via^,-12 MANUAL OF METHODS FOR PURE CULTURE STUDY 



cases no demonstrable lysis occurs, although the three substances, 

 complement, antigen, an 1 antibody, become united. If no obvious 

 visible phenomenon accompanies the fixation of complement by a 

 bacterial antigen an 1 antibody, it becomes necessary to add to the 

 primary mixture of complement, antigen, and antiboiy, an indi- 

 cator capable of detecting whether the complement is fixed or is still 

 free. 



The only available indicator is an antigen-antibody mixture which 

 undergoes visible change in the presence of free complement and 

 shows no change in the presence of fixed complement. Such an 

 indicator is a mixture of red blood corpuscles and a specific antibody 

 for these. For convenience, sheep erythrocytes are most frequently 

 used for this purpose. An antibody, called hemolysin, or anti-sheep- 

 cell amboceptor, is prepared by immunizing an animal of a different 

 species (usually rabbit) by means of injections of sheep's washed 

 red corpuscles. This hemolytic amboceptor is a thermostabile anti- 

 body which retains its potency over long periods in suitable storage. 

 When the amboceptor in the serum of the immunized animal (rabbit) 

 reaches a suitable potency, the animal is bled, and the amboceptor- 

 containing serum is preserved for subsequent use. Amboceptor is 

 freed by heat from the complement in the serum of the immunized 

 animal which produced it. The combination between red cells and 

 specific amboceptor plus complement causes hemolysis, or laking of 

 the cells. 



A mixture of this antibody and the red corpuscles for which it is 

 specific is used as an indicator of the degree of fixation of complement 

 in any other antigen-antibody combination to which complement had 

 been originally added in the right proportions. If the complement 

 has been fixed by the formation of the first antigen-antibody com- 

 bination, none will be left to bring about hemolysis of the red cor- 

 puscles: they will not be laked. But if complement is still free, the 

 red blood cells will be hemolyzed when they are added with ambo- 

 ceptor to the original mixture. The first type of reaction, shown by 

 absence of hemolysis, is called a positive reaction, indicating the 

 specific union of the antigen and antibody being tested. The second 

 type of reaction, hemolysis or laking of the red cells, is called a 

 negative reaction, indicating that the original antigen-antibody 

 mixture did not result in a specific combination. 



Innumerable practical apjjlications of the complement fixation 

 test are made, especially in diagnosing various infectious diseases. 

 Such applications do not fall within the scope of this Manual; but 

 there are various ways in which essentially the same technic may be 

 adapted for use in the pure culture study of bacteria. Antibodies, 

 in general, are quite specific in their action; in other words, they will 

 ordinarily unite only with the particular antigen inoculated into the 

 animal in which the antibodies are produced, or else with some other 

 related antigen. For this reason, the complement fixation test may 

 be employed in pure culture study by producing antibodies to 

 the various strains under investigation (by means of animal inocula- 

 tion) and then determining the probable relationship of these strains 

 by noting the action between the known antibodies and the sus- 



