1905.] 



ivilh reference to the Purification of Sewage. 



253 



Pure cultures were also obtained with more difficulty directly from less 

 pure material, by means of nitrite agar plates, but the organism isolated 

 was in every case the same. 



All attempts to isolate a nitrate organism by means of ordinary nutrient 

 agar and gelatine were unsuccessful. In no instance was nitrite oxidised 

 to nitrate by any organism separated on such plate cultures, though over 

 40 such organisms were investigated. 



Experiments with " Symbiotic " Cultures of the Nitrate-producer. — Although 

 nitrobacter, when alone, is incapable of growing in bouillon, it would appear 

 to be capable of surviving an inoculation into bouillon if not alone, but 

 growing with certain other bacteria. A very instructive set of experiments 

 was made with culture " d " (Table VII), in which this strain was inoculated 

 into bouillon through four generations. From each set of tubes nitrite 

 medium was inoculated, and it was found that the change to nitrate 

 occurred invariably in the tubes sown from the earliest bouillon generation, 

 and in two instances also from those sown from bouillon of the fourth 

 generation (Table VII, d 3 and rf 4 ). The quantities inoculated were large, one 

 or two drops, but it is impossible to believe that nitrobacter would still be 

 present in a fourth generation if no multiplication had taken place in the 

 bouillon. Pure cultures of the nitrate-producer showed no such effects ; 

 inoculated bouillon remained quite clear ; examined under the microscope it 

 showed complete absence of bacteria, and nitrite tubes inoculated from the 

 bouillon in no case showed any oxidation to nitrate. In Table VIII are 

 shown the results of further experiments in which three pure and four mixed 

 cultures were compared in this respect, and one is compelled to conclude, in 

 explanation, that the presence of the accompanying organism in some way 

 protects the nitrate bacterium from adverse influences present in the bouillon, 

 which it is unable to withstand if alone. Without further experiment, any 

 attempt to explain in what this action really consists must be pure conjecture, 

 but it is possible that the harmful organic substances present are in some way 

 altered by the accompanying organism, and it would be interesting to see 

 whether the nitrate organism in pure culture could thrive in bouillon 

 previously exhausted by its companion.* 



Phenomena which present an interesting analogy with these observations 

 are found in the case of certain anaerobic organisms, one instance of which 

 has been precisely investigated by Winogradsky, viz., that of Clostridium 

 Pasteur ianum.^ This strictly anaerobic species was found to be capable of 



* The experiment was not made in this instance because the cultures had then been 

 isolated a considerable time and their properties were enfeebled, 

 t ' Archives des Sciences biolog. de St. Petersb.,' vol. 3, 1895. 



T 2 



