616 A MANUAL OF BACTERIOLOGY 



upon the edge of the table. Pour the blood from the saucer into a 

 small conical glass. " Whip " to defibrinate with a piece of cotton- 

 wool upon the end of an iron wire. Centrifuge and pipette off 

 the serum. 



[If not more than 5 c.c. of complement are required, this may 

 be obtained by bleeding a guinea-pig from an ear vein without 

 killing the animal. By bleeding two or three animals considerable 

 quantities of complement may be obtained. Complement is best 

 used two or three hours after collection. Large buck guinea-pigs 

 should be chosen.] 



(i) Antigen. In two bottles A and B. Preparation of heart 

 extract (A). Obtain a fresh human heart. With a pair of scissors 

 cut off the muscular portions of the ventricles and elsewhere, but 

 do not take fat. Mince these pieces and weigh them. Place in a 

 mortar with a little sand and grind them up with absolute alcohol, 

 using 9 c.c. of alcohol to 1 grm. of heart. Transfer the whole to a 

 well-fitting glass- stoppered bottle, and shake occasionally for one 

 and a half hours. Filter into another well-fitting glass- stoppered 

 bottle through paper and preserve in ice chest. A deposit will be 

 found to form and this may be filtered off again. Cholesterin 

 solution (B). Take 1 grm. of pure cholesterin, such as Kahlbaum's, 

 and place in a well-fitting glass-stoppered bottle. Add 100 c.c. 

 absolute alcohol and put in the stopper tightly. Shake and heat in 

 water-bath at 40-50 C. until the cholesterin is dissolved. 



(j) Amboceptor. Serum haemolytic for sheep's corpuscles ; 

 this will usually be purchased. [The author has found Messrs. 

 Burroughs and Wellcome's hsemolytic serum quite satisfactory.] 

 III. STANDARDISATION OF THE H^MOLYTIC AMBOCEPTOR. To 

 be performed with two different guinea-pigs to ensure maximum 

 complement action. The standardisation should be repeated 

 every two months. 



(a) Materials. (1) Saline solution. (2) Tube containing 0-5 c.c. 

 guinea-pig's serum and 0-5 c.c. saline. (3) Tube containing ambo- 

 ceptor diluted 1 in 1,000 thus : Tube a = ambo. 0-1 c.c. (exact) -f 

 saline 9-9 c.c. (shake) ; tube b = I c.c. from tube a + 9-0 c.c. 

 saline (shake) this is 1 in 1,000. (4) Tube containing 1 c.c. of 

 deposited washed sheep's corpuscles -f- 19 c.c. saline (shake). 

 (5) Back with 9 test-tubes 4 x f . 



(b) Method. Fill reagents into the tubes as indicated in the table 

 on next page. Shake. Incubate for one hour at 37 C. and then 



