SEROLOGICAL METHODS vnu^AS 



pensions (antigens) of the various strains. If complement fixation 

 occurs (indicating that such a union has taken place) it is assumed 

 that the bacterium used as antigen in the test must have antigens in 

 common with the bacterium used to produce the antibody. 



The test requires careful attention to detail and the preparation of 

 a number of accurately standardized serological reagents (antigen, 

 antibodies or immune serum, complement, red corpuscles — usually 

 those of a sheep — and antibodies to red corpuscles, known as hemoly- 

 sin). A brief discussion of the methods of preparing these reagents is 

 given below, as well as the methods of making the test. If greater 

 detail is desired, it may be obtained by consulting standard text 

 books on Immunology and Serology. 



Materials Required: The glassware used for the complement fixation 

 test, as well as for other serological reactions, should be chosen with 

 care and kept scrupulously clean. Texts dealing with the Wasser- 

 mann reaction describe suitable test tubes and pipettes A con- 

 venient tube is one measuring 100X10 mm. The pipettes should be 

 serological pipettes: 10 ml. and 5 ml. pipettes graduated in 0.1 ml.; 

 1 ml. pipettes graduated in 0.1 ml.; and 0.2 ml. pipettes graduated in 

 0.01 ml. Suitable racks are necessary for holding the tubes. 



PREPARATION OF REAGENTS FOR BACTERIAL COMPLEMENT 

 FIXATION REACTION 



(a). Antigen. With 0.85% NaCl solution ("saline") wash off the growth from 

 a 24i-hour agar slant culture of the organism to be used. The amount of saline neces- 

 sary to make a satisfactory emulsion varies between 5 and 10 ml. depending upon 

 the heaviness of growth. Shake well.' Filter through cotton. Heat in a water bath 

 at 60°C. for 1 hour. Phenol, to make a 0.5% solution, may be added. This is not 

 advisable, however, as it increases the anticomplementary action. This suspension 

 may be kept for weeks in the cold without much loss of antigenic power. 



For comparative work, the density of the emulsion should be standardized by 

 nephelometric determinations or by a direct count of the number of organisms con- 

 tained in 1 ml., as it is important to use approximately similar suspensions. All cell 

 suspensions, including suspensions of bacteria, have the property of inhibiting the 

 action of complement. This non-specific property is known as their "anticomple- 

 mentary action." The titration of the anticomplementary action of the antigen is 

 given in a subsequent paragraph. 



There are a number of other methods of preparing bacterial antigens some of which 

 are better adapted to certain kinds of bacteria than the one given here. Extracts or 

 solutions of bacteria and organisms obtained from broth or special culture media may 

 be used. The Committee realizes the difficulties involved in prej^aring a satisfactory 

 antigen, but feels that a complete treatise on this important subject is outside the scope 

 of this Manual. The student must consult with instructors and refer to text bot>ks 

 for more definite suggestions. A good antigen is the most difficult of all the required 

 reagents to secure. 



(b). Immune Serum (Antibody).^ Immunize an animal against the organism to 

 be studied by repeated injections of the organism. Rabbits are especially suitable for 

 this purpose. Injections maj' be made into the marginal veins of the ears, iiitra- 

 peritoneally, or subcutaneously. For the injections, use light susi)ensions of the 

 organism in 0.85% saline, made by washing ofi' the culture from a 24-liour agar slant. 

 As little as possible of the medium should be added to the same with the organism. 

 Washed broth cultures can be employed in cases where it is desired to use an organism 



'A preferable procefhire would be the use of a shaking machine for two days; 

 centrifuge to give a clear extract. 

 -See page VI1I47-0-6. 



