352 Marine Microbiology 



and half for seven days at round C. After incubation, a sample 

 from each bottle was tested for the presence of phage active 

 against each of the inoculated bacterial strains by means of lysis 

 of the appropriate strain in a soft agar layer. Firstly, however, 

 it was necessary to remove completely tlie large numbers of 

 bacteria present in the sample without affecting unduly any 

 phage which may have been present, and consideration was given 

 to Fredericq's (8) chloroform technique for this purpose. A 

 preliminary examination of Fredericq's technique, however, 

 showed it to be unsatisfactory in the present circumstances, as 

 the chloroform which dissolved in the medium inhibited the 

 bacterial growth in the soft agar layer, and it was not practicable 

 to aerate all the samples as recommended by Adams ( 2 ) . Conse- 

 quently, the usefulness of two other organic solvents, toluene 

 and carbon tetrachloride, which are much less soluble in water 

 than chloroform, (0.057 ml/lOOg at 16 C for toluene and 

 0.077g/100g at 15-25 C for carbon tetrachloride, as compared 

 with 0.822g/100g at 20 C for chlorofonn, 15), was investigated. 

 Under the experimental conditions used, these two substances 

 were found to be more satisfactoiy than chloroform, in that 

 bacterial growth in the soft agar layers was not inhibited and 

 there tended to be less effect on the marine phages isolated 

 (Table 1) although it was subsequently found that two out of 

 the seven marine phages isolated, namely P/SW31 and P/SW34, 

 were inactivated by all three solvents. Carbon tetrachloride was 

 selected in preference to toluene because of its slightly greater 

 bactericidal effect during the shaking stage and because, as it 

 is heavier than water, sampling of the aqueous phase was 

 facilitated. The final method for the removal of bacteria was 

 as follows. The supernatant was decanted from the bottle and 

 centrifuged to remove most of the bacteria. A 50 ml amount was 

 placed in a bottle and 0.5 ml of carbon tetrachloride added. This 

 was shaken for one minute, allowed to stand for five minutes, 

 and the bacteria-free aqueous layer removed for phage assay by 

 the soft agar layer method given below. 



Where phages were isolated by the indirect method, there 

 was the possibility that they arose not from the sea water but 

 because one of the four bacterial cultures added was lysogenic. 



