456 INFECTION AND RESISTANCE 



fairly constant and very gradual rate of weakening, and is, com- 

 paratively speaking, stable. 



In the United States Hygienic Laboratory in Washington, ac- 

 cording to Rosenau, the recommendations of Theobald Smith are 

 largely followed in the production of toxin. The procedure is as 

 follows : 



The culture medium, "Smith's Bouillon," is prepared from 

 chopped beef from which fat and tendon have been cut out. This is 

 adjusted by phenolphthalein titration to 0.5 per cent, acidity. It is 

 then placed into Fernbach flasks and inoculated on the surface with 

 a Park-Williams bacillus ~No. 8. The flasks are incubated for 7 days 

 at 37.5 C. The reaction of the medium after such incubation is 

 determined, and flasks showing an acidity of 1.5 or over are dis- 

 carded. The usual reaction at the end of incubation is 0.6 to 0.8 

 per cent, acidity. This broth is filtered through Berkefeld filters or 

 porcelain candles. 



Toxin so prepared is now tested and its L and L + doses deter- 

 mined by the methods described above. Rosenau 20 states that poi- 

 sons are discarded as containing too large a proportion of toxon if 

 the difference between L and L + is greater than 15 M L D. The 

 toxin is now set aside in flasks for the process which Rosenau 

 calls "seasoning." At intervals of about a month it is retested 

 and finally it is found that the rate of toxoid formation decreases 

 and the poison reaches a period of equilibrium. It can now be used 

 for accurate determination of the L + dose, and this is done from 

 careful measurements on a large number of guinea pigs. 



Examples 21 of such measurements, abbreviated for the sake of 

 simplicity, are given in the following tables : 



Toxin Determinations of M L D or "T" 



Dose in c. c. Result 



0.03 = death in 1^ days 



0.02 = death in \y% days 



0.01 = death in 2 days 



0.008 = death in 3 days 



0.006 = death in 3}^ days 



0.005 = death in 4 days M L D 



0.004 = death in 6 days 



0.003 = death in 8 days 



. 002 = late paralysis 



0.001 = well in 16 days. 



Toxin Determination of L+ Dose 



1 Antitoxin unit -f- . 2 c. c. = 



1 Antitoxin unit -j- 0.21 c. c. = = L 



1 Antitoxin unit + 0.22 c. c. = local infiltration 



20 Rosenau. Hyg. Lab. Bull No. 21, April, 1905. 



21 Examples are taken from measurements reported by Rosenau, loc. cit. 



