450 
Journal of Agricultural Research 
Vol. VIII, No. 12 
When received at the Pathological Division these serums were in their 
native state, without preservative, and had not been filtered. Shortly 
after their receipt they were Berkefeld filtered and preserved with 0.5 
per cent chloroform in a refrigerator. 
With the analytical technic described in the previous publication by 
Eichhorn, Berg, and Kelser, 1 experiments were made on serums anthrax 
48 and 96, diphtheria 1, and tetanus 1. The experiments on anthrax 48 
and 96 were made in connection with the preparation of globulin for 
therapeutic use. After filtering the precipitated euglobulin, with or 
without heating, analyses were made of the filtrates. Obviously the 
more pseudoglobulin that is converted into euglobulin and precipitated, 
the less total protein should remain in the filtrate. A large number of 
analyses obtained on such filtrates, together with those obtained in the 
experiment in which diphtheria 1 and tetanus 1 were treated with 
ammonium sulphate, with and without heat, are omitted here because 
they were inconclusive. In analyses in which 10-c. c. portions of serum 
or filtrates corresponding to this amount are used, the unavoidable or 
unknown errors were great enough to obscure the effect of the heat treat¬ 
ment. 
HEATING THE SERUM 
In the following experiments the serums were heated in a water bath 
maintained at 61 0 C. The bath was heated by gas and was provided 
with a thermoregulator and an electrically driven stirrer. Numerous 
blank experiments (8) were made in which flasks containing water or 
one-third saturated ammonium-sulphate solution were heated. One 
standard thermometer was used for taking the temperature of the water 
in the bath, while two others were in the flasks being heated. The bath 
temperature varied from 61 0 C. by a few tenths of a degree, the bath* 
having been so adjusted that during the experimental heating of the 
serum the temperature did not rise beyond 61.2° C. nor fall below 60.5° C. 
This drop was caused by the introduction of the flasks at room tempera¬ 
ture, after which the bath temperature rose to almost 61 0 C. The bath 
contained about 15 liters of water. 
The serum was heated in 200-c. c. Erlenmeyer flasks in quantities of 
50 c. c. Two such flasks that were lightest and therefore thinnest were 
chosen from two dozen. These were always used for heating the serum 
mixtures. The object was to bring the content up to 6o° C., hold it 
there for exactly 30 minutes, and then rapidly cool it. These two 
flasks were provided with perforated rubber stoppers, each carrying a 
thermometer graduated in whole degrees and tested by the Bureau of 
Standards. The thermometer used in the water bath was tested by 
the Physikalisch-Technische Reichsanstalt, and was graduated in 
tenths of a degree. Experiment A was on anthrax 48 and 96, both 
1 Eichhorn, Adolph, Berg, W. N., and Kelser, R. A. Op. cit. 
