SULPHUR AND NITROGEN BACTERIA 227 



a result of unfavourable culture conditions, and endeavoured to aid the 

 access of air by employing larger and more extensive layers of fluid. He 

 obtained indeed a very marked increase in nitrification (to as much as 22-7 mg. 

 of nitrogen per day) on spreading a culture, which had previously oxidized 

 9 mg. of nitrogen, over a surface four times as great in extent. But the effect 

 he expected did not take place, for instead of a decrease he obtained an increase 

 in the amount of nitrite. The cause of the formation of nitrate and nitrite 

 obviously lay deeper, and WINOGRADSKY was soon able to prove that 

 the formation of nitrite takes place first, and that only after all the ammonia 

 is used up a further oxidation of the nitrite to nitrate follows. 



The next question to be answered was, did one and the same organism 

 carry out the formation both of the nitrite and of the nitrate very much as, 

 according to many authors, the acetic acid-bacteria first turn alcohol into 

 acetic acid, and then respire the acetic acid or was there a distinct organism 

 concerned in each of the two stages, one forming nitrite, the other nitrate ? 



There were certain indications that in all probability the latter view 

 might be the correct one, because when a new culture was taken from one in 

 the stage of most active nitrite formation, only nitrite formation went on in 

 it. It was certainly possible that the organism had become altered, and had lost 

 its power of forming nitrate, but it appeared more probable that by a lucky 

 accident only the nitrite organism had been transferred to the new culture, 

 and that in the previous experiments at least 

 two organisms were operating one after the < 

 other, by whose combined action the transforma- ^ 

 tion of ammonia into nitrate was effected. In his f < 

 experiments WINOGRADSKY had now advanced 

 conclusive evidence of the existence of two * 

 kinds of nitro-bacteria, one of which constructed 

 nitrites the other nitrates only. Both kept 

 to their specific functions, the one operating on Fig . 40< Nitro .bacteria : a, from Zurich; 

 ammonia, the other on nitrite ; other kinds of *, from Java ; c, from Quito, x 1000. 

 nitrogenous bodies, such as urea, asparagin, pro- Aftcr FISCHER (Vorles ' a> Bakt " * nd ed) - 

 teid, &c. (OMELIANSKI, 1899), cannot be nitrified at all, nor can the nitrate 

 organism oxidize either phosphorous or sulphurous acids (OMELIANSKI, 1902). 



Only a few words need be said as to the morphology of these organisms 

 {WINOGRADSKY, 1892). The nitrite organism is apparently similar in appear- 

 ance in all European countries, viz. an oval, at some period motile, bacterium (Fig. 

 40, ). Those obtained from Java and other non-European countries resemble 

 the European type (Fig. 40, b), but in America other forms, related to the coccus 

 have been found. The nitrate-bacteria, so far as is known, are short rods (Fig. 40, c). 



The nitrite- and nitrate-bacteria, as we have seen, behave very differently in 

 relation to the nitrogenous compounds which they can oxidize, but quite similarly 

 in relation to carbon. Both in fact not only require no organic carbon, but are 

 actually injured by it. Whence then do they obtain their carbon ? That is the 

 important question we have yet to answer. In his first experiments, in which 

 simple mineral solutions without any organic additions were used, WINOGRADSKY 

 took special care that all the materials used, the culture and vessels and fluids, 

 were absolutely free from organic impurities, and that no such substances 

 were permitted to enter from the air. In these solutions the nitro-bacteria 

 oxidized the nitrogen, and grew so markedly that he was able to determine 

 quantitatively and directly their gain in organic substance. Thus he found 

 that in four cultures carried on during about three months the organically 

 combined carbon amounted to (in mg.) : 



No. ii. No. 12. No. 26. No. 30. 



19-7 15-2 26-4 22.4 



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