Bacteriological Studies of the Fixation of Nitrogen. 37 
above solutions in the following amounts in which S represents the 
original strength: S, S-H2, S-H4, S-^-8. P 
. .,T h £ agar wa ? P laced ln 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 beyopd 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 Sh- 4 and Sh-8. S 
An inspection of the above table, No. 7, shows very clearly that the 
mann’r S Ttf CtorS ‘i t P ! gment production a « 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 transfen 
his 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 dirtv 
Tub , e reNo fr 3 and tH u St ° Ck CultUre ° f A ' chroococcum. In 
ure No. 8, there was a small amount of brown pigment at the bot- 
om of the streak, and in the water of condensation, the remainder of 
hh fr™p th - b !u g d ' rty ™ hlte ’ cuIture 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 
a te hro Y m° U eXCeptl0n ’ a11 the cultur « produced abundant choco- 
ate brown to black pigment on all the different agars except those lack 
mg mannite and sodium nitrate. I feel that wf are nofLgging 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 
of thedark ‘Y "“"f' E W3S the limitin & factor in the formation 
thekh-LLY C ° Y' i 1 3m n0t prepared t0 sa y> j»st now, whether 
the nitrate acts as a stimulant to growth, pure and simple, or whether 
- exercises an oxidizing function on certain bacterial products. 
, h T 1 ? 6 res “ lts of thls stud y were so striking and so self convincing 
iv rp 1 !tf Ve had SetS °- f the ™ lfures reproduced in colors as near? 
y like the originals as possible. These were made from 20 day cultures 
by Miss Palmer, the Station artist. The upper set in Plate T 
shows Cuhure 8, the lower, Culture 93. The lumbers 1,2, 3 etc on 
the tubes indicate the composition of the agar as follows: 
Lacking Ca SO. 5 . Lacking Na 2 SO. 
2. 
3 - 
4 . 
Lacking Na 2 COs 
Lacking Na Cl 
Lacking Na NOs 
6. Lacking Mg SO. 
7. Lacking K 2 SO. 
8. Lacking Mannite. 
