260 THE DIPHTHERIA BACILLUS 



Nicolle's method. Use beef killed the same morning. Mince the meat. Add 

 twice its weight of water and allow to stand for an hour at 10 or 12 C. Add 2 per 

 cent, peptone and 0*5 per cent, common salt. Heat to boiling point. Filter. Make 

 " sufficiently " alkaline. Heat to 120 C. for 10 minutes. Filter. Distribute in 

 sterile plugged vessels. Heat to 115 C. for 15 minutes. 



Grown in this medium for 7 days at 37 C. the diphtheria bacillus will yield, with- 

 out having a current of air passed over the culture, a toxin quite as powerful as 

 that obtainable by Roux and Martin's method. 



Mack's method. Use ordinary peptone broth containing in addition 10 per cent, 

 calcium carbonate. Distribute in litre or two litre flasks and autoclave. Incubate 

 after sowing for 4-6 weeks at 37 C. The product is said to be equally as toxic 

 as the nitrate prepared by Roux and Martin. 



Park and Williams' method. [vide ante Dean's method. ] These observers grow 

 the bacillus on ordinary peptone broth made alkaline to the extent of 7 c.c. normal 

 soda solution per litre (p. 31). 



Protein-free media. Utchinsky has shown that it is possible to get toxin by 

 growing the organism on media containing no protein and consisting merely of 

 salts and asparagin (p. 39). Hadley prefers to substitute glycocoll (1 gram per 

 litre) for the asparagin in Utchinsky's medium. 



These methods always yield a nitrate very weak in toxin. Nicolle, by adding to 

 Utchinsky's medium peptone Chapoteaut and gelatin liquefied by B. subtili-s, gets 

 a very powerful toxin. 



(b) The testing and storing of toxin. 



(a) The testing of toxin. The toxin content of the product manufactured 

 with the same bacillus under apparently identical conditions is subject to 

 considerable variation [vide Dean's results, p. 259]. It follows therefore that 

 every sample of toxin must be tested. 



To be suitable for the immunization of animals (for the purpose of preparing 

 a therapeutic serum) a toxin must kill a guinea-pig weighing 400-500 grams 

 in 48 hours or less when inoculated in quantities of 0*1 c.c. beneath the 

 skin. 



The toxins now used are often much stronger than this so that a dose 

 of yj^ or -0^(5- c.c. will kill a guinea-pig weighing 500 grams in 36 hours. 



For convenience of comparison Ehrlich has suggested the adoption of a 

 unit of toxin. A unit of toxin is the quantity necessary to kill a guinea-pig 

 weighing 300 grams in 96 hours ; and a toxin is said to contain 100, 200 etc. 

 units per cubic centimetre. 



For measuring small quantities of toxin it is convenient to make dilutions in 

 sterile water ; for example, 1 c.c. of toxin added to 9 c.c. of sterile water gives a 

 dilution of which 1 c.c. represents O'l c.c. of toxin. [Similarly 1 c.c. of No. 1 dilution 

 added to 9 c.c. of sterile water affords a dilution of which 1 c.c. represents O'Ol c.c. 

 of toxin. ] 



' (ft) To store toxin. For the purpose of storing toxin use sterile [amber- 

 coloured] bottles, which must be exactly filled, well plugged and kept in the 

 dark. Even under these conditions the toxin slowly loses its toxic properties. 



(c) Action of toxin on animals. 



The symptoms which follow the inoculation of diphtheria toxin into 

 susceptible animals are identical with those produced by the inoculation of 

 living cultures of the diphtheria bacillus. It is immaterial whether the toxin 

 be administered by inoculation beneath the skin, into the peritoneal cavity, 

 into the veins or into the brain. But given by the mouth toxin has no 

 effect whatever. 



On the guinea-pig. If a fraction of a c.c. of toxin (0' 1-0*25 c.c. the exact 

 amount depending upon the toxin content of the filtrate) be inoculated 

 beneath the skin of a guinea-pig an oedema rapidly forms at the site of 



