vin, B, 6 Barber: Infection of Achlya 375 



selected and transferred to a second cover for the permanent 

 culture. These spores produce in a few hours a thrifty myce- 

 lium with hyphae larger than those from zoospores and more 

 suitable for inoculation. Abundant zoospores were obtained 

 within twenty hours by sowing one of these resting spores in 

 a hanging drop, consisting of the original fluid in which the 

 resting spores had formed. This fluid was somewhat enriched 

 by nutriment from the mycelium crushed in the process of 

 separating the resting spores. No extended experiments were 

 made to determine the conditions which favor the formation of 

 various types of reproduction, since the primary object was 

 simply to get material suitable for inoculation. 



The following is an example of one series of isolations: 



May 21. Resting zoospores from the tip of a zoosporangium 

 of Achlya were separated, washed, and transferred to tubes of 

 distilled water plus a few drops of glucose broth. Tube 1 

 received 16 spores; tube 2, 6 spores; tube 3, 3 spores; tube 4, 

 4 spores; tubes 5, 6, 7, 8, and 9, each 1 spore. 



May 23, all showed growth of Achlya except tube 8, which 

 remained sterile. All were free from bacteria except tubes 1 and 

 2 which had received the masses of 16 and 6 spores. Tube 7 

 developed zoospores after four days' growth at room temper- 

 ature; but zoospores could not be detected in any other. Five 

 of the pure cultures, including tube 7, developed oogonia and 

 antheridia after six days' growth, and the 2 contaminated ones 

 developed them some time later. The addition of undiluted 1 per 

 cent glucose broth to 2 tubes, 1 containing zoospores the other 

 none, brought about a luxuriant vegetative growth with few or 

 no zoospores or oogonia. 



Both liquid and agar media were used for hanging drop cul- 

 tures. In making these a large cover, about 38 by 65 milli- 

 meters in size, was sterilized and placed on the glass box used 

 in isolating microorganisms. A rectangular or ovoid barrier of 

 sterile paraffin was usually made on the underside of this cover by 

 melted paraffin blown from the tip of a bent pipette. Within 

 this inclosure the medium was placed, and the spore or myce- 

 lium planted in it. Such cultures may be grown for days over 

 a moist chamber. This chamber should be 0.5 centimeter or 

 more deep, else branches of the mycelium may grow to the 

 bottom of the cell and introduce bacteria into the culture. 



Liquid culture media may be readily withdrawn from such 

 cultures and a new medium or any other liquid substance added 

 by means of a sterile capillary pipette bent at the tip. 



It is possible to inoculate a hypha so that all growth will 



