1916] 
DUGGAR AND DAVIS—NITROGEN FIXATION 427 
6. Multiplication of the experimental error through taking 
an aliquot part of the fungous mat or culture solution and 
upon the determination from this basing a calculation for the 
whole. 
7. Inadequate controls. 
Analyzed chemicals may be obtained always, but these 
should be checked by running blank experiments. Standard 
acids and alkalis should be checked up by at least two methods. 
Nevertheless, slight discrepancy in the standard affects the 
actual rather than the relative analytical results, provided the 
same solutions are used for the nitrogen determinations 
whether they grow the fungus or are used as controls. Certain 
indicators have, in the presence of ammonia, what might be 
called a ‘‘running’’ end-point; that is, the color change occurs 
through a fairly wide range of H-ion concentration. After 
trying several indicators for this work alizarin red (Alizarin 
sulfonsáure Natrium, Merck) and cochineal were found to give 
the best satisfaction. The former in .1 per cent aqueous solu- 
tion was used. 
The error due to incomplete digestion or distillation, while 
easily guarded against, may sometimes occur, if care is not 
observed. It was the practice here to continue the digestion 
15 minutes after the mixture had become colorless. А full 
hour was given to distillation, since this interval proved en- 
tirely sufficient as shown by tests from time to time. 
In the critical review of literature it has been emphasized 
that many of those investigators reporting nitrogen fixation 
forthe fungi have limited their nitrogen determinations to 
aliquot portions of the culture solution. The total nitrogen 
was then calculated. Summarizing some of the points to 
which attention should be drawn, it is found that Puriewitsch 
(295), Saida (201), Ternetz (’07), and Froehlich (208) all 
filtered the solution from the fungous mat, determined the 
nitrogen from a portion of the solution, and calculated for the 
whole solution. The mat nitrogen was determined separately. 
Stahel (211), Peklo (713), and others, after separating solu- 
tion from fungous felt, evaporated the culture medium to 
small bulk (following the addition of acid) and determined the 
