476 BACTERIOLOGY. 



The bouillon prepared as above under (1), or the mixed 

 medium described under (2) is inoculated with the diph- 

 theria bacillus and set aside at 37. A scum or pellicle 

 forms on the surface within 24 hours, and, if this is shaken 

 down, another one will form. The absence of a pellicle in- 

 dicates a weak culture or an unfavorable medium. The 

 maximum toxicity is reached on about the 5th to the 7th 

 day. The culture should then be filtered, first through 

 paper, then through porcelain. This should be done under 

 pressure (p. 469). After the 10th day the toxin gradually 

 diminishes. The filtered toxin can be transferred to sterile 

 bottles, preferably of dark-brown glass; or, it may be 

 drawn up into sterile bulb pipettes (p. 457) which are then 

 sealed in the flame. Inasmuch as air tends to alter the 

 toxin it is advisable to fill the bottle or pipette. More- 

 over, the toxin should be kept in a cool, dark place. A 

 half per cent, of carbolic acid or of toluol can be added as 

 .a preservative. The strength of the toxin must now be 

 ascertained by inoculating a series of animals. 



Determination of the minimum fatal dose. For this pur- 

 pose a number of guinea-pigs are selected having about the 

 same weight (250 g.). Four of these animals should be in- 

 jected subcutaneously with iV, ^V, TT><), and ^ c.c. of the 

 filtered toxin, respectively. The small quantities are meas- 

 ured as indicated on p. 479. The animal may die in a few 

 hours or not until after the lapse of two or three weeks. 

 In the latter case, post-diphtheritic paralysis of the ex- 

 tremities is likely to be observed for some days previous to 

 death. The animal may be considered as having recov- 

 ered, when it increases in weight and has a normal tem- 

 perature. 



The first series of guinea-pigs serves to establish ap- 

 proximately the fatal dose. Thus, if the guinea-pig that 

 received ^V c.c. of toxin died whereas the one that received 

 rta c c. survived, it is evident that the minimum fatal dose, 



