452 
Journal of Agricultural Research 
Vol. VIII, No. 12 
filled to the top. After carefully draining the flasks the small amounts 
remaining in them were disregarded. The tubes were centrifuged for 
25 minutes at about 2,500 revolutions per minute. The sedimentation 
was perfect and the euglobulin was firmly packed at the bottom of the 
tubes. The supernatant liquids were poured into 100-c. c. volumetric 
flasks. They may be poured through filter paper to make certain that 
no particles are poured off; but this is not necessary, as the euglobulin 
is sticky and firmly adheres to the bottom of the tube, which may be 
inverted without loss of precipitate. These 100-c. c. flasks should be 
weighed when dry and graduated in whole cubic centimeters on the neck, 
as the volume of liquid poured off may be more or less than 100 c. c. by 
2 or 3 c. c. The volumes should be noted to be certain that they are the 
same for both the heated and unheated mixtures of the same serum. 
The saturated ammonium-sulphate solution used was neutral to 
alizarin sulphonate, and it had been filtered through cotton and hard 
filter paper. The specific gravities of the saturated, one-half saturated, 
and one-third saturated aqueous solutions of ammonium sulphate were 
determined with a Westphal balance and found to be as follows at 26° C.: 
1.250, 1.142, and 1.089. These figures were of value in calculating from 
the weight of the flask and contents the amount of supernatant liquid 
obtained. The supernatant liquid was used for the estimation of (1) 
pseudoglobulin, (2) albumin, and (3) these two together, in the form of 
total coagulable protein. 
The precipitated euglobulin in the centrifuge tubes was dissolved in 
water and transferred to 400-c. c. beakers. These were heated up to the 
boiling point to coagulate the euglobulin, which separated out in large 
flocks. The addition of acid was not necessary, although in some 
instances 1 c. c. of N/5 acetic acid was added to favor flocculation. 
The precipitates were then filtered on weighed papers, washed free from 
sulphate, washed with small amounts of alcohol and ether, dried to 
constant weight in the air oven at ioo° C., and weighed. 1 In only one 
instance was there any difficulty in securing flocculation—namely, in 
a heated diphtheria-euglobulin precipitate. When this occurs, there is, 
of course, a loss through the passage of unprecipitated protein into the 
filtrate, and generally the filtrate is very cloudy. That the result would 
be low in the case referred to (see Tables I and II) was noted before the 
determination was completed. The heated euglobulin apparently was 
different from the unheated. The latter dissolved readily in water, 
forming a clear solution, while the heated euglobulin dissolved much more 
slowly, forming a milky suspension which became almost water-clear on 
standing overnight. 
Table I contains the results for euglobulin obtained from 50-0. c. 
portions of serum. 
1 For details see Eichhom, Adolph, Berg, W. N., and Kelser, R. A, Op. dt. 
