02 Papers from the Marine Biological Laboratory at Tortugas. 



Samples of sea-water were collected in sterilized stoppered bottles from 

 the surface and from depths of 3 and 6 fathoms from positions about 5 miles 

 from shore, where, from a consideration of the wind and tide, the water was 

 probably under truly oceanic conditions and unaffected by the neighboring 

 land. 10 c.c. of these samples were added to 1,000 c.c. of Gran's medium. 

 The cultures were kept in a moderate light and the 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 culture 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 reactions 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 72 hours many bubbles of gas were 

 being produced, and the nitrite and ammonia reactions were very slight. 

 After 86 hours the bubbling 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 gm. of potassium nitrate had been decomposed by 

 bacterial growth. If a further 0.5 gm. 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. 



An abstract of the behavior of a few of the cultures is given below. 



I. Sample collected 5 miles south of Port Royal, wind southeast, force 4, tide rising. Sample 

 taken from surface. 1,000 c.c. of Gran's medium was inoculated with 10 c.c. 

 of sample. 

 After 20 hours a slight cloud developed in the culture and faint nitrite reactions were 

 given. 



