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SEROLOGICAL METHODS 205 similarity in structure of the two molecules. In either case, it is usually- possible to remove cross-reacting antibodies by absorption. The heterologous antigen is mixed with the serum, allowed to react, and removed by centrifugation. Usually, considerable antibody for the homologous strain remains, whereas antibody for the absorbing strain can no longer be Purchase Tadalafil Online demonstrated. The technic has been widely employed for establishing the antigenic structure of bacteria, and a classic example is found in the Kauffmann- White antigenic classification of the genus Salmonella (Edwards and Ewing, 1955). To secure bacteria for agglutinin absorption, cultures are grown on a suitable agar medium. Since large quantities of cells are needed, the agar is dispensed in Roux bottles or petri dishes. The cells are washed from the agar with saline and killed by adding phenol to a concentration of 1 per cent. Heat killing may be used instead if the antigen under study is not heat labile or if it is necessary to destroy a heat-labile antigen. The cells are centrifuged from the saline and washed twice with addi- tional saline in graduated centrifuge tubes. The time and speed of centrifugation should be standardized to permit uniform packing. To 1 ml of packed cells is added 9 ml of antiserum. High-titered sera are diluted 1:5 or 1:10. The cells and serum are mixed and Purchase Tadalafil Online incubated at 37C for 2 hr mth intermittent shaking. Following this, the cells are removed by centrifugation and the serum tested for agglutinins against the absorbing strain. If agglutinins remain, the serum is absorbed again, using a second 1-ml portion of packed cells and the same incubation. When absorption is complete, the serum should fail to react with the absorbing strain but still react with the homologous strain unless the two cultures were antigenically identical. In setting up an absorption, one should include a control serum without cells Purchase Tadalafil Online to be incubated under the same conditions as the test. An absorption of the serum with the homologous strain may be included also. Absorptions are Purchase Tadalafil Online not always reciprocal, and if two cultures are to be compared, the specific antiserum for each should be absorbed with the heterologous strain (Krumwiede eial., 1925). Purchase Tadalafil Online Absorption can also be conducted with soluble antigens. One method is to add small amounts of antigen to an antiserum on successive days until Purchase Tadalafil Online a precipitate no longer occurs with that antigen. Another method is to locate the region of slight antigen excess, using the supernate test described under ^'precipitation.'^ A volume of antiserum and antigen calculated to give slight antigen excess is then mixed and incubated at 0-5C until precipitation stops. The precipitate is removed by centrifu- gation, and the supernate is tested with the absorbing antigen and the homologous antigen. 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 Purchase Tadalafil Online 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 Purchase Tadalafil Online 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. Purchase Tadalafil Online If grown on agar, the cells are Purchase Tadalafil Online 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 60C for 1 hr. In preparing antigens of the Salmonella ''O" variety it may be desirable to heat at 100C 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 37C 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. SEROLOGICAL METHODS 207 Tube agglutination test. Serum is diluted in a series of 10- by 75-mm test tubes using a twofold dilution series as illustrated in Table 21. Into the first tube 0.8 ml of saline solution and 0.2 ml of antiserum are Table 21. The Tube Agglutination Test Saline, ml
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