482 Marine Microbiology 



grew in the same medium prepared with fresh water, after the 

 cultures had been held for five months without transfer. It was 

 subsequently reported by Zobell (9) that 56 of 60 species of 

 marine bacteria had developed a capacity to grow in fresh water 

 media. Paradoxically, attempts to tiain the original cultures of 

 these organisms to grow at lower sea-water concentrations met 

 with only limited success. Since then, Stanier (6), MacLeod and 

 Onofrey (4), and Littlewood and Postgate (3) have all reported 

 difficulty or failure to train marine bacteria to grow at lowered 

 sea-water or salt concentrations. In 1959, Pratt and Waddell (5) 

 reported the selection of what appeared to be mutants of a marine 

 bacterium on a trypticase medium prepared without added NaCl 

 but containing the other ions of sea water. 



All of the reports of the growth of marine bacteria in media 

 prepared without sea water or NaCl have been based on obser- 

 vations made using non-chemically defined media which would 

 be expected to be contaminated with, in some cases, relatively 

 high concentrations of Na^ as well as other inorganic ions. Since 

 the requirement of marine bacteria for Na"^ is the one feature of 

 their cultural requirements which distinguishes them most readily 

 from land forms, and since the evolution of these bacteria to land 

 forms would represent the development of a capacity to grow 

 without Na"^, it was of interest to know more about the stability 

 of the Na"^ requirement. Attempts have therefore been made to 

 train and to produce mutants of marine bacteria that will grow in 

 media prepared with chemically pure components containing at 

 the most only traces of Na^ as a contaminant. 



Three organisms of marine origin were studied initially - Bo, 

 a Flavobacterium, Bio a Pseudomonad and Bie, a Pseudomonad 

 or Spirillum. The qualitative and quantitative inorganic require- 

 ments of these three organisms were studied previously and have 

 been reported. A salt solution containing Na\ K\ Mg^% Ca^^ and 

 Fe"^* salts and providing amounts of the ions known to be required 

 for growth by the organisms has been used as the diluent for the 

 various media. Trypticase medium was prepared by making this 

 salt solution 1 per cent with respect to B.B.L. typticase. S>Tithetic 

 medium was made by adding glucose and the three amino acids, 

 alanine, aspartic and glutamic acids to the salt solution. When 



