206 MANUAL OF MICROBIOLOGICAL METHODS 



AGGLUTINATION 



When bacteria or other particulate antigens are mixed with specific 

 antibody (agglutinin), clumping or agglutination of the cells takes place. 

 The agglutination reaction may be performed in several ways. Slide 

 tests in which a drop of antigen suspension is mixed with a drop of anti- 

 serum on a glass slide offer the advantages of speed and simplicity with 

 some sacrifice in accuracy. Such slide tests may be read either micro- 

 scopically or macroscopically. Agglutinins may also be titrated in a 

 tube test, and this is preferable to the slide tests for some uses, since it 

 affords greater accuracy. Where even greater precision is required, the 

 quantitative determination of agglutinin may be used. In this pro- 

 cedure, the amount of antibody bound to the antigen is determined by 

 micro-Kjeldahl nitrogen analysis. 



The preparation of antigen. The microorganisms are cultured on a 

 suitable agar or fluid medium. If grown on agar, the cells are washed off 

 with saline solution, filtered through cotton, and killed by the addition of 

 formalin to a final concentration of 0.3-1.0 per cent or by heating at 

 60°C for 1 hr. In preparing antigens of the Salmonella ''O" variety it 

 may be desirable to heat at 100°C for 30 min to destroy the less heat- 

 stable ''H^' and '' Vi" antigens. Broth cultures may be killed by heat or 

 by the addition of 0.3-1.0 per cent formalin directly to the culture and 

 incubation at 37°C for 12 hr. After the cell suspensions are killed, the 

 organisms are centrifuged out and resuspended in saline. This washing 

 process is repeated three to five times, and the final saline suspension is 

 standardized. 



Standardization may be accomplished by nitrogen analysis, by direct 

 count using a hemocytometer or by measurement of turbidity with a 

 photoelectric nephelometer or colorimeter or for less precise work by 

 visual comparison with barium sulfate standards (McFarland, 1907). 

 The correct density of the antigen suspension is best determined by pre- 

 liminary titration ; however, for many systems, the arbitrary selection of 

 a density corresponding to McFarland tubes 1, 2, or 3 will yield satisfac- 

 tory results. After the initial antigen concentration is chosen, it may be 

 duplicated by comparison in a photoelectric nephelometer or colorimeter 

 or by determination of the nitrogen content. 



Certain antigen suspensions have a tendency to clump spontaneously. 

 This problem may be overcome in some cases by brief exposure to sonic 

 oscillation ; however, the treatment should not be so drastic as to disrupt 

 the cells. Other measures which may be effective include adjustment of 

 the pH and ionic strength of the suspending fluid or the addition of an 

 antigenically unrelated colloid hke gelatin (0.01-0.5 per cent) to the 

 system. 



