272 MANUAL OF MICROBIOLOGICAL METHODS 



stand unmolested for 2 hr at room temperature or refrigerator (4°C) 

 temperature. The results in the antihemagglutination test will be the 

 reverse of those in the hemagglutination test. The tube in row 1 con- 

 taining the highest dilution of serum that completely prevents hemag- 

 glutination is considered the end point and is recorded as the anti- 

 hemagglutination titer. This figure means little unless it is known 

 against how much hemagglutinin the serum was employed. Tubes 1-5 

 inclusive in row 2 represent a final check on this matter under the con- 

 ditions of the experiment. If the hemagglutination end point is in 

 tube 1, you have used 1 hemagglutinin dose in the antihemagglutination 

 test; in tube 2, 2 doses; in tube 3, 4 doses; in tube 4, 8 doses; and in tube 5, 

 16 doses. Some workers like to correct the antihemagglutination titer 

 by multiplying it by the hemagglutinin doses used. This is permissible 

 only if a small number of hemagglutinin doses are employed. If large 

 doses, e.g., only slightly diluted, high-titered virus fluids, are employed, 

 the results are not comparable to those obtained by more dilute virus 

 even after correction involving the large number of doses. 



Tube 6, row 2, controls the greatest concentration of the serum used in 

 the test in regard to its ability to agglutinate the red cells by itself. 

 There should be no agglutination. Tube 7, row 2, containing only saline 

 and red cells, is a control on spontaneous agglutination of the cells. 



It is almost always desirable to run normal serum from the same 

 species from which the serum containing antibody was obtained. Since 

 some normal sera possess antihemagglutination properties, the titer of 

 the experimental serum may lose its significance. Heating either normal 

 or immune sera at 56° C for 30 min may increase its antihemagglutination 

 power. The extent to which this occurs varies considerably with the 

 serum obtained from different animals. 



The fact that hemagglutination is prevented by specific antiserum 

 permits the antihemagglutination test to be used in identifying the 

 unknown virus that possesses hemagglutinin. By the same token the 

 presence of specific antibody may be noted by using a known virus. 

 Acute and convalescent sera may be compared to detect a rise in anti- 

 body titer against a known virus for the serologic diagnosis of the disease. 

 The last technic, because of its importance, necessitates consideration in 

 some detail. 



Blood drawn during the acute phase of the disease is allowed to clot; 

 the serum is removed and stored in the frozen state. A second sample of 

 blood is drawn 10-14 days later and treated likewise. In some infections 

 weekly samples drawn for 3 weeks after the acute specimen are advisable. 

 The technic for running the hemagglutination test described above is 

 used, but it is important that the sera under comparison are run at the 

 same time, using identical reagents and conditions. Under such cir- 



