METHODS OF SERO-DIAGNOSIS. 207 



the methods of agglutination, precipitin-formation and comple- 

 ment fixation. 



General technique of these methods. 



In the performance of the agglutination test, in order to 

 demonstrate in the serum of the subject the presence or other- 

 wise of agglutinins formed in response to the presence in the 

 subject's body of the causal agent of the infection acting as an 

 antigen, there is necessary: — 



(i) The antigen, which is an emulsion of the organisms 



suspected of causing the infection. 

 (2) The serum from the subject. 



The emulsion of the organisms is sometimes made from a 

 fluid culture, but most often from a culture grown on some 

 suitable solid medium, agar, or some of its modifications, being 

 usually employed. A young culture of the organism growing on 

 the solid medium is scraped off and emulsified in physiological 

 saline solution, to which, in many cases, a bactericidal agent is 

 added, in order to kill the bacteria, since the reaction can occur 

 even when these are dead. The emulsion must, of course, be 

 uniform and contain no clumps, this latter being ensured by being 

 careful, in making the emulsion, to rub up the organisms well in 

 the fluid, to remove the clumps of bacteria, which is usually 

 effected by centrifugalizing the mixture for a short period, and 

 also to make it of a determined suitable density, which is usually 

 ascertained by comparison with a standard emulsion. 



The serum from the subject is obtained by withdrawing the 

 blood in a sterile manner from one of the veins — most often the 

 jugular — and when it has coagulated, the serum is collected. This 

 scrum is now, in varying dilutions, brought into contact with the 

 emulsion of the organisms, and evidence of agglutination may be 

 sought for either according to the microscopic or macroscopic 

 methods. In the first, the mixture of bacterial emulsion and 

 serum is examined as a hanging drop preparation under the 

 microscope, and the reaction may be clearly seen in the forma- 

 tion of clumps or groups of the bacteria in the drop of fluid, in 

 which they were in uniform emulsion at the commencement of 

 the reaction. 



The macroscopic method consists in placing the solution of 

 serum in different dilutions, together with the antigen, in small 

 test tubes, when the reaction manifests itself by the formation of 

 small flocculi, which fall to the bottom of the tube, forming 

 small heaps, and leaving the fluid in the tube quite clear instead 

 of turbid as it was when the mixture was first placed in the tube. 



In making the precipitin test for diagnosis of infection by a 

 specific pathogenic agent, it is necessary to have either an extract 

 of this agent or a filtrate of a liquid-medium in which it has been 

 growing. This is added to the serum for examination, the addi- 

 tion being made in a number of different dilutions in very small 

 test tubes, and the reaction consists in the production of an 

 opalescent appearance where the two fluids come in contact or 

 the formation of a precipitin. 



