626 A MANUAL OF BACTERIOLOGY 



fill into them the reagents as indicated in the Table, 1 unit volume 

 now corresponding to 0-1 c.c. given in that Table, e.g. 8 unit 

 volumes now correspond with 0-8 c.c. in the Table. 



For the saline, use the throttled pipette (a) ; for the ambo- 

 ceptor dilution, the throttled pipette (b) ; for the complement 

 dilution, the throttled pipette (c) ; and for the corpuscle sus- 

 pension, the throttled pipette (e) (see p. 624). Incubate the mix- 

 tures for half an hour in the water-bath and read off the minimal 

 hsemolytic dose, i.e. the first tube which is absolutely clear. 



B. PREPARATION OF THE CORPUSCLE-AMBOCEPTOR MIXTURE. 

 Each tube in the test requires 4 m.h.d. of amboceptor. But the 

 unit volume now being used is only one-fifth of the unit volume 

 of the " large volume " technique. The amount of amboceptor 

 to be used is therefore obtained by multiplying the quantity 

 arrived at by the foregoing standardisation (A) by four, and 

 dividing by five. Thus, if the fifth tube in the Table on p. 617 

 indicates the minimal hsemolytic dose of amboceptor, this corre- 

 sponds to 0-0005 c.c., and four-fifths of this = 0-0004 c.c., and is 

 the amount of amboceptor to be used for each tube. This amount 

 of amboceptor, together with 5 per cent, corpuscles, is to be con- 

 tained in every 0-1 c.c. of the amboceptor-corpuscle mixture, 

 as this is the quantity (= 5 unit volumes), used for every 

 tube of the test. For 100 doses of the mixture we should there- 

 fore require 0-04 c.c. of amboceptor in 10 c.c. of the mixture. 

 With the 0-1 c.c. pipette, measure into a test-tube 0-04 c.c. of 

 amboceptor, add 8 c.c. of saline, and 2 c.c. of a 25 per cent, sus- 

 pension of sheep's corpuscles. The mixture now contains 5 per 

 cent, corpuscles and the correct amount of amboceptor, and 10 c.c. 

 of it will suffice for twelve to twenty specimens, including the 

 preliminary standardisation of complement and the final tests 

 for positiveness. Larger quantities may be made up in a similar 

 manner, if required. For example, for 150 doses (= 15 c.c.), we 

 should take 0-06 c.c. amboceptor -f- 12 c.c. saline + 3 c.c. corpuscles. 

 C. STANDARDISATION OF COMPLEMENT. Keferto Sect. IV,p. 617, 

 and proceed much in the same way. Make up the amboceptor- 

 corpuscle mixture as in B above, and the antigen dilution (0-3 c.c. 

 heart extract -f 0-2 c.c. cholesterin solution -f 7 c.c. saline). 

 Make up with the throttled pipette (a) two dilutions of comple- 

 ment in watch-glasses, viz. ( 1 ) 1 volume complement -+- 23 

 volumes saline (= 1 in 24), and (2) 2 volumes complement-}- 



