Bacterial Viruses in the Sea 351 



B. disciformus and B- mesentericiis (12, and Kriss, personal com- 

 munication ) . 



I reported in 1955 (17) the isolation of a phage from a 

 sample of sea water collected in the North Sea some ten miles 

 off the coast of Scotland, which was active against strains of 

 Photobacterium phosphoreum, and more recently (18) the isola- 

 tion from the same area of six phages active against several 

 species of bacteria previously isolated from sea water. 



It must thus be concluded that phages do exist in sea water 

 remote from terrestrial contamination, and that they may readily 

 be isolated by recourse to suitable techniques. As, however, at- 

 tempts to isolate such phages by various workers have not always 

 been successful, the methods used with success by myself, and 

 the experiments which led up to them, will be given in detail. 



METHODS FOR THE ISOLATION OF PHAGES 

 FROM SEA WATER 



It was expected that if phages were present in sea water, 

 they would be so in only low concentrations and that it would 

 thus be necessary to examine large volumes of sea water against 

 a variety of strains of bacteria. Also, as many marine bacteria 

 have cultural requirements of, for example, temperature and 

 salinity, different from those of terrestrial bacteria, it was con- 

 sidered necessary to pay careful attention to the cultural condi- 

 tions of temperature and salinity in the isolation methods. Finally, 

 it was thought that phages found in a particular area of sea were 

 most likely to be active against bacterial strains which were them- 

 selves present in that area, and consequently that bacterial strains 

 should be isolated from the area to be examined for use in the 

 subsequent phage isolation attempts. 



An enrichment or indirect method was first developed and 

 was as follows. To each of a series of sterile bottles were added 

 100 ml of a quadruple strength sea water-based nutrient broth 

 containing 4 per cent peptone and 4 per cent Lab Lemco, 300 

 ml of aseptically collected sea water and 8 ml of broth culture of 

 four different bacterial strains. In each experiment, 6 liters of 

 sea water were examined, against 40 bacterial strains. Half the 

 inoculated bottles were incubated at 20 C for two to three davs 



