SYPHILIS 133 



former and 2 of the latter. Mix in a dry test-tube and add the neces- 

 sary a'mount of saline, mixing quickly. 



Complement. Kill a guinea-pig by a blow on the head, cut its 

 throat, and collect the blood in a sterile dish. Allow it to clot, pipette 

 off the clear serum, and use it the same day. 



Amboceptor. This is serum from a rabbit which has been injected 

 with sheep's corpuscles. It keeps, and can be obtained commercially. 



Blood Corpuscles. Sheep's blood is obtained from the slaughter- 

 house, and the corpuscles are washed three times in saline. 



Standardization of the Reagents Amboceptor. Assuming that the 

 strength of all the ingredients is unknown, it is most convenient to 

 begin with this, and to use complement in great excess i.e., fresh 

 guinea-pig's serum diluted i in 2. Add -i c.c, of amboceptor serum to 

 9-9 c.c. of saline, mix, and add i c.c. to 9 c.c. of saline, thus getting 

 a i in 1000 solution. Into a series of test-tubes place -i, -2, -3, etc., of 

 this dilution, add saline to i c.c., and -i c.c. of complement serum (i 

 in 2). Now add -5 c.c. of a 5 per cent, emulsion of washed sheep's 

 corpuscles, mix, incubate, and read off the results after twenty minutes. 

 The tube containing the lowest amount necessary for complete haemo- 

 lysis contains the minimal haemolytic dose (M.H.D.),- and three times 

 this amount is used in the actual test. 



To prepare the emulsion of sensitized corpuscles. Suppose you 

 want 20 c.c. Take a graduated measure, place 16-17 c.c. of saline 

 therein, and add i c.c. of the washed corpuscles. Now calculate the 

 amount of amboceptor serum you require, multiplying the M.H.D. of 

 the serum you are using by 3 and then by 40, since you are making 40 

 times the *5 c.c. for which your M.H.D. was estimated. Measure 

 out this amount, add, make up to 20 c.c. with saline, and mix. 



Next proceed to standardize your complement. Dilute some serum 

 i in 25 with saline (i to 24), and measure out -i, -125, -15, '175, '2, 

 ' 2 5 '3> '35> an d -4 c.c. : make up each volume to i c.c. Add to each 

 tube -5 c.c. of the sensitized emulsion prepared as above, and mix. 

 Incubate for twenty minutes, and read off the result. The lowest tube 

 which just shows complete haemolysis contains i M.H.D. of comple- 

 ment, and 2$ are used in the actual test. Do not use complement 

 which is so weak as not to give complete haemolysis with -3 c.c. 



Standardization of the Antigen. What is actually estimated is the 

 strength of antigen which completely inhibits haemolysis under stan- 

 dard conditions : one-third of this amount is used. Measure out -05, 

 075, -i, -125, -15, and -2 of the mixture of the two alcoholic solu- 

 tions previously described, make up to i c.c. with saline, and add 

 *i c.c. of complement so diluted as to contain 2| M.H.D. in this 

 amount. Incubate twenty minutes, add -5 c.c. of sensitized emulsion, 

 incubate again for twenty minutes, and read off the result, and dilute 

 the antigen for use accordingly. 



Preparation of the Mixed Antigen and Complement. Having de- 

 cided how many c.c. of the mixture will be required, prepare half this 

 amount of a dilution of guinea-pig serum in saline of such a strength 



