118 THE ROYAL SOCIETY OF CANADA 



An observation which suggested the optimum salinity was that 

 the red colour which developed on 16 per cent, salt media most 

 nearly approached the colour found on the fish itself — on 14 per cent, 

 it was a paler pink, while on 18 per cent, it was more red. Con- 

 sequently, new medium was made up, using 3^ lb. shredded salt cod 

 to a litre of distilled water, 2 per cent, agar, and 16 per cent, solar 

 salt. The cod was digested overnight in water, and then cooked in 

 the Arnold steamer for twenty minutes or half an hour. After 

 straining this through a coarse cloth the salt and agar were added to 

 the broth, and the whole heated until salt and agar were thoroughly 

 dissolved, then tubed. At first very little of the cod was removed in 

 straining the broth, but as it made a very dense medium, and was 

 very awkward to get into test tubes, it was thought advisable to use 

 a finer strainer. This medium proved to be entirely successful for 

 the development of colour, and consequently has been used extensively. 

 Possibly a deeper colour developed when less of the codfish was 

 removed in straining, or the deeper colour which seemed to be present 

 may have been due to the density of the medium. However, as the 

 growth was quite as abundant, and the colour a yery satisfactory red 

 on the clearer codfish agar, and as mechanical difficulties were over- 

 come by its use, the fine straining seemed the better method. 



In order to obtain isolated colonies, plates were poured and 

 allowed to harden. The surface was then stroked with a camel's 

 hair brush charged with a heavy suspension of the red material in 

 16 per cent, sterile salt solution. The plates were incubated at 

 37° C. for at least four days before any red colour developed, and 

 sometimes as long as six or eight days. To prevent plates from drying 

 out 15 c.c to 20 c.c. of the fish agar was used for each plate. It was 

 only to be expected that brush plate No. I would be so covered that 

 there would be no discrete colonies, but the fourth, fifth or sixth 

 plates of each series were more successful. The best one varied to 

 some extent because of the fact that sometimes the suspension was 

 more heavily inoculated than at other times. As a rule, brush plate 

 No. 4 was the first of the series where the colonies were well separated, 

 though not infrequently No. 5 or No. 6 even, was the only one where 

 the colonies were sufficiently isolated to permit transfers from single 

 colonies. On these plates various coloured colonies developed, in 

 addition to the desired red ones, such as sulphur yellow, waxy yellow, 

 luteus, orange, Isabellinus, black, flesh, salmon pink, and two different 

 white ones — one a punctiform colony and the other a small round one. 

 All of these were sub-cultured and will be referred to later, but at 



