262 THE DIPHTHERIA BACILLUS 



down in the precipitates which can be produced in the solutions in which it 

 is dissolved (Miahle's reaction). 



By adding a solution of chloride of calcium drop by drop to diphtheria toxin 

 phosphate of lime is precipitated as the result of the combination of the calcium 

 with the phosphates present in the liquid. This precipitate when collected on a 

 filter and washed, is very toxic ; the sub-cutaneous inoculation of a mere trace 

 of it rapidly causes death in a guinea-pig accompanied by a swelling at the site of 

 inoculation and the formation of a small greyish false membrane. The precipitate 

 is more toxic in the moist than in dry state. Nevertheless after desiccation it 

 retains most of its toxic properties, and in this condition it is more resistant to the 

 action of heat and can be raised to a temperature of 70 C. without losing any of 

 its toxicity ; and further a very small amount of the desiccated precipitate if inserted 

 beneath the skin will kill three guinea-pigs in succession if transferred from one 

 animal to the other as each dies. 



After filtering off the first precipitate the clear filtrate is still toxic, and precipi- 

 tates may be produced time after time ; every time the precipitate contains toxin 

 but in a progressively diminishing quantity, until finally the nitrate will no longer 

 produce a precipitate though it is still slightly toxic, as is shown by the fact that 

 when inoculated into guinea-pigs in very large doses, it sets up a chronic intoxication. 



Diphtheria toxin is soluble in water but again like the diastases is precipi- 

 tated by alcohol : but precipitation with alcohol diminishes its toxicity. 



To precipitate the toxin it is best to evaporate the filtrate first to one-tenth its 

 volume in vacua at 25 C., and then to add -to the liquid extract 4-5 volumes of 

 strong alcohol : the toxin mixed with numerous impurities is carried down in the 

 precipitate. Toxin may also be precipitated by ammonium sulphate. 



Toxin obtained by filtration can be dried in vacuo to the consistency of a 

 dry extract : this extract is soluble in water and contains the true toxin 

 mixed with a very large proportion of impurities. On dialysis the watery 

 solution quickly loses the mineral salts in solution, but the toxin is only 

 removed with great difficulty. This method may be used for purifying diph- 

 theria toxin. 



The toxic content of diphtheria toxin is considerable. 



1 c.c. of filtered cultures yields O'Ol gram of dry residue : thus, if 0'005 c.c. of 

 filtered culture suffice to kill a guinea-pig the lethal dose of the dry residue is f^ 

 gram (0'00005 gram) and of this small quantity the greater part consists of mineral 

 salts, peptone etc. This will give some idea of how infinitely small is the fatal 

 dose of the real toxic substance. 



Many chemical substances alter the toxic nature of diphtheria toxin. 

 For instance, toxin is destroyed by peptic ferments, while alcohol, acids, and 

 antipyrin diminish the toxin content. Oxidizing agents, again, are remark- 

 able for the capacity they exhibit of changing its character : thus, hydrogen 

 peroxide, alkaline hypochlorites and especially iodine and iodine terchloride 

 lower its toxicity considerably. The action of these substances is turned to 

 practical account in attenuating toxin which is to be used for the immuniza- 

 tion of animals. 



4. Vaccination. 



(i) In laboratory animals it is difficult to produce immunity by repeated 

 inoculation of very small doses of diphtheria toxin because the toxin accumu- 

 lates and the animals become cachectic and die. 



(a) G. Hoffmann was the first to successfully immunize guinea-pigs. He 

 inoculated them first with cultures attenuated by keeping, and later with 

 fully virulent cultures. Subsequently, Behring and Wernicke employed a 

 similar method. 



(b) Frsenkel immunized guinea-pigs by inoculating them sub-cutaneously 

 with cultures heated for an hour to 65-70 C. : altogether the animals 





