On the Influence of Ozone on the Vitality of some Bacteria. 61 



virulence; 0-25 c.c. of the broth culture, injected into the peritoneal sac, 

 killing the animal within twenty-four hours in typical fashion.* 



Experiment IV. — Although it seemed to have been conclusively 

 proved by the experiments of Ohlmiiller, already referred to, that ozone 

 was capable of considerable bactericidal action when the organisms 

 were suspended in certain fluids, we determined to carry out a single 

 experiment, using milk as the medium. We used milk because we 

 considered that it would, as containing a large quantity of organic 

 matter, test the bactericidal action of the gas severely. 



Five flasks, each containing 125 c.c. of milk, were prepared as fol- 

 lows : — 



Flash 1 contained sterilised milk which had been inoculated with a 

 culture of B. anthracis (sporing). 



Flask 2 contained sterilised milk which had been inoculated with 

 a non-sporing culture of B. anthracis. 



Flash 3 contained ordinary fresh unsterilisecl milk, to which a 

 quantity of a broth culture of B. prodicjiosus had been added. 



Flash 4 contained ordinary fresh unsterilisecl milk. 



Flash 5 contained a sample of commercial " sterilised " milk which 

 had " gone bad " owing to the presence in pure culture of an anaerobic, 

 sporing, butyric acid forming bacillus. 



A current of oxygen containing the same proportion of ozone as that 

 used in Experiment III was passed through the milk in each of the 

 flasks for a period of twenty minutes at the rate of 1*5 litres per minute. 

 Loopfuls of milk were then taken from each flask, transferred to 

 various culture media, and incubated under both aerobic and anaerobic 

 conditions ; the flasks with the bulk of the milk still remaining in them 

 were also incubated. 



In the result, it was found that the contents of flasks 1, 2 and 5 

 were sterile of bacteria. The milk used for flasks 3 and 4 was taken 

 from the same sample, and on incubation of the sub-cultures after 

 ozonisation a growth of a mould-fungus was obtained from each flask ; 

 from flask 3 a very free growth of the mould was obtained, but neither 

 B. procligiosus or any other bacterium ; from flask 4 a few colonies of a 

 coccus were obtained in both aerobic and anaerobic cultures in addition 

 to the mould which was present apparently in less quantity in the con- 

 tents of flask 4 than it was in the contents of flask 3. In the case of 

 the sub-cultures from flask 3, the growth of the mould was very rapid, 

 and soon covered the surface of the medium, and so possibly checked 

 the growth of the coccus which appeared on the sub-cultures from 

 flask 4, in which the mould growth was less abundant. 



A loopful of the milk used for flasks 3 and 4, taken before ozonisa- 

 tion and smeared over nutrient agar, gave, on incubation at 22° C, 



* We are indebted to Mr. Wood Smith, F.I.C., for assistance in the working of 

 the ozonising apparatus iised in this experiment. 



