324 THE TUBERCLE BACILLUS 



Auclair extracted tubercle bacilli with ether and with chloroform. On 

 inoculation into the trachea of a guinea-pig, these extracts produced lesions 

 of tuberculous pneumonia. 



Borrel also extracted a toxic substance from tubercle bacilli with xylol. 



8. Koch's old tuberculin. 



The tuberculin prepared by Koch in 1890 by a method at first kept secret 

 is prepared in the following manner at the Pasteur Institute in Paris. 



A culture of the tubercle bacillus on glycerin-broth is grown in a flask. 

 (Bacilli of mammalian and avian origin yield the same tuberculin.) [Certain 

 experiments recorded by the English Commission however show that tuber- 

 culin of avian origin cannot be relied on to produce a reaction in animals 

 suffering from mammalian tuberculosis. ] The growth must be on the surface 

 of the broth. The film appears after incubating for 15-20 days at 38 C. 

 and is complete about the 40th day. 



The whole is sterilized at 110 C. for 15 minutes, then evaporated on a 

 water bath to one-tenth its volume, and filtered through filter paper. The 

 filtrate constitutes crude tuberculin. 



Tuberculin is a brownish syrupy liquid with a faint, pleasant, characteristic 

 smell. It has no definite composition but is a simple extract prepared from sterilized 

 cultures on glycerin-broth, and contains in addition to the products secreted by the 

 bacilli the substances originally present in the broth. The active principle has not 

 yet been extracted. Tuberculin is very resistant to heat but is destroyed at 150 C. 



Attempts have been made to purify the crude product : 



(a) On the addition of 20 volumes of strong alcohol a brown precipitate is thrown 

 down which contains the active principle and a number of other extraneous sub- 

 stances. Tannin, picric acid, metallic salts, ferrocyanide of potassium and acetic 

 acid also form an albuminoid precipitate which carries down the active principle. 

 Koch, Hunter and Klebs have failed in their attempts to purify this precipitate. 



(6) Koch precipitated crude tuberculin with three volumes of 66 per cent, alcohol 

 and obtained a flocculent precipitate which on drying formed a white powder. This 

 constitutes purified tuberculin : it contains numerous extraneous substances but 

 is a very toxic product and kills guinea-pigs when inoculated in doses of 1 mg. This 

 is a very expensive method of preparation, as nine-tenths of the tuberculin remain 

 in solution and are lost. Calmette precipitates crude tuberculin with 95 per cent, 

 alcohol. 



'Except for Calmette's ophthalmo-reaction no advantage is to be gained 

 by using purified tuberculin it has the same properties as crude tuberculin. 



1. The effect of Koch's old tuberculin on man and animals. 



1. Healthy (non-tuberculous) subjects. Crude tuberculin inoculated into 

 healthy animals in small doses has no untoward effect except, possibly, a 

 very slight rise of temperature. Guinea-pigs can be inoculated with 2 c.c. 

 of tuberculin without harm. Rabbits stand an injection of 5 c.c. of crude 

 tuberculin very well, there is a slight rise of temperature and a transitory 

 loss of weight but the animal quickly recovers. Cattle and dogs do not 

 react to doses of 10 c.c. 



In man an injection of 0'25 c.c. into a healthy adult leads to somewhat 

 severe symptoms : rigors, diarrhoea and vomiting with a rise of temperature 

 to perhaps 39 C. (Koch). As small a dose as O'Ol c.c. may produce a slight 

 rise of temperature. Man is therefore about 1000 to 1500 times as sensitive 

 to tuberculin as the guinea-pig. 



The toxicity of tuberculin can be considerably diminished by adding to it a calcu- 

 lated amount of anti-tuberculous serum (vide infra), the toxicity of the mixture is 

 then due to toxones. Tuberculin neutralized in this way gives no better results in 



