172 ANTIGENS AND THE TECH NIC OF SERUM REACTIONS 
pension; one part to one-sixth, and one part to one-twelfth part of 
suspension. 
Shaking of the Ingredients.— Agitation of the mixture of antigen sus- 
pension and syphilitic serum markedly hastens precipitation. In the 
case of strongly potent senmi a few seconds of agitation is sufficient 
for precipitation. Weaker sera require agitation for some minutes 
for complete precipitation. The optimum shaking speed for these sera 
was found to be about 275 oscillations per minute. Experiments 
further showed that a shaking period of three minutes meets the 
requirements for a practical test. 
In summarizing it appears that for optimum results in precipitation 
reactions in syphilis, it is essential that the element of dilution in a 
precipitation mixture be reduced to a minimum ; that the antigen-saline 
suspension be of high instability; that this suspension be mixed with 
serum in the proper proportions; that the final mixture be agitated to 
hasten contact between the particles. It is of interest to note that 
these requirements for precipitation in syphilitic sera correspond in 
some measure to the requirements of precipitation in colloidal systems 
in general. 
Apparatus.— Test tubes for performing this test are about 7.5 cm. in 
length and 1 cm. in diameter. 
Vials (with straight wall and flat bottom) for preparing antigen 
suspension are about 5.5 cm. in length and 1.5 cm. in diameter. 
Pipettes: 10 cc. graduated to 0.1 cc; 1 cc. graduated to 0.01 cc. 
and 0.2 cc. graduated to 0.001 cc. or 0.25 cc. pipettes graduated in 
0.0125 cc. 
Test-tube rack: made of sheet copper, 3 inches wide, 11| inches long, 
2f inches high. It consists of three shelves, the upper and middle ones 
containing 3 rows of holes, each of approximately | inch diameter. 
The center row holes are offset ^ inch. 
The shaking apparatus may be of any construction that will hold the 
test-tube racks employed. The required speed is 275 oscillations per 
minute with a stroke of H inches. 
The water-bath (56° C), centrifuge and centrifuge tubes may be of 
any make which will be found convenient in the particular laboratory. 
Re&gents. Antigen .—The antigen consists of an alcoholic solution of 
coordinated amounts of ether and alcohol extractives of powdered 
heart muscle, containing 0.6 per cent cholesterol. 
Powdered Beef Heart— A given amount of fat-free heart muscle is 
cut out from at least three beef hearts and passed four times through 
a meat grinder. The ground material is spread into a thin layer on 
a porcelain platter or glass plate and dried by means of one or two 
revolving fans. After six or eight hours, when the exposed surface is 
relatively dry, the material is turned over and drying continued over 
night. When the layer of beef heart is in the form of a dry plate, it is 
broken into small pieces and drying is continued until the material is 
