2 BACTERIOLOGICAL EXAMINATION OF WATER. 



in sterile test tubes and sterilised by heating to 65 C. for ten 

 minutes on four successive days ; (b) placed in test tubes and 

 sterilised in the autoclave at 120 C. for fifteen minutes ; (c) 

 filtered through a Berkefeld bougie into sterile test tubes. All 

 the test tubes were inoculated with one loopful of a forty-eight 

 hours growth of the B. typhosus, and then kept in the laboratory 

 cupboard at a temperature which varied between 16 and 22 C- 

 The day following one loopful was removed from each of the 

 tubes and plated out in gelatine; after seventy-two hours 

 incubation at 22 C. all the plates were crowded with typhoid 

 colonies. Four days later a loopful was again removed and 

 rubbed over an agar slope ; after twenty-four hours at 37 C. a 

 growth appeared which, when made into an emulsion, was found 

 to be completely agglutinated by anti-typhoid serum. At the 

 end of three weeks similar results were obtained. After two 

 months a loopful was removed and plated out in gelatine ; very 

 few colonies appeared, and those which did grow were black in 

 colour with granular contents, and the margin was only slightly 

 wavy. In fact they appeared so unlike the original colonies 

 that it was feared some pollution had occurred. However, one 

 of the colonies was fished and inoculated on an agar slope ; after 

 twenty-four hours a growth appeared which, when tested with 

 anti-typhoid serum, was completely agglutinated by the serum 

 diluted 1-1000. Besides the change in the appearance of the 

 colonies the B. typhosus appeared to have less resistance to 

 carbolic acid, for while the original culture gave a copious 

 growth in broth containing 0'05 per cent, carbolic acid after 

 twenty-four hours incubation at 37 C., the agar growth prepared 

 from the discoloured colonies showed no growth after twenty- 

 four hours incubation at 37 C. in 0'05 per cent, carbolic acid 

 broth, but after forty-eight hours there was a slight loss of 

 transparency, and after seventy -two hours there was a marked 

 growth. Experiments on the same lines were made with the 

 same sewage diluted 1 in 10 ; identical results were obtained. 

 These experiments appeared to show that the B. typhosus will be 

 found alive after sixty days immersion in both strong and diluted 

 sewage containing its usual toxines and salts, but freed from 

 other living organisms. The power of reacting to anti-typhoid 

 serum will still be present, but the colonies may present a dark 



