Culture of Single Species 37 



tus should be adjusted so that the velocity of the vertical current in the 

 left arm is less than the rate of sinking of the diatoms. Free swimming 

 flagellates and organisms lighter than diatoms escape while the latter 

 accumulate in the area (D) just above the mouth of the capillary tubing. 



Cultures of a single species of diatom obtained by the method of isola- 

 tion and subsequent washing usually are contaminated with bacteria, 

 the presence of which apparently does not interfere with the growth 

 of the diatom. Allen designates them as "persistent" cultures, reserving 

 the name "pure" only for bacteria-free cultures of a single diatom species. 

 The elimination of bacteria is a very difficult and time consuming process 

 which consists of repeated washings in sterile media followed by frac- 

 tional subdivision. Purification of cultures by a method of differential 

 poisoning was attempted by Allen ( 1914) with only a measure of success. 

 Cultures of Thalassiosira gravida were treated by adding 1 mg. of 

 CUSO4.5H2O to each 100 cc. After an interval of 12 minutes a fresh 

 medium was inoculated with 1 cc. of the first one. In this way the num- 

 ber of bacteria was materially reduced but complete sterilization was 

 not obtained. 



Chlorination produced by electrolysis of water was applied also only 

 with partial success (Allen, 1914). An electric current varying from 

 1.7 to 1.5 amperes was passed between the two sterile carbon electrodes 

 immersed in seawater. The electrolysis was continued for about 3 min- 

 utes; then the water was allowed to stand for one hour. Fifty cc. of 

 chlorinated water were added to an equal amount of sterile medium and 

 the solution was inoculated with a small amount of a Thalassiosira cul- 

 ture. In this way the number of bacteria was materially reduced. 



In healthy cultures the presence of bacteria does not interfere with 

 the propagation of the diatom (Nitzschia) but as soon as conditions 

 become unfavorable the bacterial growth is promoted and the diatoms 

 suffer (Galtsoff, et al., 1935). The unhealthy state of such cultures is 

 easily noticeable for the cells stick together forming large clumps which 

 settle on the bottom and which sink almost immediately after stirring. 

 Microscopical examination of a stained preparation showed that every 

 Nitzschia cell was covered with a large number of bacteria closely ad- 

 hering to its body. 



In old cultures Nitzschia has a tendency to develop teratological forms. 

 This condition may be remedied by subculturing and it usually disap- 

 pears in a short time. 



Natural seawater enriched by the addition of nutrient salts appears to 

 be the best medium for the cultivation of diatoms. It is of interest that 

 according to Allen (1914) Thalassiosira failed to grow in artificial sea- 

 water to which nitrates, phosphates, and iron were added according to 

 Miquel's method. Excellent results were obtained, however, when less 



