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



obtained were to all intents and purposes pure cultures, showing pure 

 pictures of an oval, almost spherical organism, resembling the nitrosomonas 

 of Winogradsky, except that it seemed to be somewhat smaller in size. 

 It appeared constantly in the form of zooglcea embedded in the particles of 

 magnesium carbonate at the bottom of the culture tubes, and it stained 

 easily and well. The individual bacteria were often found to be well 

 separated in a culture, but an actively motile stage was not observed. 



These cultures, however, still gave a growth, though extremely slow, in 

 bouillon, and this consisted of the other quite inconspicuous organisms 

 present. By means of the dilution method, pure cultures were obtained 

 which yielded absolutely no growth in bouillon when preserved indefinitely 

 either at 37° or at the room temperature. These pure cultures were not, 

 however, robust, and they nitrified very feebly; attempts are now being 

 made to obtain vigorous pure cultures. 



Isolation of the Nitrate-prodticer. — The dilution method was also employed 

 for the isolation of the nitrate organism, the original material being a 

 culture obtained during the enumeration experiments (Table V), which 

 showed active oxidation of nitrites. A culture was separated which consisted 

 of the nitrate bacterium mixed with one other species, and the combination, 

 referred to in future as culture " d," formed a very interesting symbiosis. 

 Pure cultures were obtained from culture " d " by making surface plate 

 cultures, in great dilution, on nitrite agar.* These pure cultures showed a 

 small non-motile bacterium, agreeing in essentials with Winogradsky's 

 organism, though somewhat larger in size. It was a bacterium very thick 

 in comparison with its length, so that it often appeared to be almost a 

 coccus ; stains were badly taken up, and it frequently appeared imperfectly 

 and irregularly stained. These pure cultures rapidly changed nitrite to 

 nitrate, when growing in nitrite-containing medium,f the nitrite present 

 being sometimes completely oxidised in less than two weeks. Bouillon on 

 the other hand remained indefinitely sterile ; the tubes were kept under 

 observation for seven weeks without there being any sign of growth. 

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



t Winogradsky's nitrite culture solution was invariably employed, and the cultures 

 were tested from time to time for the production of nitrates. When time enough had 

 elapsed and all nitrite had disappeared, then, on testing the culture liquid, a negative 

 reaction with starch-zinc-iodide, and a positive with diphenylamine proved the presence 

 of nitrates. But if all nitrite were not oxidised, the remainder was decomposed by 

 evaporating to dryness with a little NH 4 C1, and the residue dissolved in water and tested 

 for nitrates with diphenylamine. This method has been shown to be quantitative when 

 such substances as sugar and peptone are present (Frankland, ' Journ. Chem. Soc.,' 1888), 

 and it is possible it might also prove a useful method of estimating nitrates in presence of 

 nitrites in sewage effluents. 



