146 



LABORATORY COURSE IN SERUM STUDY 



reaction of 0.5 per cent acidity to phenolphthalein. (Various modifica- 

 tions of this medium may be used. Smith recommends a broth which 

 has been made sugar free by inoculation with colon bacillus, and Rosenau 

 recommends the addition of 1 per cent glucose to the broth.) A strain 

 of B. diphtheriae known to produce strong toxin (preferably the Park- 

 Williams bacillus No. 8) is used for inoculation. Before it is planted on 

 flasks it should be transferred by the instructor daily through a series 

 of several broth tubes to obtain vigorous and rapid growth. The flasks 

 are incubated for seven days and one or two which show the best growth 

 filtered through a Berkefeld filter. The filtrate is stored in a sterile 

 dark bottle on ice until used. 



The class, working as a group, will carry out one or more of the 

 following determinations on one such toxin, each student injecting one 

 of the pigs in a given series. (In actual class work, in order to save 

 guinea pigs, usually only III, the determination of the antitoxic value 

 of an unknown serum, is carried out.) 



I. DETERMINATION OF THE MLD OF A TOXIN 



Guinea pigs are injected subcutaneously with varying amounts 

 of this toxin until the amount is determined which will regularly 

 cause death in from four to five days. For a satisfactory toxin the 

 MLD must be less than 0.01 c.c. The toxin is diluted in salt solution 

 so that the desired amount will be contained in 2 c.c. This amount is 

 injected subcutaneously, with a Rosenau syringe, 1 the needle of which 

 is inserted in the flank of the guinea pig and passed subcutaneously in 

 the flank of the pig till the point is near the linea alba before the diluted 

 poison is injected. After injection 1 c.c. of salt solution is placed in 

 the syringe without removing the needle and injected to wash out the 

 last traces of the poison. The following example of such a determination 

 is taken from Rosenau's toxin No. 5. The MLD in this case was .002 c.c. 



1 Ordinary Luer or other all-glass syringes are used in many laboratories. 



