128 BACTERIA IN THE SOIL 



was inoculated with a small portion of the soil under investigation, 

 and after four or five days sub -cultured on the same medium in fresh 

 flasks, and repeated half a dozen times. Now, as this inorganic 

 medium was unfavourable to the ordinary bacteria of soil, it was 

 supposed that after several sub-cultures the nitrous organism was 

 isolated in pure culture. Winogradsky employed for sub-culturing 

 upon a soHd medium a mineral gelatine, silica jelly.* Upon this 

 medium it is possible to sub-culture a pure growth from the film at 

 the bottom of the flasks in which the nitrous organism is first 

 isolated. In 1899 Winogradsky showed that the nitrous organism 

 (nitroso-bacterium) was able to grow in the presence of large amounts 

 of organic matter, and since that date Fremlin has carried this branch 

 of work to a further stage of advancement. He has shown that 

 cultures developed in inorganic solutions become eventually pure 

 cultures of this species of nitrifying organism, and when inoculated 

 into solutions containing small quantities of organic matter they were 

 able to oxidise the ammonia present. Fremlin has also demonstrated 

 that the nitrous organism grows. well on silica jelly and ammonia agar, 

 and colonies from these media transferred to beef-broth agar and 

 gelatine also grew well. From these experiments he concluded " that 

 the nitroso-bacterium grows well on any ordinary medium " but " that 

 in the presence of large percentages of organic matter the nitroso- 

 bacterium, although growing very profusely, loses for a time the power 

 of converting ammonia into nitrites."f 



The NitFie Organism. — ^It was soon learned that the nitrous 

 organism, even when obtainable in large quantities and in pure 

 culture, was not able entirely to complete the nitrifying process. As 

 early- as 1881 Professor Warington had observed that some of his 

 cultures, though capable of changing nitrites into nitrates, had no 

 power of oxidising ammonia. These he had obtained from advanced 

 sub-cultures of the nitrous organism, and somewhat later Wino- 

 gradsky isolated and described this companion of the nitrous 

 organism. It develops freely in solutions to which no organic matter 

 has been added ; indeed, much organic matter will prevent it growing.^ 



The temperature for incubation is 30° 0. Winogradsky con- 



* Two per cent, of dialysed silicic acid mixed with neutral salts and magnesium 

 carbonate in order to solidify it. 



t Jour. ofHyg., 1903, pp. 378, 379. 



J Compt. Bend., 113 (1891) p. 89 — Winogradsky isolated it from soils from various 

 parts of the world on the following medium : — Water, 1000 '0 ; potassium phosphate, 

 I'O; magnesium sulphate, 0'5; calcium chloride, a trace; sodium chloride, 2'0. 

 About 20 c.c. of this solution was placed in a flat-bottom flask, and a little freshly 

 washed magnesium carbonate was added. The flask was closed with cotton-wool, 

 and the whole sterilised. To each flask 2 c.c. of a 2 per cent, solution of ammonium 

 sulphate was subsequently added. Recently, the following medium has been used 

 for cultivation of the nitric organism : — Sodium nitrite, 1 -0 ; sodium carbonate, 1 '0 ; 

 sodium chloride, 0'5 ; potassium phosphate, 0"5 ; magnesium sulphate, 0'3 ; ferrous 

 sulphate, 0'4, in 1000 parts of distilled water. 



