THE NATURE OF TOXIN 115 



production are always present when bacteria are permitted to 

 undergo lysis in fluid culture media, and that actual toxin pro- 

 duction depends first on the characters of the emulsified fat 

 antigen complex and second upon its colloidal arrangement. 



The mixtures of hemoglobin and broth, and of casein and 

 broth did not show toxicity whereas these emulsions containing 

 the fat antigen were toxic. 



Still another factor instrumental in the making of suitable 

 protein-antigen broth" colloids is that of the color of the broth. 

 The pigment of broth appears to be a distinct aid to emulsifica- 

 tion. Very Ught colored broth is a much poorer colloidal medium 

 than one which is dark. Besredka^^ noted that the filtrates of 

 his streptococcus lysin which had lost some of the color during 

 the filtration were impaired in hemolytic power. Connell and 

 Holly showed that a broth which had been passed through a 

 Berkefeld filter prior to the addition of artificial megatherium 

 antigen had practically lost its hemolytic power as compared 

 with the unfiltered broth containing the antigen. Very shght 

 alterations in broth lead to great colloidal changes. 



A very unportant factor also is the maturation of the protein- 

 fat antigen-broth colloids. A period of time of at least one 

 hour at 20°C. after the mixing of the ingredients is essential to 

 the development of maximum hemolytic and toxic power, after 

 which time, at 4°C. the activity remains stationary for a con- 

 siderable period and then gradually declines. Heat inactivates 

 these mixtures in a manner similar to true hemolysin and toxins. 



The toxicity of the artificial fat-protein colloids is shown in 

 the specimen protocol given in table 6. All the injections were 



TABLE 6 



Artificial colloid no. 1: 35 cc. of standard broth to which was added in divided 



doses 10 mgm. of typhoid protein dissolved in 5 cc. salt solution. 

 Artificial colloid no. 2: The same, to which the protein solution was added in a 



single dose. 

 Artificial colloid no. 3: The same, to which was added in divided doses 5 cc. of a 



salt solution emulsion of 10 mgm. of typhoid protein with 4.8 mgm. of K salt 



diphtheria antigen. 

 Artificial colloid No. 3A: The same, with 5 mgm. of typhoid protein. 



" Ann. de I'lnst. Past., 1901, 15, 880. 



