250 



Dr. H. Chick. The Process of Nitrification [Apr. 1, 



slowness, due to the sluggish growth of the organisms and to the extremely 

 small numbers of them introduced in the higher dilutions. The method 

 might, perhaps, give useful comparative results even without allowing the 

 maximum time for development in the subcultures, and the rate of growth 

 could be accelerated by a temperature of 28° to 30° C. 



The enumerations were made as follows : — The nitrate was successively diluted with 

 sterile water to a tenth degree six times ; of these six dilutions (viz., 1 in 10, 1 in 100, 

 1 in 1000, 1 in 10,000, 1 in 100,000, 1 in 1,000,000) 1 c.c. was used in every case for 

 inoculation into bouillon and into Winogradsky's ammonia and nitrite-containing media 

 respectively,* which were distributed in test-tubes, each containing about 5 c.c. The 

 ammonia tubes were subsequently tested for production of nitrites with acidified starch- 

 zinc-iodide solution, and the nitrite tubes for nitrates with diphenylamine sulphuric acid 

 (after evaporation to dryness with NH 4 C1 if any nitrite remained unoxidised). 



The numbers in which the nitrifying bacteria are present are surprisingly 

 large (see Table V), and it will be seen that there is no strict relation 

 between the numbers present respectively of nitrite and nitrate producers ; 

 but the latter would appear to be present generally in less amount even 

 when the filtrate shows complete oxidation of its nitrogen to nitrates. The 

 filtrates used were in all cases from the Munich continuous filters. 



Isolation of the Nitrite Producer— The isolation of a nitrite-producing 

 bacterium in pure culture was found to present considerable difficulty and 

 many unsuccessful trials were made. Attempts were first made to isolate it 

 directly from the coke of the filters by the method of dilutions. This 

 method, originally invented by Lister, was formerly employed by Waringtonf 

 and P. and G. FranklandJ for the same purpose, but with only partial 

 success. The method here employed was similar to that used by the 

 Franklands, except that much higher dilutions were made, and a larger 

 number of tubes (about 200) containing appropriate culture media§ were 

 sown with small amounts of liquid from the higher dilutions, bouillon tubes 

 being also similarly inoculated as controls. Kepeated attempts to isolate 

 from the coke of the filter were only partially successful. || A culture 

 was, however, obtained which was comparatively, but not absolutely, pure ; 

 this was used for further isolation experiments, and will be referred to as 

 culture " a." 



* Omeliansky, 'Centralbl. f. Bakt.,' 2 abt., 5, 1899. 

 t See footnote, p. 247. 



\ P. and G. Fraukland, ' Phil. Trans.,' B, vol. 181, 1890. 



§ Culture solutions used were diluted urine and Winogradsky's solutions. 



|| Starting from such very impure material, the dilution method does not give an 

 adequate return for the great labour it entails. The filtrates might have proved better 

 original material, as in them the nitrifiers sometimes predominated (see Table V). 



