﻿346 MARSHALL. 



recognize the early stages of reaction by proving the presence of minute 

 traces of soluble anti-bacterial substances in diseased tissues. They then 

 went one step further and made a novel application of this principle, 

 applying it in the detection of minute traces of even unknown infections 

 agents in the infected body. 



They perform the experiment in testing for anti-tubercnlin and 

 tuberculin in organs as follows : 



A specific hemolytic serum is first obtained and this is used with the 

 corresponding anti-genetic blood corpuscles as an indicator of deflection 

 of complement ; for. if a specific serum and the corresponding red cor- 

 puscles are brought together and fresh serum is added, haunolysis occurs 

 if the fresh serum contains free complement, it does not occur if no 

 free complement is present. Haemolysis is therefore the index of 

 whether or not free complement is present in the fresh serum. If a 

 fresh serum which has been shown to contain complement loses its com- 

 plementary action upon being treated with a mixture of tuberculin and 

 anti-tuberculin, the complement is said to be deflected, provided suitable 

 controls indicate that its disappearance is directly due to the union 

 occurring between tuberculin and anti-tuberculin, and is not due to 

 the tuberculin alone, to the anti-tuberculin alone, or to any other agent 

 involved in the experiment. 



It having been established that the union of tuberculin and anti-tuber- 

 eidin deflects complement, any given tissue extract is tested for its 

 content in tuberculin by noting whether the suspected extract and a 

 stock preparation of anti-tuberculin will block the action of a serum 

 known to contain active complement. On the other hand, a given tissue 

 extract is tested for its content of anti-tuberculin by bringing together 

 a stock preparation of tuberculin, the suspected extract and the com- 

 plementary serum, and afterwards testing for ha?molysis. 



The technique of the experiment is elaborate, a large number of controls are 

 required and the test tubes must be scrupulously cleaned. The organs to be 

 tested for tuberculin are removed under aseptic precautions and ground in a mortar 

 with normal salt solution containing 0.5 per cent of carbolic acid. Five cubic 

 centimeters of normal salt per 1 gram of organ are employed. The mixture is 

 shaken in a machine for twenty-four hours at room temperature, centrifugalized 

 clear of particles and the supernatant fluid drawn off and tested by mixing vary- 

 ing amounts with anti-tuberculin and fresh guinea pig serum. These three are 

 brought together in a test tube and kept for one hour at 37° C. Finally, the 

 mixture is added to test tubes containing 1 cubic centimeter of a 5 per cent 

 suspension of serum-free sheep's corpuscles and twice the dissolving dose of in- 

 activated rabbit's serum specifically haemolytic for sheep's corpuscles. The volume 

 of fluid in each tube is brought to 5 centimeters of salt solution. The test tubes 

 are placed in the thermostat at 37° C. for two hours, are kept on ice over night, 

 and in the morning are examined to determine the extent of haemolysis which they 

 mav exhibit. 



