494 G. H. DKEW. 



was experienced from this cause. No apparatus for obtaining deep 

 samples was available, but samples were taken from a depth of 

 six fathoms by means of a bottle from which the stopper was pulled 

 by a line, and then allowed to fall back into place. A measurement 

 of the rate of denitrification in fluid culture media inoculated with 

 samples of sea-water was made, but isolation of the bacteria on solid 

 media was not attempted. 



The following method was employed : — 



Samples of sea-water were collected in sterilized stoppered bottles 

 from the surface, and from depths of 3 and 6 fathoms, from positions 

 about five miles from shore, where, from a consideration of the wind 

 and tide, the water was probably under truly oceanic conditions and 

 unaffected by the neighbouring land. 



10 c.c. of these samples were added to 1000 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 colour produced in 

 this reaction was very intense, the culture became cloudy, and on 

 testing with Nessler's Eeagent 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 some- 

 what 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 gramme of 

 Potassium nitrate had been decomposed by bacterial growth. If a 

 further 0*5 gramme 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, or the 



