506 A MANUAL OF BACTERIOLOGY 



details of the "small quantity" method here given I am indebted 

 to my friend and colleague, Dr. F. E. Taylor, who has elaborated 

 it in this form. 



For a single serum six small quill tubes are required, five being con- 

 trols and the sixth the test, and for every additional serum two more 

 tubes are required. The six tubes (and the additional ones also 

 when more than one serum is being tested) are arranged in two rows 

 in a metal rack which is immersed in a water-bath and maintained 

 throughout the test at a temperature of 38-40 C. To each tube 

 in the back row run in 4 volumes of saline solution with the marked 

 Wright's pipette, into each tube of the front row four volumes of 

 the antigen suitably diluted as ascertained by the standardisation 

 of the antigen. Then commencing from the left hand, add to each 

 of the first two tubes (back and front) one volume of diluted known 

 normal inactivated serum (these form the negative control mixtures). 

 To the next two tubes add one volume of diluted known syphilitic in- 

 activated serum (these form the positive control mixtures). Then 

 to each of the next two tubes add one volume of the inactivated 

 serum to be tested, diluted 1 in 2, and repeat this with as many 

 series of tubes as there may be sera to be tested. Next to every 

 tube add one volume of suitably diluted complement, and leave in 

 the warm bath for five minutes. After this add five volumes of the 

 prepared haemolytic system and leave in the bath for fifteen minutes. 

 The haemolytic system is prepared by mixing in bulk four volumes 

 of suitably diluted inactivated haemolytic serum and one volume of 

 20 per cent, suspension of washed sheep's corpuscles. The pipette 

 used for the additions of the reagents should be rinsed with saline 

 solution between each constituent of the test. 



At the end of fifteen minutes the tubes are centrifuged and the 

 pressure or absence of haemolysis noted. If haemolysis has occurred, 

 the fluid in the tubes form a clear red solution without deposit of 

 corpuscles, whereas if fixation is complete the corpuscles are 

 deposited at the bottom of the tube while the fluid above is colourless 

 and transparent. 



All the tubes in the back row should show haemolysis as they 

 contain no antigen, tube 1 in the front row should also show haemo- 

 lysis as it contains antigen and a negative serum, tube 2 in the front 

 row should show no haemolysis as it contains both antigen and 

 positive serum. In the remaining front row tubes, haemolysis or 

 fixation will occur according as the sera are negative or positive 

 respectively. 



If it be desired to obtain some idea of the amount of syphilitic 

 amboceptor present in a positive serum, a quantitative estimation 



