210 MANUAL OF MICROBIOLOGICAL METHODS 



inhibited. Generally speaking, a satisfactory electrolyte level can be 

 assured by preparing antigen solutions in 0.9 per cent sodium chloride 

 solution (''saline"). This is approximately equivalent to 0.15M sodium 

 chloride. If the electrolyte strength is unknown, the sample should be 

 dialyzed against saline solution of known strength. The pH should be 

 maintained near neutrality, at least not lower than 6.5 or higher than 8.0. 



The antigen dilution method. Prepare a series of tenfold dilutions of 

 the antigen ranging from 1 : 10 through 1 : lO'^. Place 0.2 ml of each dilu- 

 tion in small serological tubes (10 by 75 mm), then add 0.2 ml of anti- 

 serum to each tube. The proper dilution of antiserum may be estab- 

 lished by preliminary trial, or undiluted serum may be used arbitrarily. 

 The necessary control tubes consist of antigen plus preimmunization or 

 ''normal'^ serum and saline plus antiserum. All the tubes are mixed by 

 shaking and placed at 37°C for 2 hr. The strength of the reaction may 

 be indicated by plus signs, faint cloudiness being recorded as + . The 

 tubes are placed at 0-5°C for 24 hr and read again. With weak anti- 

 serum, the test may be left in the cold up to 7 days; however, a preserva- 

 tive such as 0.01 per cent merthiolate should be present to prevent con- 

 tamination and "precipitates" should be checked microscopically to 

 ensure that they are not bacterial growth. When desirable, the test may 

 be repeated, using closer intervals between dilutions of the antigen. 



Since the antibody is held constant, this test is actually a titration or 

 measurement of the amount of antigen present and should not be used to 

 indicate the antibody content of a serum. For that purpose, the serum 

 dilution procedure is preferable, or if a higher degree of accuracy is 

 desired, the quantitative determination of antibody nitrogen is used. 



The serum dilution method. In a series of 10- by 75-mm test tubes 

 place serial twofold dilutions of serum ranging from 1:2, 1:4, 1:8 through 

 1 :4096. A volume of 0.2 ml is satisfactory for most tests. Add 0.2 ml 

 of antigen solution to each tube. The antigen dilution is held constant, 

 usually at a level found to be optimal in a preliminary antigen dilution 

 titration. Controls of antigen plus preimmunization serum and saline 

 plus antiserum (1:2) are included. The tubes are shaken and incubated 

 as in the antigen-dilution procedure. 



Since precipitation is inhibited by an excess of antigen, if the antigen 

 concentration chosen is too great, the serum will appear to have a low 

 antibody level. Martin (1943) suggested a preliminary antigen titration 

 at a constant serum level. The highest dilution of antigen which yields a 

 precipitate is then used as an antigen dose in titrating the serum. In the 

 hypothetical example presented in Table 22, the antigen dilution chosen 

 would be 1 : 10^ and the titer of the serum with this dilution would be 

 1 : 160. Had an antigen dilution of 1 : 10^ been chosen, it can be seen that 

 the serum would not have precipitated beyond a dilution of 1 :20. 



