Jan., 1921] 
WANN — FIXATION OF FREE NITROGEN 
II 
microscopical examination. This precaution was therefore omitted in 1919, 
the microscopical examination being relied upon to detect contaminations 
not apparent to the naked eye. In all cases, however, such contaminations 
as occurred were perfectly evident from macroscopic examinations, the 
majority of them appearing soon after inoculation. 
After considerable prehminary work with total nitrogen determinations, 
the Gunning-Kjeldahl method was adopted for media free from nitrates, 
and in the presence of nitrates the Forster modification of this method was 
used. In the former method the digestion mixture consists of a solution of 
20 grams phosphorus pentoxide (P2O5) in 500 cc. sulphuric acid of sp. gr. 
1.84, 20 cc. of this solution being added to each culture flask. After the 
addition of 10 grams potassium sulphate to each, the flasks were heated 
slowly over a low flame. The presence of the agar made the digestions 
particularly trying, as it was necessary to watch the flasks constantly at the 
beginning of the process in order to prevent the contents from foaming up 
into the necks. It was found advisable to start with a very low flame and 
to shake the flasks occasionally until sufficient agar had gone into solution 
to allow the ready escape of bubbles from below. At this point the full 
flame was used, and the water boiled off vigorously until foaming began. 
The flame was then turned very low again for about 30 minutes or until the 
appearance of dense, white fumes, at which time foaming gradually ceased. 
The fire was then slowly increased to full capacity and the digestion con- 
tinued for 15 or 20 minutes after a clear liquid resulted. About I J hours 
were required for digestion after foaming ceased. In all cases the flasks 
at the end of the digestion were perfectly clean and the liquid was entirely 
transparent. 
The distillation was carried out in the usual way. About 150-200 cc. 
of distilled water was added to each flask when cool, the neck of the flask 
being thoroughly washed down in the process. The solution was made 
alkaline with 50 cc. concentrated sodium hydroxide, and, after the addition 
of a gram of granulated zinc, the flask was immediately connected to the 
still. The ammonia was distilled over through block tin tubes into a 500-cc. 
Erlenmeyer flask containing 30 cc. standardized tenth-normal sulphuric 
acid, diluted with enough distilled water to cover completely the end of 
the delivery tube. Standard traps were used between the Kjeldahl flask 
and the condenser. Distillation was continued until the contents of the 
flask began to "bump." About 125 cc. of water was distilled over in this 
process, and it was usually completed in about 40 minutes. During the 
latter part of the distillation the receiving flask was drawn away from the 
still sufficiently to uncover the end of the delivery tube, the inside walls 
of which were washed down by the remaining distillate. At the end of the 
distillation the delivery tube, as well as the inside wall of the receiving flask, 
was washed down with a small amount of distilled water. The excess 
acid was titrated against tenth-normal sodium hydroxide, using cochineal 
as an indicator. 
