Nutritional Patterns in Marine Bacterial Populations 689 



sterilized aged sea water and refiltered as before. The washed 

 concentrated sample (together with the filter and well) was then 

 transferred aseptically to a jar and made up to 100 ml with sterile 

 aged sea water containing 10 p. p.m. of surface-active agent 

 "Tween 80." After shaking the jar to resuspend the microor- 

 ganisms and as far as possible disperse cell aggregates, dilutions 

 were prepared and plates poured with mud extract agar in quad- 

 ruplicate from each dilution. 



From the sediment cores, 1 g quantities of surface mud were 

 aseptically transferred to jars similarly containing sterile aged 

 sea water and surface-active agent, and plates were poured 

 from dilutions as above. 



The isolation medium used in all cases was prepared from 

 the same batch of marine mud extract. 



Isolation of Cultures 



Plates were incubated for fourteen days at 18-20 C and 



counted. To avoid haphazard selection of organisms, all colonies 

 were systematically picked off selected plates of known dilutions 

 carrying well spaced colonies. These isolates were successively 

 streaked twice on mud extract agar and finally inoculated as stab 

 cultures in mud extract semisolid agar containing 0.1 per cent 

 yeast extract to serve as stock cultures which were stored at 4 C. 

 Approximately 80 isolates were obtained from each of the original 

 samples. 



Nutritional Requirements 



The nutritional requirements of the isolates were evaluated 

 by observing their growth response in a series of five media of 

 varying complexities. Organisms were thus differentiated ac- 

 cording to their requirements for maximum growth into the fol- 

 lowing groups: 



1. Bacteria with Simple Requirements. These organisms 

 grow well in a basal medium (Medium B° ) of synthetic sea 

 water enriched solely with phosphate, glucose and inorganic ( am- 



* Basal medium B. As a synthetic sea water substitute, the ASP2 medium of 

 Provasoli et al. (19) was used. From the formula there given, silicate and 

 vitamin B12 were omitted, phosphate was increased from 0.5 mg to 2.0 mg 

 K-.HPO4/100 ml medium, nitrate was replaced by 0.1 g. (NH4)2SOj, and glucose 

 was added at 0.1 per cent. The complete trace metal solution was retained at 

 3 ml/100 ml. pH = 7.4 



