236 



FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE 





This dehydration process can be partly overcome 

 by keeping the agar slants in a cold room. How- 

 ever, to make this experiment feasible, it was 

 necessary to accelerate the growth of Nitzschia. 

 At this time it was discovered that by transferring 

 a small piece of agar on which Nitzschia was 

 growing (as described above) as much growth 

 could be obtained in 1 week as was previously 

 obtained in 4 to 6 weeks with transfers made by 

 the conventional bacteriological streaking method. 



In 3 to 5 days after Nitzschia was transferred 

 to the agar slants containing Chlorella, it had 

 grown as close to Chlorella as it would. It was 

 quite evident that Nitzschia would not extend its 

 growth so as to make contact with Chlorella. A 

 small area remained between Chlorella and 

 Nitzschia as shown at C in figure 11. 



Another agar-slant experiment was run in 

 which Chlorella was placed at the opposite end of 

 the agar slant. Results were similar to those just 

 discussed. Nitzschia- grew most rapidly in the 

 central portion of the slant. This was the area on 

 which Nitzschia first reached a point beyond which 

 it would grow no closer to Chlorella. Later Nitz- 

 schia covered the area to both sides of this point, 

 leaving the same relative distance between it and 

 Chlorella. 



It might be thought that lack of nutrients pre- 

 vented Nitzschia from growing until it came in 

 contact with Chlorella; however, Detmer's me- 

 dium diluted with distilled water to two-thirds 

 its normal concentration still contains more of 

 each nutrient than the liquid standard culture 

 medium, and 16 times the total concentration of 

 nutrients in that medium. It thus seems improb- 

 able that lack of nutrients could be responsible. 

 Another possible cause was the use of test tubes 

 instead of Petri dishes. The surface of the agar 

 was not horizontal in the test tube, and this might 

 have prevented Nitzschia from growing until con- 

 tact was made with Chlorella. However, since 

 Nitzschia is motile, and since it grows as rapidly 

 upgrade as down, this factor can be disregarded. 

 Further, as the agar was uniform in composition 

 throughout, the sudden cessation of growth in the 

 rapidly growing Nitzschia when it reached the 

 proximity of Chlorella must have been due to 

 some change in the agar so that Nitzschia would 

 not grow on it. The failure of Nitzschia to grow 

 on this area is believed to he due to the presence 



of chlorellin, the antibiotic substance shown by 

 Pratt (1940) and in this report to be produced 

 by Chlorella. 



INHIBITION OF GROWTH RATE IN 

 CONDITIONED MEDIA 



INHIBITORY EFFECT ON CHLORELLA OF 

 JV/TZSCHL4-CONDITIONED MEDIUM 



It was found that the medium in Nitzschia cul- 

 tures became conditioned by an accumulation of 

 an antagonistic substance as the culture aged, so 

 that greater inhibition in the growth rate of 

 Chlorella occurred when that species was added to 

 aged cultures than when it was added initially 

 with Nitzschia to new medium. Nitzschia cul- 

 tures with a concentration of 10 million cells per 

 liter were prepared in six flasks with medium con- 

 taining twice the concentration of nutrients used 

 in the standard culture medium. At the end of 

 the second day three cultures were removed from 

 the illuminated shelves, and Chlorella was added 

 in sufficient quantity to give a concentration of 

 70 million cells per liter; three cultures were left 

 as controls. Also three Chlorella controls con- 

 taining 70 million cells per liter were prepared 

 with fresh culture medium. 



Chlorella added to the Nitzschia cultures 

 reached a population size about 45 percent of that 

 obtained in the control cultures (fig. 12) and 65 



5000 



2 3 



TIME IN DAYS 



Figure 12. — Comparison of growth curves of Chlorella 

 in control and in tfiteseftia-conditloned medium. Dots 

 represent growth curve in control cultures; circles, 

 growth curve in JVi'r:scftia-conditioned medium. 



