Mar. 19,1917 Transformation of Pseudoglobulin into Euglobulin 451 
being run at the same time. Experiment B was on diphtheria 1 and 
tetanus 1, both also run at the same time. On account of the breaking 
of a centrifuge tube containing a heated diphtheria mixture, the heating 
was repeated next day in duplicate, so that data were obtained on the 
heated and unheated single portions of anthrax 48, 96, and tetanus 1, 
and in duplicate portions of diphtheria 1. 
The procedure with a single serum was as follows: Into each of two 
Erlenmeyer flasks of 200-c. c. capacity, 50-c. c. portions of the serum were 
pipetted. One of these flasks was always a thin flask used specially for 
heating. To each flask 25 c. c. of water and 32 c. c. of saturated ammo¬ 
nium-sulphate solution were added, making a total volume of 107 c. c. 
This procedure was then repeated with the second serum, as it was 
found convenient to run two together. The two flasks not to be heated 
were stoppered and set aside. The two to be heated were stoppered 
with stoppers carrying the thermometers. In the meantime the water 
bath had been in readiness at 61 0 C., and the two flasks were introduced. 
All three thermometers were carefully watched and the temperatures 
recorded at intervals not exceeding five minutes. The temperatures 
inside the flasks rose from room temperature, about 27 0 C., to 56° in 
the first five minutes of the heating, then to 59 0 in the next five minutes. 
After 10 minutes' heating the temperatures inside the flasks were exactly 
at 6o° or below it by only a small fraction of a degree. The heating was 
then continued for exactly 30 minutes, during which time the tempera¬ 
ture inside the flasks did not exceed 6o°. It dropped a fraction of a 
degree for a few minutes on three occasions, but the bath temperature 
during this time was between 60.7° and 61 °, and consequently for all 
practical purposes the serums may be regarded as having been heated 
for exactly 30 minutes at exactly 6o°, with a preheating lasting 10 
minutes. At the end of the heating period the flasks were transferred 
to a pan containing cold water. This brought the temperature down 
to that of the room in about five minutes. It is believed that this is as 
severe heating as is necessary in the preparation of globulin. In prepar¬ 
ing large amounts of antitoxin, Banzhaf 1 brings the temperature of the 
mixtures up to just 6o° which required two hours' heating in a water 
bath kept at 66°. 
METHOD OF ANALYSIS 
The next step is the separation and estimation of the precipitated 
euglobulin. In the following scheme of analysis the precipitations of 
euglobulin and pseudoglobulin are made under exactly the same con¬ 
ditions as in the antitoxin (pseudoglobulin) preparation, and conse¬ 
quently the analytic data may be applied to the corresponding globulin 
or antitoxin preparations without error. All four mixtures were trans¬ 
ferred to 100-c. c. centrifuge tubes, which held about no c. c. when 
banzhaf, E. J., 1913* Op. cit., p. 115. 
