408 VENOM HEMOLYSIS 



pulverized venom and dissolving this in 10 c.c. of normal saline solution 

 (1 : 1000). This stock dilution is best preserved in amounts of 1 c.c. in 

 sealed ampules, kept in the frozen state in the ice chest in a wide-mouthed 

 well-stoppered vacuum bottle containing salt and ice (Schwartz). 



Each cubic centimeter is sufficient for making three tests, so that the 

 10 ampules will be enough for 30 tests, or 1 gram of venom for 3000 re- 

 actions. Or the dried venom may be weighed out in amounts of 

 0.0005 gram in test-tubes, and diluted, just before being used, with 1 c.c. 

 of normal salt solution (1 : 2000). Immediately before the tests are 

 conducted subdilutions are prepared of the stock dilution (1 : 1000), 

 using separate pipets for each, as follows: 



Solution A: 1 : 10,000 = 1 c.c. stock solution + 9 c.c. normal saline 



solution. 

 Solution B: 1 : 15,000 = 2 c.c. solution A + 1 c.c. normal saline 



solution. 

 Solution C: 1 : 20,000 = 1 c.c. solution A + 1 c.c. normal saline 



solution. 

 Solution D: 1 : 30,000 = 1 c.c. solution B + 1 c.c. normal saline 



solution. 

 Solution E: 1 : 40,000 = 1 c.c. solution C + 1 c.c. normal saline 



solution. 



These amounts are sufficient for making three tests; if more tests 

 are to be made, larger amounts of the various dilutions will keep fairly 

 well in a good refrigerator for several days, but it is always well to plan 

 the work so that the exact amount will be prepared and no waste occur. 

 Each lot of stock solution should be tested occasionally with the cells 

 of known normal and positive persons, to make certain that the venom 

 is active in these dilutions. These titrations are conducted in the 

 same manner as the test. 



Preparation of Blood-cells. With a sterile syringe blood is drawn 

 from a vein at the elbow and 2 c.c. placed in an accurately graduated 

 centrifuge tube containing 5 c.c. of a 2 per cent, solution of sodium 

 citrate in normal saline solution. The suspension should be shaken gently 

 to insure mixing and the prevention of coagulation, but defibrination 

 by means of whipping should never be practised. The cells may be pre- 

 pared at once or placed in the ice-chest overnight. Sufficient normal 

 saline solution is added to bring the total volume to 15 c.c. Mix gently 

 and centrifuge at low speed until the supernatant fluid is clear. Draw off 

 the fluid, add more normal saline solution, mix up the cells, and centri- 

 fuge again until clear. Repeat this process once more so that all traces 



