208 MANUAL OF MICROBIOLOGICAL METHODS 



more dilute, the agglutination becomes weaker. The *' titer " of antiserum 

 is the highest dilution at which clumping can be readily detected. Occa- 

 sionally, antiserums will be found which fail to agglutinate until the 

 liigher dilutions are reached. This range of more concentrated serum 

 where agglutination fails to occur is called the '^prozone." 



In instances where it is desirable to compare several antiserums or 

 several antigens, all determinations should be made at the same time. 

 Even in the hands of skilled individuals, the agglutination test may yield 

 variations of one tube among tests done on different days. 



Microscopic slide test. The microscopic method offers the advantage 

 of requiring only small amounts of serum, but at the sacrifice of some 

 precision. 



Antiserum dilutions and antigen are prepared as described for the 

 macroscopic tube test. A loopful of antiserum at a given dilution is 

 mixed with a loopful of antigen on a cover glass rimmed with petroleum 

 jelly, and over this is inverted a hollow ground (''hanging-drop") slide. 

 Controls of antigen plus saline and antigen plus preimmunization serum 

 are also included. The hanging-drop preparations are allowed to incu- 

 bate for 15-30 min and examined with low-power objective of the micro- 

 scope. With some antigens there is a tendency toward spontaneous 

 clumping; however, this should be detected in one or both of the control 

 slides. 



Macroscopic slide test. This procedure is most useful for the serologic 

 identification of cultures and is widely used for enteric bacteria (Edwards 

 and Ewing, 1955). When used for this purpose, it is essentially a quaU- 

 tative test. 



The antigen is prepared by emulsifying growth from a slant culture in a 

 droplet of saline on a glass slide. The suspension should be moderately 

 dense, but precise standardization is not necessary. A droplet of pre- 

 diluted antiserum is mixed with the antigen, and the slide is rocked back 

 and forth. With a good antiserum, agglutination should occur rapidly 

 and the clumps should be clearly discernible to the naked eye. 



The quantitative measurement of agglutinin. While the methods 

 described previously are entirely adequate for many studies of microbial 

 antigens, it is also possible to measure agglutinin with greater precision. 

 This is done by adding thoroughly washed bacterial suspension to meas- 

 ured volumes of antiserum and allowing the agglutinin to combine with 

 the bacteria. After an equilibrium is reached, the cells are washed free 

 of extraneous protein, analyzed by a micro-Kjeldahl procedure, and the 

 weight of agglutinin nitrogen calculated. 



For certain applications, such a method is greatly superior to the less 

 quantitative technics; however, the proper evaluation of all factors 

 involved may well require more information than it is possible to impart in 



