ENDOTOXINS AND AGGRESSINS 899 



(e) Autopsy the animal, paying particular attention to the kidneys 

 and gastro-intestinal tract? What changes have occurred? 



EXPERIMENT 16. ZOOTOXIN (COBRA VENOM) 



1. Collect about 2 c.c. of normal human blood and place in 5 c.c. of 2 per cent, 

 sodium citrate in 0.85 per cent, sodium chlorid solution. This mixture must not be 

 shaken and the cells should be washed at least four times with normal salt solution, 

 after which they are made up to a 4 per cent, suspension. 



2. Prepare a 4 per cent, suspension of sheep corpuscles in the same manner. 



3. Prepare a stock dilution of cobra venom by dissolving 0.005 gm. dried venom 

 in 10 c.c. of normal salt solution. This solution will keep about one week in the re- 

 frigerator. 



4. Prepare a solution of venom 1 : 10,000 by placing 2 c.c. of the stock solution in 

 8 c.c. salt solution. Prepare a 1 : 20,000 dilution by placing 2 c.c. of dilution 1 : 10,000 

 in 2 c.c. salt solution. 



5. Place 1 c.c. of each corpuscle suspension into two small test-tubes and add 1 

 c.c. and 0.5 c.c. eac^h venom dilution. Shake the tubes gently and place in the incu- 

 bator for an hour and then in the refrigerator overnight. 



6. Inject 2 c.c. of the 1 : 10,000 dilution intravenously into a rabbit and 1 c.c. 

 intravenously into a guinea-pig. 



7. Observe the animals closely, particularly the urine. 



(a) Does the venom show a hemotoxic action? Does it act the 

 same on rabbit and guinea-pig corpuscles? If not, why not? 



(b) How do you explain venom hemolysis? 



(c) What are the evidences of venom hemolysis in vivo? 



(d) Is the hemotoxic poison the most dangerous constituent of venom? 



EXERCISE 6. ENDOTOXINS AND AGGRESSINS 



EXPERIMENT 17. ENDOTOXINS 



1. Incubate eight agar slants with Bacillus typhosus and cultivate at 37 C. for 

 seventy-two hours. 



2. Wash off the growths with 5 c.c. distilled water for each tube. 



3. Place the emulsion in a sterile bottle with glass beads and shake for twenty- 

 four hours at room temperature. At the same time inoculate six more slants of agar 

 and wash off the growths at the end of twenty-four hours with 3 c.c. of normal salt 

 solution for each tube. 



4. Filter the emulsion, which has been shaken, through a Berkefeld filter. 



5. Inject two rabbits intravenously with 2 c.c. each of the nitrate (endotoxins) 

 and unfiltered culture. 



6. Observe influence on body-weight and temperature and for symptoms of 

 toxemia. 



7. Inject three pigs intraperitoneally as follows: 



1st pig: 2 c.c. of emulsion of typhoid bacilli. 



2d pig: 2 c.c. of nitrate. 



3d pig: 1 c.c. each of emulsion and filtrate. 



