SEROLOGICAL METHODS viit4,-9 



group agglutinins. In dealing with a bacterial division such as that 

 cited above, group agglutinogens and agglutinins are encountered 

 in addition to strain-specific agglutinogens and agglutinins. 



AGGLUTININ ABSORPTION 



Agglutinin becomes attached to bacteria which are mixed with 

 an homologous antiserum, and can be removed from the fluid by the 

 removal of the bacteria. This is known as the absorption of agglu- 

 tinin. Some inagglutinable organisms retain the capacity to link up 

 with the antibody (agglutinin) and hence, like agglutinated bacteria, 

 are capable of absorbing agglutinin. The absorption of agglutinins 

 with agglutinable and inagglutinable strains of bacteria has become 

 an extremely important serological procedure for determining 

 identity of bacterial strains and for establishing group relationshijjs. 

 The scope of this Manual does not permit consideration of all the 

 factors involved in this reaction nor the description of the several 

 technical procedures which have worked well in the hands of differ- 

 ent investigators. It is to be emphasized that highly significant 

 results in pure culture studies can be obtained by the application 

 of this method after the user has become thoroughly conversant 

 with the technic and is familiar with the conditions which influence it. 



Two principles govern the application of the test for the absorption 

 of agglutinin. According to one principle, the ability of individual 

 strains to absorb agglutinins from type antisera is tested. A given 

 organism is considered to be identical with the type strain when it 

 completely absorbs the agglutinins from the type antiserum and 

 when the type organism completely removes the agglutinins from 

 the antiserum for the organism being studied. According to the 

 second principle, the agglutination of organisms by type sera from 

 which group agglutinins have been previously removed is tested. 

 Each method has its special advantages. The first method gives 

 the more precise results and will be described below, as it includes 

 the chief procedures which would be used in the application of the 

 second method. 



Procedure for Absorption of Agglutinin: At the start, the agglutinating antisera are 

 prepared according to the method described. The antigens are prepared in the same 

 manner as those used in the agghitination test. Dense suspensions are used for the 

 absorption of agglutinins, while the usual type of suspension (0.5 on McF.arland scale) 

 is employed in the test? with the absorbed sera. 



To prepare the absorbing antigen, wash off the bacteria from agar slants or petri 

 dishes into a small amount of saline. Filter through absorbent cotton if necessary to 

 obtain a smooth suspension. Absorption is accomplished by adding the concentrated 

 antigen to serum diluted 1:20 or l:-iO and removing the bacteria by centrifugation 

 after a period of incubation at room temperature for one half hour or at 37°C for 1 hour. 

 The minimal absorbing dose of bacteria for a given volume of serum can be determined 

 by varying the absorbing dose and selecting the smallest one which completely removes 

 the agglutinins for the absorbing strain. Successive absorptions with 2 or 3 doses are 

 more efficient in removing antibodies than a single absorption with the same total 

 amount of bacterial suspension. In identifying unknown strains, doses 2—1 times the 

 minimal dose are used. After absorption the serum is tested for its ability to aggluti- 

 nate the homologous strain, and any other strains of Iiactcria used in the study. These 

 agglutination tests are. set up with dilutions covering the original serum range and 

 eviending as low as 2.5% of the original titer of the serum. It is important to cover 

 the entire range of the titer of the scrum. At times pre-zone phenomena occur which 

 would lead to a false result if only a single dilution were used in the final test for 

 agglutination. 



