Bacteriological Studies oe the Fixation oe Nitrogen. 37 
above solutions in the following amounts in which S represents the 
original strength: S, S-^-2, S-^4, S-t-8. 
The agar was placed in test tubes, using about 7 c.c. per tube, and 
sterilized for 5 minutes in the autoclave at I20°C. 
Streak cultures were next made on each agar of each strength, 
using cultures Nos. 3, 8, 93 and a stock culture of A. chroococcum. It 
will be remembered that No. 3 had retained its pigment producing 
power since isolation, No. 93 had lost it and No. 8 had shown little if 
any color beyond a dirty white. The results of these inoculations, when 
observed after fifteen days, are given in Table No. 7. 
The best growth as well as pigment was secured on those agars 
represented by the formulae S^-4 and S^8. 
An inspection of the above table, No. 7, shows very clearly that the 
two limiting factors in the pigment production are sodium nitrate and 
mannite. The growth obtained on the agar lacking mannite was so 
very slight that it was, indeed, difficult to say whether there was any 
actual growth or whether it was just the line of the original transfer. 
This was not the case in the agar lacking sodium nitrate. The line 
of inoculation was well defined in all and in two there was a moderately 
heavy growth. There was absolutely no brown color but only a dirty 
white with culture No. 3 and the stock culture of A. chroococcum. In 
culture No. 8, there was a small amount of brown pigment at the bot¬ 
tom of the streak, and in the water of condensation, the remainder of 
the growth being dirty white; culture No. 93 contained a few brown¬ 
ish specks in the water of condensation, which under ordinary circum¬ 
stances would have been overlooked; the streak proper was dirty white 
in color. Without exception, all the cultures produced abundant choco¬ 
late brown to black pigment on all the different agars except those lack¬ 
ing mannite and sodium nitrate. I feel that we are not begging the 
question when I make the statement that the reason we obtained no 
pigment in the absence of mannite was because we had no growth. To 
me, it was perfectly clear from the results of this series, that given 
a source of energy, the nitrate was the limiting factor in the formation 
of the dark brown color. I am not prepared to say, just now, whether 
the nitrate acts as a stimulant to growth, pure and simple, or whether 
it exercises an oxidizing function on certain bacterial products. 
The results of this study were so striking and so self convincing 
that I have had two sets of the cultures reproduced in colors as near¬ 
ly like the originals as possible. These were made from 20 day cultures 
by Miss Palmer, the Station artist. The upper set in Plate I, 
shows Culture 8, the lower, Culture 93. The numbers 1, 2, 3, etc. on 
the tubes indicate the composition of the agar as follows: 
1. Lacking Ca SO* 
2. Lacking Na 2 COs 
3. Lacking Na Cl 
4. Lacking Na NOs 
5. Lacking Na 2 SO* 
6. Lacking Mg SO* 
7. Lacking K 2 SO* 
8. Lacking Mannite. 
