[HARRISON & KENNEDY] DISCOLOURATION OF CODFISH 117 



the entire surface of the fish, and even the salt crystals adhering to 

 the coloured parts were decidedly pink. Two pieces of fish, where 

 discolouration had started, were cut ofï and placed in moist chambers, 

 one kept at 22° C, the other at 37° C. In both cases colour spread 

 over the entire surface, the only difference being that at 37° C. the 

 colour was much deeper than at 22° C. Numerous attempts to 

 culture the organisms on artificial media, however, were, at first, 

 unsatisfactory. Ordinary laboratory media, such as nutrient agar, 

 sugar agars, nutrient gelatine, beef broth, sugar broths, cider, milk, 

 and potato slants in varying percentages of salt were inoculated 

 directly from the fish, and also from dilutions of the pink material 

 in 16 per cent, solar salt solution, but no growth developed. Fish 

 slants, similar to potato slants, were tubed in varying percentages 

 of solar salt — (2, 4, 6 to 18) sterilized, and then inoculated, one set 

 left at 22° C, the other at 37° C. At the latter temperature growth 

 was slow, no colour developing in less than a week, and at 22° C. 

 growth was still slower, and there was none earlier than two weeks. 

 Even at the end of this time no colour was present on fish in tubes 

 which contained less than 6 per cent, brine, and here colour was very 

 pale. As the percentages of salt increased, deeper colour developed, 

 so that on 16 per cent, it was rosy red, while on 18 per cent, it was a 

 more vivid red. The effect of temperature seemed to be a difference 

 more of degree than of kind, as tubes containing equal percentages 

 of salt showed a deeper colour when kept at 37° C. than when kept at 

 22° C. 



Fish agar was made up in the proportion of 100 grams salted 

 codfish to a litre of distilled water, 2 per cent, agar, and varying 

 percentages of solar salt. Using this medium, slants were inoculated 

 and three methods of plate culture tried: (1) plates made in the 

 ordinary way by inoculating melted agar, and pouring the plate; 

 (2) by pouring the plate, and, after media had hardened, washing 

 the surface with a suspension of the pink material in salt water; 

 and (3) by making streak cultures on the hardened agar with a 

 platinum needle, charged with the infected parts of the fish. No 

 growth appeared when the plates were made in the ordinary way, 

 nor when the surface was washed with a suspension. Streak cultures 

 on either fish agar slants or fish agar plates were only fairly successful, 

 for no growth developed on agar containing the lower percentages of 

 salt, and, on the higher percentages, when growth did appear, there 

 were no isolated colonies; so that it seemed impossible to get pure 

 cultures. 



