178 ANTIGENS AND THE TECH NIC OF SERUM REACTIONS 
Physiological Salt Solution.— A solution is prepared of 0.9 per cent 
sodium chloride (chemically pure) in distilled water. 
Routine Diagnostic Test With Serum.— In this test three tubes are 
employed, each containing a different proportion of serum and antigen 
suspension. It is well to have everything arranged before mixing the 
antigen with salt solution for the test. The racks should be set up, 
tubes numbered, sera heated, and pipettes ready for measuring antigen 
suspension and serum. For measuring the 0.05 cc. quantities of 
antigen suspension, mark off these amounts on a 1 cc. pipette (gradu- 
ated to 0.01 cc.) with a wax pencil. For measuring the 0.025 cc. and 
the 0.0125 cc. quantities, use 0.2 cc. pipettes (graduated to 0.001 cc.) 
on which these amounts are also indicated with a wax pencil. 
Preparation of Standard Antigen Suspension.— Mix antigen with salt 
solution according to required titer. Thus, if the titer is 1 cc. antigen 
plus 1.1 cc. normal saline, mix the antigen as follows: (a) Measure 
1.1 cc. saline into a standard antigen suspension vial; (6) measure 1 cc. 
antigen into a similar vial; (c) pour the salt solution into the antigen, 
and as rapidly as possible (without waiting to drain the vial) pour the 
mixture back and forth six times to insure thorough mixing; (d) allow" 
the antigen suspension to stand for ten minutes before using. The 
suspension should not be used after thirty minutes' standing. One 
may mix more than 1 cc. of antigen with proportionally larger amounts 
of salt solution, but not less than 1 cc. This amount when mixed with 
saline will be sufficient for about 15 tests. Two cubic centimeters of 
antigen mixed with saline will be sufficient for about 35 tests. 
Measuring Antigen Suspension and Serum.— After the antigen suspen- 
sion has stood for ten minutes, shake it well and measure 0.05, 0.025, 
and 0.0125 cc. amounts for each serum, delivering the suspension to 
the bottom of the tubes. When employing the standard rack which 
contains 30 tubes, measure 0.05 cc. amounts in the tubes of the first 
row; 0.025 cc. amounts in the tubes of the second row and 0.0125 cc. 
amounts in the tubes of the third row. The serum should be added as 
soon as possible after the antigen suspension has been pipetted, to 
avoid undue evaporation from the suspension. When examining 
large numbers of sera, it is well for one worker to measure the antigen 
suspension, and for another to follow with the sera. Add 0.15 cc. 
serum to the 0.05, 0.025 and 0.0125 cc. amounts of antigen suspension, 
and shake the rack of tubes vigorously for a few seconds to insure 
thorough mixing of the ingredients. The rack can now be set aside 
until the remaining tests are ready for the regular three-minute 
shaking period. 
Controls. At least one positive and one negative serum control should 
be included with each series of tests. Every serum giving a positive 
reaction should be examined to demonstrate that it is free from red cells 
or foreign particles which might be confused with a specific precipitate. 
Dilute 0.1 cc. serum with 0.3 cc. saline. Shake well and examine for 
