1905.] 



with reference to the Purification of Sewage. 



251 



Culture " a," and all cultures showing production of nitrites* invariably 

 contained in quantity a small oval bacillus or coccus, which was recognised 

 as the nitrite-producing organism. 



Attempts to obtain a pure culture were further made with the use of 

 ammonium agar as medium,f but without success. The plate cultures 

 showed vigorous formation of nitrites,* but all nitrifying subcultures were 

 found to be impure. The employment of a similar medium composed of agar 

 and diluted cows' urine was equally unsuccessful. 



Ordinary gelatine plate cidtures were made and bouillon was inoculated 

 from the impure cultures ; none of the colonies separated from the former 

 were able to nitrify, although 40 were investigated. From the growths in 

 bouillon, plate cultures were also made on nutrient gelatine and agar, and 

 70 of the organisms separated were further investigated, but in no case did 

 nitrification occur. This seemed to show that the nitrifying organisms 

 in filters resembled those of Winogradsky very closely. Therefore, in order 

 to decide if the nitrifying organism was or was not able to live in the 

 bouillon, an arnmoniacal medium was directly inoculated from the growths 

 in bouillon. Usually there was no nitrite-production (e.g., Table VI, 

 culture " a "), and indeed the oval bacillus could in no case be traced in 

 the bouillon growths. In one instance, however (Table VI, culture " b "), 

 inoculation from a bouillon growth led to nitrification, but this property was 

 lost after a second generation in bouillon ; it therefore seemed probable that, 

 if the nitrifier had not been killed in the bouillon, it certainly had not been 

 able to multiply there. 



It thus was evident that, contrary to expectation, the nitrite-producing 

 organisms of sewage filters were also unable to grow upon media containing 

 organic matter ; recourse was then had to silica plate cultures, which were 

 made and inoculated according to the directions given by Omeliansky.| 

 This operation was accomplished with comparative ease if the original 

 sodium silicate was quite pure ; the study and isolation of the separate 

 colonies was, however, found to be exceedingly difficult. The sub-cultures 



* The test for production of nitrites was usually made by allowing a little of the 

 culture fluid, withdrawn with a sterile pipette, to drop into a small quantity of acidified 

 starch-zinc-iodide in a porcelain dish. This was preferred to the similar test with 

 diphenylamine, partly because of its specific nature, and partly because the ferric salt 

 present in the sediment of the culture-tubes also yielded a slight blue colour with 

 diphenylamine. 



t (NH 4 ) 2 SO„ 2-0 gr. ; NaCl, 2*0 gr. ; K 2 HP0 4 , 1*0 gr. ; MgS0 4 , 0"5 gr. ; MgC0 3 , in excess ; 

 agar-agar (purified by washing, Beyerinck's method, ' Centralbl. f. Bakt.,' vol. 19, 1896), 

 20 gr. ; distilled water, 1 litre. 



J See footnote, p. 250. 



VOL. LXXVII. — B. T 



