DEPARTMENT OF MARINE BIOLOGY. 137 



phenylene diamine hydrochloride to 25 c.c. of the culture. The production 

 of a brown coloration (due to the formation of Bismarck brown) is an mdi- 

 cation of the presence of a nitrite, and is an extremely delicate reaction. 



The formation of ammonia was similarly tested for by the addition of 5 

 c.c. of 10 per cent potassium hydrate and 5 c.c. of Nessler's reagent; the 

 white precipitate formed on the addition of the potassium hydrate does not 

 appreciably interfere with the test, though it renders it somewhat less deli- 

 cate. 



The cultures were kept in a moderate light, and the room temperature 

 varied from 25° to 31.5° C. The average temperature during the growth of 

 each culture was noted. 



In a typical culture made from surface-water, and for which the average 

 temperature was 29° C, the first indication of the formation of a nitrite, as 

 given by the metaphenylene diamine reaction, appeared after 27 hours ; after 

 38 hours the brown color produced in this reaction was very intense, the cul- 

 ture became cloudy, and, on testing with Nessler's reagent, slight ammonia 

 formation was apparent. After 48 hours the culture became very cloudy 

 and a scum of bacterial growth developed. The nitrite and ammonia reac- 

 tions remained unaltered. After 63 hours the nitrite reaction was somewhat 

 less marked, the ammonia reaction was unaltered, and bubbles of gas began 

 to appear. After ']2 hours many bubbles of gas were being produced, and 

 the nitrite and ammonia reactions were very slight. After 86 hours the bub- 

 bling had ceased, and no nitrite or ammonia was present in the culture. 

 Testing the culture for nitrates by the brucine and diphenylamine reactions 

 then showed that no nitrate was left in the solution. 



In the absence of a gas-analysis apparatus the nature of the gas evolved 

 could not be determined, but considering that it was non-inflammable, did 

 not turn lime-water milky, and that the nitrate originally present had been 

 destroyed, it seems strongly probable that this gas was pure nitrogen. Thus 

 in 86 hours 0.5 gram of potassium nitrate had been decomposed by bacterial 

 growth. If a further 0.5 gram of potassium nitrate were then added it was 

 rapidly decomposed, and this could be repeated many times until the other 

 constituents of the culture medium were used up. 



It was found that the rate of denitrification varied somewhat with the 

 temperature, and that in cultures kept at a temperature of between 10° and 

 12° C. no growth or denitrification occurred. The denitrification was always 

 more rapid in cultures from water taken from a depth of 3 or 6 fathoms 

 than from the surface. It was also more rapid with samples taken from 

 the thick muddy water of a mangrove swamp, where organic matter was 

 plentiful. 



The bacteria present in the cultures were very minute, actively motile 

 bacilli with rounded ends. 



The results of similar experiments performed with samples of water taken 

 from the English Channel near Plymouth, in the autumn of 1909, show that 

 there the process of denitrification is very much slower, and was never com- 

 plete at the room temperature. The first trace of the formation of a nitrite 

 in cultures in the modified Gran's medium, as detected by the metaphenylene 

 diamine reaction, occurred about the fifth day, and a large proportion of 

 nitrite always remained, even in the oldest cultures. 



Samples of water taken from the neighborhood of the Tortugas showed 

 about the same rate of denitrification in cultures as those from Jamaica. 



