374 The Philippine Journal of Science wis 



with the capillary pipette while swarming in the nearly or quite 

 bacteria-free end of the drop, or after they have come to rest and 

 germinated. Either method enables one to obtain a culture 

 from a single zoospore, but the latter sometimes facilitates 

 freeing them from bacteria. Before finally transferring to the 

 test tube, each isolated zoospore may be examined in a small 

 droplet of water with a higher power in order to make sure of 

 the absence of bacteria. 



In the absence of zoospores or resting spores in the pure 

 culture, one may make subcultures by transferring the myce- 

 lium grown in the liquid medium to sloped agar tubes. When 

 growth is established there, new cultures may be made from 

 detached pieces of agar containing living hyphse. 



For purposes of inoculation it is necessary to have a thrifty 

 mycelium in a hanging drop of nutrient medium. For obtain- 

 ing this there are several convenient methods. 



From a young subculture growing in a thin layer of agar in 

 a Petri dish one may cut with sterile knife or platinum spatula 

 small blocks of agar from the margin where growth is most 

 vigorous. These are transferred to the sterile cover glass, and, 

 if desired, fresh nutrient medium is added to the margin of the 

 block. 



Another method would be by transferring bodily a small 

 mycelium to a liquid medium in a hanging drop. 



Zoospores in this species were formed much less freely in 

 pure culture than in ordinary water cultures with bacteria. 

 But sufficient zoospores for hanging drops or for making sub- 

 cultures were obtained from a three to ten days' growth in test 

 tubes containing about 10 cubic centimeters of distilled water 

 with 0.05 to 1 cubic centimeter of 1 per cent glucose broth 

 added to each tube. In one culture, glucose broth, made 5 + 

 acid and added in the proportion of 0.1 cubic centimeter to 5 

 cubic centimeters distilled water, gave an abundant crop in from 

 two to three days. Apparently some degree of starvation facil- 

 itates the formation of zoospores in this species, since a vigor- 

 ous growth in undiluted glucose broth or on sterilized insects 

 in water usually failed to produce them. 



The best mycelia for the purposes of these experiments were 

 obtained from the large resting spores formed vegetatively at 

 the tips of hyphse. These usually occurred abundantly in old 

 cultures in distilled water plus glucose broth. These are sepa- 

 rated from the mycelium with the platinum loop, and when 

 settled to the bottom of the test tube may be transferred by a 

 pipette to a hanging drop. From this drop one or more are 



