46 
Journal of Agricultural Research 
Vol. VIII, No. 2 
probably great. It is believed that the use of the centrifuge, as de¬ 
scribed below, for the purpose of separating the globulin precipitate from 
the “filtrate” is a marked improvement over the method of filtering. 
The electrically driven centrifuge used had a revolving head of 12 inches 
diameter and could carry eight tubes of slightly more than 100 c. c. 
capacity. To effect a good separation, it must be run for 30 to 40 minutes 
at about 3,000 revolutions per minute. 
In order that the results obtained may be comparable with those of 
Banzhaf, Gibson, and other investigators, the general method of analysis 
was similar to that used by them. The results contained in Table V 
and represented graphically in figure 1 were obtained by the following 
methods: 
Total coagulablE protein. —Ten c. c. of serum are pipetted into a 
400 c. c. beaker, 300 c. c. of distilled water added and heated not quite 
to the boiling point. Two and one-half c. c. of N/$ acetic acid (1.2 per 
cent) are added. This flocculates the proteins at once. On account of 
the higher protein content of the globulin preparations, less than 10 
c. c. may be taken, 5 c. c. being a convenient quantity. Less acetic 
acid should be used; 0.5 to 1 c. c. will flocculate the protein. After the 
flocculation has taken place, the solution is brought up to the boiling 
point for a minute, is allowed to cool, filtered on dry, weighed papers, 
then washed with small amounts of alcohol and ether, dried to constant 
weight at ioo° C., and weighed. (Further details are given on page 48.) 
This is a simple, easy determination, and duplicates seldom differ more 
than 3 or 4 mgm. The filtrates should be water-clear or nearly so. 
Total globulin. —With a pipette 10 c. c. of serum (or globulin prep¬ 
aration or other product) is transferred to a centrifuge tube having a 
capacity of about 105 c. c. (size of tube 165 by 30 mm.). Forty c. c. 
of water and 50 c. c. of a saturated ammonium-sulphate solution are 
then added and centrifuged for 40 minutes at about 3,000 revolu¬ 
tions per minute. If the centrifuge runs smoothly, the precipitated 
globulin will be firmly packed to the bottom of the tube, leaving the 
supernatant fluid clear or faintly opalescent. Generally the superna¬ 
tant fluid was not clear enough; it was then poured off into a second 
centrifuge tube and run again. On inverting the tube for complete 
drainage, none of the precipitate should be lost; it packs easily and 
firmly to the bottom. The volume of the supernatant fluid was noted, 
and the fluid was then rejected. This was done so that, if desired, cor¬ 
rections could be made for the fluid inclosed in the precipitate. The 
volume poured off was generally near 85 c. c., and varied between 80 
and 90 c. c., according to the amount of precipitate. The corrections 
were not calculated, as their use at this stage would have been prema¬ 
ture. The method can be still further improved before such corrections 
will be useful. 
The globulin precipitates in the bottom of the centrifuge tubes are 
dissolved by the addition of distilled water, transferred to 400 c. c. 
