ACTION OF DIPHTHERIA TOXIN. 69 



the bouillon culture of both diphtheria and tetanus. As most cultures show 

 only slight tendencies to toxin formation, the evidence of a virulent toxin 

 may necessitate a special strain of the bacterium. 



The length of time required by cultures for the production of moderate 

 amounts of toxins is by no means constant. With diphtheria this varies 

 from several days to 2 to 3 weeks. As a general rule, if toxin is hot liberated 

 within the first four weeks it will most probably not appear after that time. 

 On its appearance, it is isolated by filtering the bouillon culture first through 

 filter paper to remove the pellicle, and then through a bacterial filter to get 

 rid of the bacteria. A layer of toluol i to 2 cm. is added for the purposes 

 of sterilization and it is advisable to agitate the toxin and toluol thoroughly 

 every day to prevent contamination. 



It does not fall within the scope of this book to take up the various 

 methods proposed for obtaining and preserving the various toxins. It is 

 the object merely to review the details associated with their mode of action 

 and standardization. 



The first and most important member of this group is the diphtheria 

 toxin. 



The diphtheria toxin is first tested by subcutaneous injections into 

 Action of guinea-pigs 250 mg. in weight. The action of the toxin is entirely de- 

 Diphtheria pendent upon the dosage; the more toxin injected the more rapidly does 

 Toxin. death occur. This, however, is not to be taken in mathematically correct 

 proportions i.e., twice the dose does not produce the same action in 

 one-half the time. A certain period of time must always elapse before death can take place, the 

 minimum being about one day. The interim is known as the period of incubation and 

 it is the existence of this that goes to make one of the essential characteristics of a true 

 toxin. A toxin requires a definite period of time for its action to become manifest; and 

 even the largest dose of toxin cannot diminish the length of this period below a certain 

 minimum. On the other hand, the length of the incubation time can be increased by the 

 injection of a smaller dose, so that ultimately a dose small enough is obtained which is 

 not instrumental in producing death (Dosis subletalis). 



If a guinea-pig is inoculated with a quantity of toxin sufficient to kill it in three to four 

 days, nothing abnormal is evident the first day; various manifestations of illness, however, 

 follow soon after. 



Edema appears at the site of inoculation. The animal stops eating, sits in a corner, 

 and reacts poorly to sound. Gradually it becomes weaker, so that when placed upon its 

 back it does not resume its normal position; the temperature which at first rose some- 

 what, falls abruptly and then death takes place. 



At autopsy, a gelatinous and strongly hemorrhagic edema is found which starts at 

 the site of the injection. On opening the abdominal cavity one finds but very little peri- 

 toneal exudate, strongly injected vessels of the mesentery, and especially characteristic, 

 markedly reddened adrenal glands. In -the thorax are found bloody pericardial and 

 pleural exudates, and consolidated areas in the lungs. 



After the injection of smaller doses, edema likewise arises and becomes larger in 

 extent the slower the case progresses. Besides this, the animal loses in weight. With 

 sublethal doses, edema or infiltration is confined to the site of injection, and finally, with 

 the minutest doses, no edema occurs, but the hair falls out at the place of injection. 



