416 IMMUNOLOGY 



1. The glassware, which includes pipettes, test tubes, volumetric 

 flasks and graduates, should be chemically clean since minute 

 traces of acids, alkalies, dyes, and many other substances 

 may interfere with the successful performance of the tests. 



2. The size of the test tubes should follow ]U'oferably Kolmer's 

 recommendations, i.e., 15 mm. by 85 mm. 



3. Sterile, cold, properly prepared physiological saline contain- 

 ing 0.1 gm. of magnesium sulphate per liter should be 

 used uniformly as a diluent. 



4. The final volume in each tube is to be 3.0 c.c. 



f). One unit of cell suspension is contained in 0.5 c.c. of a 2 

 per cent suspension of sheep cells prepared from waslicd 

 packed cells. 



6. A unit of hemolysin (antisheep) is contained in 0.5 c.c. of 

 the highest dilution that brings about complete hemolysis 

 of a unit of red cells in the presence of 0.3 c.c. of a 1 :3() 

 dilution of complement when incubated for one hour at 

 37° C. 



7. An exact iniit of complement is the lea.st amount oL" a stand- 

 ard dilution 1:30 of complement that will comi)letely hemo- 

 lyze a unit of red cells in the presence of 2 units of hemolysin 

 when incubated for one hour at 37° C. in a water bath. A 

 full luiit is 0.05 c.c. more than a unit, (complement should 

 be titrated in the presence of a test dose of antigen contained 

 in 0.5 c.c. of saline. 



5. Pooled complement from 3 or more guinea pigs is ])referable 

 to complement from one pig. 



}). (^om])lement nuiy be preserved either hy adding 10 limes the 

 normal concentration of dry NaCl or by freezing. A modi- 

 fied Sonnenscheine method as suggested by Boerner and 

 Ijukens (1940) has been successfully used in this laboratory. 

 Ijyophil or cryochem complement is preferred. 



10. High titered inactivated antisheep hemolysin is preferably 

 preserved by adding an equal amount of C.P. glycerin. 



11. For antigen titration 2 full units of complement and two 

 units of hemolysin are recomnu^ided. 



12. It is recommended that six to fifteen hours at 6° to 8° C. 

 be used as the time and temperature for primary incubation. 



13. For the second incubation it is recommended thai one hour 

 in a 37° C. water bath be employed. 



