159 



Dunham's peptone solution : — Growth as in bouillon ; no indol produced ; 

 no pigmentation. 



Fermentation tests, from material grown on agar : — Observations were made 

 daily for four days with each of the following: dextose, inosite, glucose, 

 dextrin, saccharose, lactose, maltose, mannite, dulcite, arabinose, sorbite, adonite, 

 amygdalin, laevulose, salicine, erythrite, raffinose, inulin. In no case did fer- 

 mentation occur. 



Temperature reaction : — The optimum when grown on agar was at about 

 25° C, while growth was slightly inhibited at 37° C. 



Motility : — When examined direct from blood the organism was non- 

 motile, but after 18 to 24 hours' growth in +15 bouillon at 25° C. showed a 

 very feeble translatory movement when examined in hanging drop, and on the 

 succeeding day the motility was more pronounced, but was never active. Most 

 activity was obtained by using galactose broth as a medium. 



Spore formation : — In old cultures the organism assumed an ovoid form 

 suggestive of spore formation, but as the staining was quite uniform it probably 

 does not form spores. 



Inoculations : — A pure culture from an agar slope was diluted with sterile 

 physiological saline and standardized to approximately 1,000 million organisms 

 per C.C. A half C.C. of the mixture was injected into the peritoneum of a 

 guinea pig without any apparent effect during the succeeding two months when 

 the animal was kept under observation. 



Another culture was similarly treated and made up to 500 million per C.C, 

 T C.C. of the mixture being injected subcutaneously into a goldfish, which 

 died in four days, while the controls all remained alive. This experiment was 

 repeated on another goldfish, death occurring in four days. Post-mortem exam- 

 ination revealed conditions exactly similar to those seen in the naturally infected 

 fish from the Brisbane River, and gram negative diplobacilli were isolated in 

 pure culture from both of the experimental fish. 



An agar culture was scraped into a large bowl containing four healthy 

 goldfish, and in order to prevent the organisms from being washed away by the 

 flowing water and to allow some degree of acidity in the water and thus stimulate 

 stagnant conditions, the supply of fresh water (from the city water supply) was 

 withheld for two days, the maximum acidity reached being 4 per cent, (using 

 phenolphthalein as an indicator). On the third day the bowl was washed out 

 and the normal supply of oxygenated water restored. On the eighth day all 

 the fish spent most of their time at the surface, gulping air. On the fourteenth 

 day one of the fish died. Post-mortem examination revealed a general 

 septicaemic condition, and the gall-bladder was considerably enlarged. Blood 

 smears showed the presence of some gram-negative diplobacilli. Cultures were 

 made from the heart and spleen, and the organism recovered from them. The 

 remaining fish eventually became apparently normal. 



This evidence showed that when introduced directly into goldfish the 

 organism was lethal, but when infection occurred through the alimentary canal 

 or gills the effect was more slowly produced and might not be so virulent. 



The growth reactions of the diplobacillus showed that a high degree of 

 acidity in the water favoured its propagation. Consequently any conditions which 

 caused either stagnation of water (i.e., insufficient aeration) or a considerable in- 

 crease in the animal life of a mass of water /^.^., dwindling of a river into a series 

 of waterholes and the consequent increased density of the fish population in such 

 waterholes) would favour the growth of the organism. Then, again, the in- 

 creased density of the fish population would favour the outbreak of an epidemic 

 which would probably cause the greatest havoc amongst the least-resistant species. 



We have no proof that the organism found by us in the Brisbane River is 

 the same as that producing the epidemics in Western Queensland rivers, but 



