of large num- 

 bers of typically chytridiaceous thalli.The culture was maintained in crude 

 form for several weeks by repeated streaking of pieces of attacked filter 

 paper on fresh plates, but such treatment did little to reduce the large number 

 of contaminating organisms originally present, and it became clear that 

 other methods of purification would have to be adopted. 



When a piece of filter paper heavily invested with mature thalli was placed 

 in water, spore discharge occurred almost simultaneously from numerous 

 zoosporangia after a period of from 20 minutes to half an hour. This phe- 

 nomenon suggested a convenient method of purification. Large pieces of 

 attacked filter paper were removed from a gross culture and put in the bot- 

 toms of test tubes containing 10-15 cc. of sterile tap water, where they were 

 left undisturbed until after spore discharge had begun. Since the zoospores 

 move more rapidly than bacteria and are in addition strongly aerotactic, 

 they soon accumulated in large numbers in the upper layers, accompanied 

 by only a few bacteria. At this stage, microscopically controlled, loopsful 

 of the surface water were removed (care being taken to avoid agitation of 

 the tube) and streaked on mineral dextrose agar plates, which were incu- 

 bated at 28° C. After 24 hours the plates were examined under a dissecting 

 microscope; numerous small thalli were seen interspersed among the bac- 

 terial colonies. The positions of uncontaminated thalli well separated from 

 bacterial colonies were marked on the bottoms of the plates with India 

 ink, and the cultures reincubated until spore discharge had taken place (this 

 took on the average an additional 12-36 hours at 28° C). At the time of 

 discharge a small amount of liquid was exuded from the zoosporangia but 

 rapidly reabsorbed by the agar, after which the spores lay motionless in small 

 fields surrounding the empty spore cases. . . . Under the dissecting micro- 

 scope a mass of such spores was picked up on a sterile needle, transferred to a 

 drop of sterile water on another mineral dextrose agar plate, mixed with the 

 water and streaked. With proper precautions, the air contamination inci- 

 dental to manipulating an open plate under the microscope is negligible; 

 thus the second plates yielded either pure cultures or else cultures whose 

 further purification presented no difficulties. One point concerning the isola- 

 tion technique deserves special mention. For both the first and second streak- 

 ings it is highly desirable, if not essential, to use well-dried agar plates, 

 so that after inoculation no water remains on their surfaces. If this is not done, 

 isolation of the chytrid may be impossible due to the rapid spreading of motile 

 bacteria always present in the enrichment cultures. 



