36 AQUATIC PHYCOMYCETES 



collect a hundred or more zoospores. They are contained in such a small 

 amount of water that the number of bacteria included is small if the initial 

 preparation is relatively clean. These spores can be deposited in another 

 small dish of sterile water and with another pipette can be picked up singly 

 or in twos or threes and transferred to agar plates. (7) The agar used [corn- 

 meal, prune] for initial isolations should be as transparent as possible and 

 poured in thin layers, not over 2 or 3 mm. thick, in Petri dishes. It is allowed 

 to dry for a day or two until all surface-moisture has disappeared. Four or 

 five drops of sterile water, large enough to spread out to a diameter of several 

 millimeters, are then distributed separately on the agar surface. Before this 

 water has been absorbed into the agar, washed zoospores are transferred in 

 small numbers to the drops on the agar plate. The site of each inoculation 

 is marked on the bottom of the Petri dish. A few minutes later, by turning 

 the plate upside down and examining it microscopically, the zoospores can 

 be detected, swimming about in the remaining surface-film of water or already 

 rounded and quiescent. (8) Germination takes place within 24 hr. at room tem- 

 perature, and by 48 hr. little germlings can be found with well-developed 

 rhizoidal systems and swollen hyphal tubes. ... As soon as discrete colonies 

 of bacteria appear, and if they are not too numerous or close to the fungus 

 germlings, the latter, embedded in little pieces of agar, can be lifted out with 

 needle-tweezers and transplanted to fresh agar plates. Sometimes the washing 

 is so effective that there are no contaminating bacterial colonies and the 

 growth is pure from the start. 



The formula for the basic glucose-yeast medium that Emerson and 

 Cantino used in their cultivation and physiological studies of Blasto- 

 cladia pringsheimii is given below. 



Salts - - K 2 HP0 4 , 0.1 %; MgS0 4 .7H 2 0, 0.02 %; NaCl, 0.01 %; CaCl 2 

 .2H 2 0, 0.01 %; FeCl 3 .6H 2 0, 0.002 %. Glucose -- 0.3 % Powdered yeast 

 extract (Difco) - - 0.1 %. pH, adjusted with sterile M 15 phosphoric acid 

 and M/10 sodium hydroxide to 6.5. 



Owing to excessive acid formation, healthy growth in culture was 

 maintained only when alkali was added. By keeping the pH of the 

 medium near the starting level viable material for transplants was 

 derived from such cultures up to seven weeks after their inoculation. 



Stiiben (1939), Harder and Sorgel (1938), and Cantino (1951a) have 

 all grown various species of Blastocladiella in pure culture. 



Of the Monoblepharidales, Monoblepfiarella offers no particular prob- 

 lems of cultivation of the vegetative phase on various media, but no 



