1903.] 



On the Culture of the Nitroso-hacterium. 



357 



always been tested for the presence of oxides of nitrogen, and a 

 control set up when batches of the solution were inoculated. 



The presence of nitrites was judged by a solution of diphenylamine ■•■ 

 in sulphuric acid, Ilosvay's solution being used as a control when 

 necessary. 



When I commenced work I obtained cultures of the nitroso- 

 bacterium by inoculating ammonia solutions with small quantities, . 

 0*2 gramme or less, of various kinds of soil ; rich garden soil, humus, . 

 sand, &c. Tubes of ammonia solution so inoculated were kept at 

 room temperature and placed in a dark cupboard in order to avoid 

 exposure to light. 



The evidence of the growth of the nitroso-bacteria was found in the 

 conversion of the ammonia in the solution into nitrous acid. This is 

 at first a slow process and does not commence in the tubes for some 

 3 weeks as a rule; usually it requires another week or two to be 

 completed. 



I inoculated ammonia tubes with soil 43 times. Of these 70 per 

 cent, showed oxidation of the ammonia. 



Gelatine plates poured from these tubes showed moulds, yeasts,, 

 liquefying and non-liquefying bacteria, and also a micro-organism 

 morphologically similar to the nitroso-bacterium. 



1st Dilutions. — From the ammonia tubes which showed oxidation 

 sub-cultures were made in like media. Of these 77 per cent, showed 

 nitrites. This occurred in 8 weeks. 



2nd Dilutions. — From the first dilutions sub-cultures were made. 

 Of these 85 per cent, showed oxidation in from 2 to 3 months. 



3rd Dilutions. — From the second dilutions sub-cultures were made.. 

 All these showed formation of nitrite in one month. 



Uh Dilidions. — Sub-cultures from the third dilutions exhibited for- 

 mation of nitrite in 93 per cent, of the tubes inoculated. These, as a 

 rule, required 6 weeks before this was completed. 



5th Dilutions. — Sub-cultures were made from the fourth dilution 

 tubes. Of these 77 percent, showed the formation of nitrite; the 

 time required being 2 months. 



With regard to the microscopical specimens made from the several 

 tubes; the 1st, 2nd, and 3rd dilutions showed, associated with the 

 nitroso-bacterium, rod-shaped micro-organisms, in gradually diminish- 

 ing numbers in succeeding dilutions. The 4th and 5th dilutions gave - 

 almost pure culture of the nitroso-bacterium apparently. 



Ammonia Solution containing no Carbonate. 



A medium containing simply ammonium sulphate and potassium 

 phosphate was tried. 



The nitroso-bacterium was able to grow in this, and to produce- 

 nitrite ; but the micro-organism did not develop in sub-cultures. 



