BACTERIAL PROTEIN; PTOMAINS 901 



growth with a sterilized platinum wire, being particularly careful not to remove frag- 

 ments of agar. 



3. Pipet the heavy emulsion to a large centrifuge tube. Another cubic centi- 

 meter or two of water is added to each bottle to remove the balance of the growth. 



4. Centrifuge at high speed until the bacilli are thoroughly settled. Decant off 

 the supernatant fluid and add 50 per cent, alcohol; mix and centrifuge. Decant and 

 add 95 per cent, alcohol; mix and centrifuge. 



5. Remove the sediment of bacteria to a small flask with 50 c.c. of absolute 

 alcohol and set aside at room temperature for a day. Decant off the alcohol and add 

 50 c.c. of ether. Mix and set aside for another twenty-four hours. Decant off the 

 ether that remains and remove sediment to a porcelain or agate mortar. Place in 

 the incubator for a few hours until thoroughly dry. 



6. Grind the dry mass very thoroughly, the operator wearing a mask, until a 

 fine powder is produced. Place the powder of bacterial substance in a wide-mouthed 

 dark-glass bottle and preserve in a dark closet. A portion will be needed later for 

 experiments in anaphylaxis. 



7. Mix 0.02 gm. of the dry powder with 10 c.c. salt solution. 



8. Inject 2 c.c. of this emulsion intraperitoneally in a 300-gram guinea-pig. 



9. Heat 2 c.c. of the emulsion at 60 C. for two hours and inject intraperitoneally 

 in a guinea-pig. 



10. Inject a third pig intraperitoneally with 2 c.c. of a four-day bouillon culture 

 of Bacillus coli. 



(a) Could endotoxins withstand these various manipulations? 



(b) Does the heated extract kill the animal more quickly than the 

 unheated, and if so, why? 



(c) What are the symptoms produced? 



(d) Autopsy the animals. Are there evidences of peritonitis? Are 

 there differences in the lesions of the three animals? If so, how do you 

 explain them? 



(e) The bacterial split portion will be studied later under Anaphylaxis. 



EXPERIMENT 20. PTOMAINS 



1. Procure 4 ounces of beef and mince. 



2. Place in a flask with 250 c.c. tap water and inoculate with a culture of Bacillus 

 coli. Fit the flask with a rubber stopper with a glass tube to carry off gases. 



3. Inoculate a flask of neutral bouillon at the same time with the same culture. 



4. Cultivate both at 37 C. for two weeks. 



5. Filter both through a coarse Berkefeld filter. 



6. Concentrate both filtrates to one-half their volume at a low temperature on a 

 water-bath. 



7. Inject two guinea-pigs with each preparation, giving 1 c.c. subcutaneously 

 and 0.5 c.c. intraperitoneally. 



8. Observe the four animals closely. 



(a) What symptoms develop in both sets of animals? 



(b) What are ptomains? What role do they play in the production 

 of disease? 



(c) Can antibodies be produced for true ptomains? 



