Studies on the Stability of the Na* Requirement 483 



solid media were required, 1.5 per cent agar was added. Special 

 precautions were taken to purify the chemicals used in the prep- 

 aration of the chemically defined medium. All glassware was 

 specially washed in a mixture of concentrated HNO:-, and Hi;SOi 

 and then rinsed with glass distilled water. In running responses of 

 the organisms to Na' the usual procedures of microbiological as- 

 say were used. 



Efforts were first made to train cultures to grow either at 

 lower Na"^ concentrations or in the absence of added Na"^ in the 

 chemically defined medium. 



We have shown previously that Na"^ affects both tlie rate and 

 extent of growth of marine bacteria, Table 1. Organism B-16 

 which grew rapidly and to the maximum extent in the presence 

 of 0.20M Na^, also grew at 0.03M Na+, though not maximally, af- 

 ter a sufficiently long incubation period. 



TABLE 1 



The Response of Marine Bacterium B-16 to Na+ in a Na+ Deficient 

 Medium (from MacLeod and Onofrey, 1957) 



Na+^ Incubation Time (Hrs.) 



M 48 72 120 312 



Percent Incident Light Transmitted'^ 







0.01 



0.03 



0.05 



0.07 



0.10 



0.13 



0.20 



0.30 



1 Added as NaCl. 



2 Evelyn colorimeter readings, 660 m/i filter. Uninoculated medium = 100. 



In the present study, efforts were made to acclimatize cul- 

 tures to grow at still lower Na"" concentrations by serially subcul- 

 turing into media containing Na^ lowered progressively in steps of 

 0.005M. Organism B-16 which grew initially, after long incuba- 

 tion at 0.03M Na"^ could not be induced to grow at less than 

 0.02M Na^, At this Na^ concentration, the organism became pro- 



