[HARRISON & KENNEDY] DISCOLOURATION OF CODFISH 119 



present we shall continue to outline the methods adopted to culture 

 the organisms producing red colouration. 



From single red colonies transfers were made to sloped fish agar. 

 After four days at 37° C. red growth developed along the line of 

 inoculation — never spreading, except at the lower part of the slant, 

 where the condensation water gathered, and here red was noticed 

 around the edge. Microscopical preparations made from the growth 

 on these agar slants showed what was considered to be the presence 

 of two organisms^ — a rod and a torula or amoeboid form. Other 

 series of brush plates were made from the agar slants, and again rod 

 and torula-like forms were present. These experiments, which were 

 repeated very frequently from other samples of fish, gave similar 

 results, and as it was thought that two organisms were present, 

 further experiments were started in an attempt to separate the two 

 organisms. 18 per cent, solar salt had been the greatest amount of 

 salt used in making up media for the growth of red organisms, and 

 as it was thought possible that varying percentages of salt might 

 tend to separate the organisms, codfish agar was made up containing 

 varying percentages of salt, 5, 10, 15 and so forth, up to concentration 

 of 35 per cent. On salted codfish agar, to which no solar salt had 

 been added, absolutely no colour developed; on codfish agar con- 

 taining 5 per cent, solar salt, there was still no colour; on codfish 

 agar containing 10 per cent, solar salt the colour was very faint; 

 on 15 per cent, the colour was distinctly red, and on each succeeding 

 percentage the colour increased in intensity. The amount of growth, 

 from 10 per cent, up, also increased in direct proportion to the in- 

 creasing amounts of salt. As the agar dried out the salt crystallized 

 at the top of the slant — and even these crystals were coloured red. 

 From this series one may reasonably conclude that the organism or 

 organisms causing red discolouration are halophilic — apparently pre- 

 ferring a saturated solution. Instead of one of the organisms being 

 eliminated it was found that both forms developed on codfish agar 

 containing 35 per cent, salt- — -that is to say, a saturated solution. 



The suggestion that the two organisms might have different 

 thermal death points olïered another possible means of obtaining 

 pure cultures. A suspension was made from red growth on an agar 

 slant, in 16 per cent, solar salt solution, and heated in a water bath for 

 ten minutes at 50° C. With a 1 mm. loop drops were placed on the 

 surface of salted codfish agar plates. Other suspensions were made 

 in the same way, one heated for ten minutes at 65° C, a second for 

 ten minutes at 80° C, and the third for ten minutes at 100° C, and 

 plates were inoculated the same as the first one. Growth developed 



