40 
Colorado Experiment Station. 
sodium nitrite. With the exception of the peptone, a solution of each 
was prepared containing the nitrogen equivalent of a ten per cent, 
solution of Na NCh. This was done so that the various agars would be 
comparable to the sodium nitrate series in point of nitrogen .content. 
'I'he percentage equivalents for the salts mentioned, corresponding to a to 
per cent solution of Na NOa are as follows: 
Asparagin 
NIL Cl - - - 
(NH 4 ) 2 SO, 
Na NO. 
- 8.8231 per cent 
- 6.2887 per cent 
6.5894 per cent 
8.1189 per cent 
The proteid (peptone) agar was made by adding to the stock glu¬ 
cose agar, standard nutrient broth, neutral in reaction, in the 
following proportions: 0.0, .1, .2, .5, .8, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 
8.0, 9.0, and 10.0 per cent. The other agars were prepared by adding to 
respective lots of stock glucose agar the above solutions in amounts 
corresponding to 0.0, .01, .03, .05, .08, 0.1, 0.3 and 0.5 per cent of 
Na NOs. For every 50 c. c. of the stock glucose agar the following 
quantities of these solutions were required to give the above per¬ 
centages: 0.0 c. c., .05 c. c., .15 c. c., .25 c. c., .4 c. c., .5 c. c., 
1.5 c. c. and 2.5 c. c. respectively. The six different agars were placed 
in test tubes using about 7 c. c. each, sterilized in the autoclave for five 
minutes at I20°C. and slanted. Stroke cultures were made on these 
employing cultures Nos. 3. 8, 93 and our stock culture of A. chroo- 
coccum. 
At the end of eighteen days, there was no brown pigment pro¬ 
duced by any of the cultures on the proteid nitrogen agar containing 
beef broth, although there was luxuriant growth in all of the tubes. No 
pigment whatever, was made by any of the cultures on either the amido 
nitrogen agar, containing asparagin, or the ammonia nitrogen agars, 
containing respectively NIL Cl and (NILE S 0 4 . The growth ranged 
from slight to moderate. On the nitrite nitrogen agar, with cultures 
Nos. 8 and 93, we obtained a decided chocolate brown in the tubes 
which contained the NaNCh corresponding to .01 per cent NaNOs. All 
of the inoculations with these two organisms grew, but without color. 
Culture No. 3 gave a brown and chocolate brown pigment with NaNCh 
corresponding to .01 and .03 per cent Na NO a respectively. The stock 
culture of A. chroococcum grew very feebly on this agar as on the others, 
and produced no pigment. Control cultures on the stock glucose agar, 
to which no nitrogen was added, were carried along with the above. 
Growth took place but there was no evidence of any pigmentation. 
The results of this work indicate that in the presence of nitrates, 
A. chroococcum produces an intensely brown to black pigment; that 
nitrites in certain proportions, exercise this influence to a less degree; 
and that nitrogen as NIL Cl, (NH 4 ) 2 S 0 4 , asparagin, and peptone has 
no effect upon this function. 
