220 



MANUAL OF MICROBIOLOGICAL METHODS 



free from erythrocytes and lipid. Although a slight amount of hemolysis 

 does not harm the serum, excessive hemolysis should be avoided, since 

 this may make the serum anticomplementary. Contaminating bacteria 

 may also render the serum anticomplementary. 



It is necessary to inactivate complement present in the serum by heat- 

 ing in a water bath at 56° C for 30 min. Some serums may possess lytic 



* Various dilutions (see text). 



The dilutions shown for the antigen control and antiserum control may differ from 

 the values indicated, but each should be the lowest dilution in the series used. 



activity because of their content of natural antisheep hemolysin. If this 

 difficulty is encountered, the natural antibody may be absorbed out by 

 adding one drop of washed packed sheep erythrocj^tes per milliliter of 

 serum. The mixture is stirred with an applicator stick and allowed to 

 stand at room temperature for 30 min. The cells are then removed by 

 centrifugation. 



The complete test. If several tests are to be made with each anti- 

 serum, greater accuracy can be obtained by preparing the serum dilutions 

 in small flasks. Using separate pipets for each dilution, 0.5-ml quantities 

 are added to a series of test tubes as shown in Table 25. The dilutions 

 shown are arbitrary and may be changed if antiserum of greater or lesser 

 potency is being titrated. 



The optimal concentration of antigen, contained in 0.5 ml, is then 

 added to each tube, followed by the saline and complement as in Table 25. 

 The reagents are mixed by shaking, and the primary incubation is carried 

 out in the 37°C bath for 0.5-2 hours or at 0-6°C for 4-24 hr. The time 

 and temperature of the primary incubation may be varied to find the con- 

 ditions best suited to the system under study. In the serodiagnosis of 

 syphilis, the primary incubation is conducted in the refrigerator for a 

 period of 15-18 hr followed by 0.5 hr at 37°C. When using 37°C incuba- 

 tion, a fixation time of 1 hr is usually satisfactory and in any case should 

 not exceed 2 hr, since complement deteriorates much faster at this tem- 

 perature than in the refrigerator. 



