376 



HEMOLYSINS 



healthy pig under ether anesthesia into a Petri dish or centrifuge tube. 

 This serum is diluted 1 : 20, making a 5 per cent, solution, by adding 1 

 c.c. of serum to 19 c.c. of salt solution. Each cubic centimeter of this 

 dilution contains 0.05 c.c. of undiluted serum, which experience has 

 shown is a satisfactory amount to use. 



The corpuscle suspension is then prepared. The blood used depends 

 upon the kind of amboceptor that is to be titrated. With sheep and ox 

 blood, a 2.5 per cent, suspension of washed corpuscles may be employed. 

 With antihuman amboceptor, the corpuscles are usually used in 1 per 

 cent, suspension. (See Noguchi Modification of Wassermann Reac- 

 tion.) After the corpuscles have been washed three times, 1 c.c. is 

 placed in 39 c.c. of salt solution, or sufficient salt solution is added to 

 2.5 c.c. of the corpuscles to make the total volume equal 100 c.c. 



To a series of six sterile test-tubes increasing doses of the diluted 

 immune serum are now added, together with 1 c.c. of complement dilu- 

 tion, 1 c.c. of corpuscles suspension, and sufficient normal salt solution 

 to make the total volume in each tube about 3 or 4 c.c. The follow- 

 ing table shows the method of preliminary titration of a hemolytic 

 serum: 



TABLE 10. PRELIMINARY TITRATION OF A HEMOLYSIN 



In this instance the unit of amboceptor is about 1 : 500, which is too 

 low for a satisfactory antisheep serum. The rabbit should, therefore, 

 receive another dose or two of corpuscles, and the serum be titrated again 

 in from four to seven days after the last injection has been given. In 

 this titration it will be well to use a higher dilution of the inactivated 

 immune serum, as 1 : 1000. This may be prepared by adding 1 c.c. of 

 a dilution of 1 : 100 with 9 c.c. of normal salt solution and mixing well. 

 The titration is then proceeded with as follows: 



