432 Major P. S. Lelean [April 23, 



1,000 million enteric organisms weighed only • 113 milligramme. A 

 nseful datum for men who are alarmed at the 1,000 germs is that 

 they only weigh ^^^ of a grain. 



(b) Xext, the technique adopted was to dilute the vaccine until 

 1 c.c. contained so few organisms that they might be counted as 

 they grew into separate colonies on solid media, on which 1 c.c. of 

 the diluted vaccine had been distributed. This means proved un- 

 satisfactory owing to the tendency of the organisms to form clumps 

 which however many organisms they contain, produce only a single 

 colony per clurhp. In any event the process of diluting 100 million 

 times does not lend itself to extreme accuracy. 



(c) The next procedure was that of counting the numbers of red 

 corpuscles and bacilli in a stained smear made from a mixture of 

 equal parts of blood and bacterial emulsion. As the blood is known 

 to contain, with remarkal)le constancy, live million corpuscles per 

 c.mm., the number of bacilli per c.mm. can be readily calculated by 

 their proportionate ratio to the corpuscles as counted under the 

 microscope. 



{d) The method now in use is the simpler and quicker one of 

 diluting the emulsion and making a direct count of the organisms on 

 a hsemacytometric cell under dark-ground illumination. 



The laboratory i^recautions concern us closely because questions 

 are often asked as to how the killing of all organisms in the emulsion 

 is ensured. 



The succession of precautionary measures speaks for itself, as to 

 the security afforded by this chain of defences. 



(«) Vegetative organisms are killed off by first heating the 

 vaccine at 53° C. for seventy minutes and adding to the resulting 

 emulsion, after it has cooled, O'-l per cent of lysol. Subjection to a 

 higher temperature would destroy the efficacy of the antigen. 



{b) Samples of the emulsion are then plated to test for sterility 

 under both aerobic and anaerobic conditions. 



(c) Absence of spores is finally proved by inoculating animals 

 with the emulsion and observing the effects for ten days subsequently. 

 In order to afford an ample margin for considerations of massive 

 inoculations, the animals get thirty .times the proportional dose given 

 to man. 



2. The injection. There are certain important precautions to be 

 observed when inoculating, but it is unnecessary to refer in detail 

 now to the routine which is adopted with such care and success. 



3. Besredka's alternative method. Before passing on, it is 

 interesting to note that Besredka advises the use of a true vaccine — 

 one containing living organisms. 



His emulsion consists of living culture sensitized by contact with 

 highly immune serum the complement of which has been destroyed 

 by heat, thus leaving only amboceptor. 



The rationale of the method is that when the sensitized livinsr 



