PLeuro-Pnrwmonia of Cattle. 171 



degrees of dilution with Martin's peptone bouillon (previously fil- 

 tered without the addition of serum). The broth and virus 

 were thoroughly mixed, and the mixture was then passed through, 

 a Chamberland F. filter at a pressure of 600 mm. of mercury. 

 After all the broth-virus mixture had passed through the filter,, 

 the requisite quantity of normal unheated ox serum was filtered 

 through the same filter into the broth-virus filtrate. The result- 

 ing filtrate was then placed in the incubator at 37°C. 



The whole operation of filtration was usually accomplished, 

 in under 1£ hours, but the time depended, of course, on the quan- 

 tity of material to be filtered. Usually not more than 250 c.c. of 

 broth-virus mixture, followed by the requisite amount of serum 

 was passed through the one filter at the one period of filtration. 



A certain amount of difficulty was experienced in demonstra- 

 ting the filtrability of the micro-organism, and a number of ex- 

 periments had to be made before it was accomplished. Previ- 

 ous workers 10 have established the fact that, unless properly 

 diluted in a definite proportion (not exceeding 2 per cent.), in 

 a medium without the addition of serum previous to filtration, the 

 virus will not pass through the filter, and a sterile filtrate will 

 result. Although this percentage was not exceeded in any of 

 my experiments (excepting for a special purpose in one series of 

 experiments), a sterile filtrate often resulted, and it would ap- 

 pear that the passage of this organism through Chamberland F, 

 filter candles, is not as easily accomplished as the statements of 

 previous workers, would imply. It is to be regretted that, owing 

 to the war, Berkfeld filters could not be obtained in Melbourne 

 for this experimental work, because the passage of the organism 

 through Berkfeld filters is apparently not attended with the 

 same difficulties as the passage through the finer-grained filters 

 such as the Chamberland F. 



In order to prove that absence of growth in the filtrate was 

 not due to changes in the medium brought about by the filtration,, 

 the filtrate of one experiment, which had shown no growth on 

 incubation was distributed into four sterile flasks, two of which 

 were then inoculated from a sub-culture of the organism of 

 pleuro-pneumonia, the remaining two flasks being kept as con- 

 trols 



In the two flasks inoculated with the sub-culture, the character- 

 istic opalescent growth was obtained after four days' incubation 

 at 37°C, the two control flasks remained sterile. 



10. Nocard, Roux, and Dujardin-Beaumetz. 



