88o Mockeridge. — Some Conditions influencing the Fixation of 
ever, that even after the expiration of eighteen days there is no loss of nitrogen, 
and the evidence goes to show that the organisms are capable of utilizing 
the last traces of carbohydrate supplied to them, and, under the most 
favourable conditions, are able to continue the process of nitrogen- fixation 
until the food supply is exhausted. Exactly why the organisms should not 
have shown greater activity as regards the fixation of nitrogen in the cul- 
tures in Ashby’s medium which had been left to grow for eighteen days is not 
clear, unless it be that the accumulation of certain products, either of partial 
decomposition of the mannite, or of excretion of the Azotobacter , or both, 
may have, in this rather less favourable medium, put an end to the nitrogen- 
fixing activity of the organism for the time being. Upon inoculation of 
a fresh portion of the culture solution with Azotobacter from flask No. 12 
a good and strong growth was obtained, so that evidently the organisms 
were in a perfectly healthy condition. Since these results have been 
obtained, an account of an investigation conducted by Koch and Leydell 
has come to hand, in which they state that nitrogen-fixation comes to 
an end after five to eight days, the remainder of the carbohydrate being 
utilized for respiration and other purposes. This would account for the 
presence of carbohydrate for eighteen days, during the last eleven of which 
no fixation of nitrogen took place in Ashby’s medium, but under the 
best conditions Azotobacter appears to fix nitrogen vigorously while the 
carbohydrate lasts. 
Three series of cultures were taken in order to determine whether, 
in the same percentage solution, the fixation of nitrogen is strictly propor- 
tional to the amount of carbohydrate consumed ; i. e. whether the increase 
in nitrogen consequent upon the consumption of 1 grm. of mannite is 
essentially twice that on \ grm., or four times that on J grm. Each 
series consisted of four cultures ; the flasks of the first series, numbering 
from 1 to 4, each containing 100 c.c. of 1 per cent. Bottomley’s solution 
and 100 grm. of sand to form a slope ; those of the second, numbering from 
5 to 8, each containing 50 c.c. of the same solution and 50 grm. of sand ; 
while those of the third, numbering from 9 to 12, each contained 25 c.c. of 
solution and 25 grm. of sand. When all had been inoculated, and Nos. 1, 
5, and 9 afterwards sterilized in the autoclave in order to serve as controls, 
all were incubated at 28° C. Nos. 4, 8, and 12 were tested from time 
to time for the presence of mannite, by evaporating a drop to dryness and 
examining for the crystals, and when one of these cultures no longer showed 
evidence of the presence of carbohydrate, the contents of the three remaining 
flasks of the series were analysed for their combined nitrogen, with the 
following results : 
