VIII47-8 MAN UAL OF METHODS FOR PURE CULTURE STUDY 



Readings and Residfs: At the end of the period of incubation, 

 for the test to be satisfactory, the control tube should show a uni- 

 form cloudiness without sedimentation or flaking. A positive reac- 

 tion will vary in appearance with the tyipe of agglutination which 

 has taken place. With progressive dilutions the reduction in the 

 quantity of agglutinins is accompanied by less and less complete 

 agglutination. This is observed in the tube as decreased amounts 

 of sediment and less marked granulation or clumping. Conversely, 

 it is associated w^ith correspondingly increased turbidity of the 

 supernatant fluid and closer and closer approximation to the ap- 

 pearance of the control tube. The titer of the agglutinin is taken 

 as the highest dilution in which agglutination takes place. Certain 

 immune sera agglutinate only in the higher dilutions. The failure 

 of relatively concentrated serum to cause agglutination has been 

 designated by such terms as "prezone," "prozone", and "zone of 

 inhibition." Example: If in Table 1 (it is to be empJiasized that the 

 residts set down in this table are arbitrarily chosen to serve as an example 

 only) no agglutination resulted in tubes Nos. 1 to 3, partial clumping 

 in tube No. 4, complete agglutination in tubes Nos. 5 to 7, while in 

 the succeeding tubes the reactions were less and less complete, then a 

 zone of inhibition would be indicated in the concentrations of the 

 sera employed in tubes Nos. 1 to 3. When absence of clumping is 

 seen in one or more tubes other than at the beginning of a series it is 

 usually due to an error in technic. Zones of inhibition should always 

 be guarded against by using a sufficient range of dilution of the 

 antiserum, lest a false negative result appear. Great care in carrying 

 out the steps in agglutination technics is essential if accurate results 

 are to be obtained by such methods. 



The macroscopic slide agglutination test is performed on a glass 

 slide using a drop of serum dilution plus a drop of heavy bacterial 

 suspension (density of McFarland 7-8). Serum and antigen are 

 mixed over a surface of about 1 cm. diameter and mixing is continued 

 by rocking the slide. The degree of clumping is read after about 2 

 minutes. While the slide technic has advantages of simplicity and 

 speed, the macroscopic tube test provides a more reliable and 

 adaptable technic for pure culture study. 



For the complete identification of a bacterial strain, agglutination 

 to titer should be secured with an antiserum produced with organ- 

 isms of known type; and, furthermore, the organism in question, 

 if used in sufficient quantity, should absorb all of the agglutinins from 

 such an antiserum, thus leaving the antiserum devoid of agglutinating 

 power against both the organism in question and the organism used 

 to produce the antiserum. Partial agglutinin absorption may indicate 

 a degree of relationship. In order to establish the identity of two 

 bacterial strains complete cross-agglutination and cross-absorption 

 should take place between the two organisms and the two antisera. 



Attention may be directed here to the phenomenon of "group 

 agglutination" which results from common agglutinins acting on 

 bacterial species which are closely allied to each other. An example 

 is to be found in the colon-paratyphoid-typhoid-dysentery group. 

 The absence of exact specificity in agglutination reactions is due to the 



