Substances Produced in Bacterial Cultures. 53 



With Gram-negative bacilli like typhoid, we felt that we had 

 to be particularly careful to eliminate the extractive substances 

 spoken of as endotoxins. In consequence, we grew typhoid bacilli 

 on various media for the shortest possible periods, consistent with 

 good growth using filtrates of 4, 6, and 12 hour cultures. These 

 always gave definite sickness in rabbits intravenously injected, 

 when 4 or more cubic centimeters were administered, and in all 

 cases the incubation time and the symptoms were in every way 

 similar to those produced by streptoccus and influenza filtrates. 

 Comparison of such young 6 hour culture filtrates with the 

 filtrates of 6 and 10 day cultures showed that the 6 hour filtrates 

 made the rabbits quite as sick within the first 2 or 3 hours as did 

 the extraction posions from the old cultures. The symptoms were 

 somewhat different, however, rabbits receiving those from young 

 cultures usually recovering in a short time, whereas, the others 

 went on to a typical endotoxin death. 



We may say in passing that we do not believe it possible even 

 with 6-hour growths to eliminate the presence of extractive sub- 

 stances because rabbits treated with 6-hour filtrates always de- 

 veloped agglutinins and complement-fixing bodies, a fact which 

 shows that investigators who have worked with filtrates of 5- or 

 6-day cultures of typhoid and dysentery could never have worked 

 with pure exotoxins. And the sera produced with such 5-day 

 filtrates must have contained considerable amounts of sensitizer 

 to the bacterial protein to which some, at least, of their always 

 limited protective action must be attributed. This is rendered 

 still more likely by the fact that such sera have been strongly 

 protective only after incubation with the antigen in vitro before 

 injection. In general, as far as we have gone, it has seemed that 

 the filtrates of young typhoid cultures were very much more toxic 

 for rabbits than for guinea pigs, being, in this, similar to strepto- 

 coccus and influenza filtrates. Filtrates of old cultures on the 

 other hand were relatively more toxic for guinea pigs. 



As to deterioration of the 6-hour typhoid substances at room 

 temperature, our results have been irregular, except that it has 

 seemed that after 24 hours' standing, there was distinct loss of 

 toxicity in isolated instances. 



In one experiment there was marked deterioration of toxicity 



