AGf~ICULTURAL RESEARCH, PUSA.

IMMUNOLOGICAL METlIODS 207
the method can he applied to small amounts,
but it has the disadvttntage that it can become
grossly inaccurate ill the hands of other than
skilled workers.
The preparation of the reagents is essentially the
same in the differcnt methods.
Two widely used examples of the different systems
of measurement are given in detail.
METHOD I. (reagents measured in ('aJaet pipetted
amounts)
ANTiGEN
(1) 20 grams of sheep heart-muscle, carefully freed
from fat and fibrous tissue, are finely ground with
sand in a mortar, and extracted for four days at
room temperature with 100 c.c. of 96 per cent. alcohol.
In this way lecithin and similar substances are
extracted from the tissue. The extract is filtered,
and pure cholesterol is dissolved in it to thc
point of saturation. The cholesterol acts by intensifying
the "antigenic" properties of the tissue
extract.
For the test, a suspension is prepared by adding
1 part of the alcoholic extract to 12 parts of nonnal
saline solution. In preparing the emulsion the
maximum turbidity should be obtained by running
the extract slowly on to the salt solution in a
cylindrical measure (or test-tube), and then mixing
slowly by rotation of the cylinder held in a slanting
position.
(2) 'rhc following alternative antigen is also recomrnended:
human hcart-mu8cle from the left ventricle
(eanse of death immaterial) is freed from visible fat,
minced finely and ground for a minute in a mortar
with absolute alcohol (1 gram of heart to !) c.c. of
alcohol) and dean sand. The mixture is shaken in a
shaking machine for I-tr hours and then nItered. '1'0
1·5 volumes of this extract is adde(l I volume of
1 per cent. eholcstcTol in absolute alcohol. For use,