(So loc;al tisste reactivity 



iiiu Icoprotein extract oi B. coli was injected iutradeniially. This 

 was followed twenty lioms later by intravenous injection of an 

 active Vibrio clwlerae cidtiire in a qtiantity of i c.c. per kilo of 

 body weight. Strong reactions were elicited in the prepared skin 

 sites. Equally severe reactions coidd be ol)tained when B. coli 

 nucleoprotein was used for the intravenous injection. 



Andervont and Shear (1936) attempted to purify B. coli active 

 filtrates by the use of methods of Felton, Kauftmann and Stahl 

 (1935) developed by the latter authors for the precipitation of 

 the solidole specific polysaccharide from pneimiococcus broth cid- 

 tures, as follows: 



Phosphate was added to a filtrate of one w^eek old broth cultine 

 of B. coli. The active principles ^vere carried down with a basic 

 calciimi phosphate precipitate. In some cases in which precipita- 

 tion failed to remove completely the active principles from the 

 supernatairt fluid, more phosphate ^vas added and the pH read- 

 justed to 9.4 with lime water. The precipitates w^ere dissolved in 

 hydrochloric acid and the insoluble material filtered off and dis- 

 carded. Reprecipitation was then obtained by the addition of 

 sodiimi hydroxide to pH 9.5. The supernatant sohuion was inac- 

 tive. The precipitate 'vvas again dissolved in acid and again pre- 

 cipitated with sodiimi hydroxide. The reprecipitations were done 

 in order to remove the protein carried down in the original 

 precipitation. 



The final calcium phosphate precipitate was then treated Avith 

 acid and alcohol in order to remove the calcium. Enough hydro- 

 chloric acid was added to dissolve the precipitate and to bring 

 the solution to pH 2.0. The insoluble matter ^vas filtered off and 

 discarded. The active material was precipitated from solution by 

 addition of ethyl alcohol; the calcimn remained in sohuion. Solu- 

 tion in acid and precipitation Avith alcohol was repeated luitil the 

 precipitate ^vas free from calciimi. The fraction ^vas dissohed in 

 water, neutralized with sodium hydroxide and evaporated to dry- 

 ness. Finally, excess sodium chloride was removed by repeated 

 treatment Avith methyl alcohol. 



The original filtrate and the purified material were tested by 

 these authors for the phenomenon-producing potency. Each of 

 four rabbits received preparatory intradermal injections of three 

 dilutions of the original filtrate and of three dilutions of the puri- 

 fied fraction. Twenty-four hours later, two of the rabbits each 



