38 
Colorado Experiment Station. 
By a system of elimination, we have shown above, that in the ab¬ 
sence of nitrates there is practically no pigment formation. I was in¬ 
terested next in knowing just how necessary the other salts were to the 
production of the brown color, and whether the nitrates alone might 
not give the desired result . In order to determine this last point, a 
stock glucose agar was prepared as follows: 
Stock Glucose Agar. 
Tap water 
Glucose 
Agar-agar 
1000 c. c. 
20 grams 
20 grams 
Glucose was substituted for mannite since two of my cultures 
produced pigment on the standard mannite agar, and it was determin¬ 
ed experimentally that if this substitution was made in the stock agar, 
practically no color resulted with any of the cultures. By doing this 
all brown pigment producing factors were eliminated, and I had a 
medium which would support growth and to which the limiting com¬ 
pounds could be added. 
A io per cent solution of Na NOs was prepared in distilled water 
and sufficient quantities of this were added to different lots of the 
stock glucose agar to give them a Na NCb content of o.o, _.oi, .03, .05, 
.08, 0.1, 0.3 and 0.5 per cent respectively. In a 10 per cent solution 
cf Na NCb, 0.1 c. c. contains .01 grams of Na NOs. In order to ob¬ 
tain the above percentages, the following amounts of this 10 per cent 
solution were added to respective 50 c. c. lots of liquified stock glucose 
agar: 0.0, .05 c. c., 0.15 c.c., 0.25 c.c., 0.4 c.c., 0.5 c.c., 1.5 c.c., and 2.5 c.c. 
The agar was placed in test tubes, sterilized for five minutes at I20°C. 
in the autoclave and slanted. Agar stroke inoculations were made 
on these with cultures Nos. 1, 3, 4, 8, 10, 93 and stock A. chroococcum 
Our results with the series were gratifying beyond expectation. 
At the end of fourteen days, we had secured either an intense chocolate 
brown or a black pigment with every one of our cultures on those 
agars which contained the Na NOs, but absolutely none on the control. 
The pigment varied in intensity with the amount of Na NOs pres¬ 
ent, the optimum quantity for the darkest pigment being between .05 
and .08 per cent. In the early growth of the cultures a very nice 
gradation could be seen in the intensity of the colors, beginning with 
none in the control, a light brown in the .01 per cent, and a shade 
darker in each tube as the amount of Na NOs increased until the deep 
chocolate brown or black was reached at .05 and .08 per cent after which 
the shade of brown became somewhat lighter and remained almost con¬ 
stant. With age, this gradation of color was lost, a'l tubes except 
.01 and .03 per cent, showing an almost uniformly dark chocolate brown 
or black pigment. Plate II, prepared from twenty day cultures, illus¬ 
trates culture No. 8 of this series. Beginning at the left hand side, 
the tubes contain 0.0, .01, .03, .05, .08, 0.1, 0.3 and 0.5 per cent of 
