1890.] The Nitrifying P?vcess and its Specific Ferment. 297 



soil, was carried on through, twenty-four generations, a minute 

 quantity on the point of a sterilised needle being introduced from one 

 nitrifying solution to the other. From several of these generations, 

 gelatine-plates were ponred and the resulting colonies inoculated into 

 identical ammoniacal solutions, to see if nitrification would ensue ; 

 but, although these experiments were repeated many times, on no 

 occasion were they successful. 



It appeared, therefore, that the nitrifying organism either refused 

 to grow in gelatine, or that the authors had failed to find it, or that, 

 growing in gelatine, it refused to nitrify after being passed through 

 this medium. 



Experiments were, therefore, commenced to endeavour to isolate 

 the organism by the dilution method. For this purpose a number of 

 series of dilutions were made by the addition to sterilised distilled 

 water of a very small quantity of an ammoniacal solution which had 

 nitrified. It was hoped that the attenuation would be so perfect that 

 ultimately the nitrifying organism alone would be introduced. 



After a very large number of experiments had been made in this 

 direction the authors at length succeeded in obtaining an attenuation 

 consisting of about too£ooo" °^ the original nitrifying solution em- 

 ployed, which not only nitrified, but on inoculation into gelatine- 

 peptone refused to grow, and was seen under the microscope to 

 consist of numerous characteristic bacilli hardly longer than broad, 

 which may be described as bacillo-cocci. 



These results are the more striking, for in the case of the two 

 other bottles similarly diluted, one had not nitrified, but on inocula- 

 tion into gelatine-peptone produced a growth already on the second 

 day, whilst the remaining bottle not only produced a growth, but 

 had also nitrified, thus clearly showing that the number of 

 organisms had been reduced to two, i.e., one which nitrified and did 

 not grow in gelatine, and another which had nothing to do with 

 nitrification, but which grew in gelatine. In the case where nitrifica- 

 tion took place and a growth also appeared in the gelatine-tube, it 

 was obvious that both the nitrifying and non-nitrifying organisms, 

 were present. These inoculation tests, together with the microscopical 

 appearances, were confirmed by repeated experiments with invariably 

 the same results. 



It is, however, very remarkable that, although this bacillo-coccus 

 obstinately refuses to grow in gelatine when inoculated from these 

 dilute media, yet in broth it produces a very characteristic growth, 

 which, although slow in commencing, often requiring three weeks 

 before it makes its appearance, is very luxuriant. 



The authors have, moreover, been successful in inducing nitrifica- 

 tion in ammoniacal solutions inoculated from such broth cultivations, 

 the extent of which has been quantitatively determined. 



