342 IMMUNO-CATALYSIS 



Heyningen separated quantitatively a- from ^-toxin present in a culture 

 filtrate by adsorption of the latter toxin on red blood cells. The a-toxin 

 content of the supernatant, as measured by the turbidometric method 

 (van Heyningen, 1941b), was unchanged, or slightly diminished, 

 but the hemolytic activity when measured in saline containing calcium 

 ion, was high. When the toxic filtrate was left with the red cells for 

 longer than five minutes, more a-toxin was adsorbed, but the adsorp- 

 tion of the a-toxin did not begin until all the ^-toxin had been adsorbed. 

 The hemolytic activity of the ^-free preparations was completely 

 inhibited by low concentrations of Glenny's a-antitoxin and unaffected 

 by high concentrations of his ^-antitoxin. 



b. Preparation of Toxins. MacFarlane and Knight (1941) used 

 the following method. The medium on which CI. welchii was grown 

 was composed of 3.3 per cent peptone (800 ml.), Na2S04 muscle- 

 extract (100 ml.) from horse and beef and 0.5 per cent glucose. It was 

 sterilized by filtration and adjusted to pH 7.6. Incubation for five to 

 six hours produced a maximum toxin concentration. 



Several preparations of dried toxin were made by 2/3 or full satura- 

 tion of the filtrate with ammonium sulfate. The average yield was I g. 

 per I. of medium. The M.L.D. of the toxin preparations used in this 

 study were as follows: Dried toxin— 0.008 mg.; glycerinated toxin— 

 0.0025 mg.'' 



c. The Lecithinase Activity of the Toxin. The activity of the 

 diluted toxin on lecithin was followed by allowing it to act on egg yolk 

 under standard conditions. The reaction was stopped by addition of 

 excess antitoxin and the turbidity measured in a colorimeter against 

 that developed simultaneously by a known amount of toxin under 

 the same conditions. The total trichloracetic acid soluble phosphorus in 

 the reaction mixtures was determined by preliminary ashing with 

 perhydrol and H2SO4, and colorimetric estimation of the inorganic 

 phosphorus. The results showed that by the action of the toxin the 

 original lipid phosphorus was converted into a trichloracetic acid- 

 soluble form. Most of the phosphorus originally present in the protein 

 component also became acid-soluble, although the protein nitrogen was 

 only slightly decreased. A comparison of the relative degree of hy- 

 drolysis and of turbidity showed that at pH 7.1 and 5.9 the rate of 



*Logan et al. (1945) described growth conditions of CI. welchii capable of produc- 

 ing 800-1000 L.D.50/ml. of type A a-toxin. 



