148 



LABORATORY COURSE IN SERUM STUDY 



II. DETERMINATION OF THE L+ DOSE OF TOXIN 



A dilution of a carefully standardized antitoxic serum is prepared 

 in sterile salt solution so that each cubic centimeter contains one unit 

 of antitoxin. A preliminary test with the dilutions spaced widely 

 should be carried out by the instructor to determine approximately 

 the L+ dose. A series of dilutions of toxin increasing by about T V 

 of the smallest amount should then be prepared, the total volume of each 

 dilution being brought up to 2 c.c. (The smallest amount is the amount 

 just below the minimal dose fatal when mixed with one unit of antitoxin 

 in the preliminary experiment.) 1 c.c. of antitoxin is then mixed with 

 2 c.c. of each dilution of toxin in a Rosenau syringe and allowed to stand 

 for one hour. The mixture is then injected into a 250-gram pig, as in 

 determining the MLD, and the syringe washed with 1 c.c. of salt solution. 



If the preliminary test has shown the dose to be between 0.2 and 0.3 

 c.c. the test should be set up as follows : 



If the pigs injected with the last two mixtures die on or before the 

 fourth day, and those injected with the first four mixtures survive but 

 possibly develop late paralysis, or if they die after the fifth day, the L+ 

 dose is 1.4 c.c. of 1 in 5 toxin, or 0:28 c.c. 



III. DETERMINATION OF THE ANTITOXIC VALUE OF AN UNKNOWN 



SERUM 



The L+ dose of a toxin having been carefully determined, this toxin 

 may be used for standardizing an unknown antidiphtheritic serum by an 

 analogous procedure. In this case the toxin is diluted so that 2 c.c. 

 contain precisely the L+ dose. Preliminary tests should be made to 

 determine approximately the unit. Then an accurate test is carried 

 out as follows : 



If the serum is found in the preliminary determination to contain 

 between 200 and 400 units per c.c. the dilutions are made as follows : 

 A stock dilution of 1 c.c. of serum plus 19 c.c. of salt solution is prepared 



