SEROLOGICAL METHODS vin„-7 



known conditions associated Avitli the composition of the bacterial 

 cell. Spontaneous flocculation can at times be avoided l)y proper re- 

 gard to the pH of the suspending fluid with the use of buft'er mixtures, 

 by passing the organism through several transfers immediately before 

 the final culture to be used in making the suspensions, and by the 

 growth of the organisms in media which favor diffuse growth. Wash- 

 ing the organisms in distilled water, ether, and chloroform, and taking 

 the supernatant fluid from heavy suspensions which have been 

 allowed to sediment are procedures which may make it possible 

 to obtain a smooth suspension of an organism which originally 

 flocculated spontaneously in saline. 



Procedure for Macroscopic Agglutination Test: The test is per- 

 formed by mixing a constant amount of the bacterial suspension 

 (antigen) with decreasing amounts of the antiserum, according to 

 the protocol in Table 1. 



TABLE 1 



COMPLETE AGGLUTINATION TEST WITH RESULTS IN A TYPICAL INSTANCE 



*The contents of each tube should be thoroughly mixed by sucking up the fluid in the 

 pipette and blowing it back into the tube several times before transferring the 0.5 ml. 

 to the next tube. After mixing, 0.5 ml. is discarded from tube No. 9. A one jnl. pipette 

 graduated to tip is the most convenient size. Tubes of about 10 mm. inside diameter 

 are suitable for this volume of fluid. 



Starting with the 1 :10 dilution of this antiserum, the series of dilu- 

 tions can be made readily in the same tubes in which the test is to 

 be done. Tube 10 is used as a control for the smoothness of the 

 bacterial suspension. It should be free from clumps. After the 

 antigen is added, shake well and incubate for 2-4 hours at .50 to 52°C.^ 

 After this period of incubation readings may be taken at once, or 

 the tubes may be allowed to stand overnight at room temperature 

 or preferably in the refrigerator. 



'The time and temperature of incubation is not the same for all bacteria. Agglutina- 

 tion proceeds more rapidly witli motile than with non-moti!e bacteria. Agglutination 

 of non-motile bacteria may be accelerated by shaking or by taking advantage of the 

 convection currents set up in the tubes where the level of the water is below the level 

 of the liquid in the tubes. 



