184 A MANUAL OF BACTERIOLOGY 



is known as a " haemolytic system." The following example illus- 

 trates the method of carrying out the test: The cholera-immune 

 serum is heated to 56 C. for half an hour. An eighteen hours old 

 agar culture of the organism to be tested is suspended in 2 c.c. of 

 sterile physiological salt solution. The complement is fresh guinea- 

 pig serum ; a portion of this is also heated to 56 C. (= non-immune 

 serum). The following mixtures are prepared in three small 

 test-tubes : 



Tubes 1 and 2 each contain 0-2 c.c. microbic suspension + 0-6 c.c. 



heated immune serum + 0-1 c.c. complement. 

 Tube 3 contains 0-2 c.c. microbic suspension +0-6 c.c. heated 



non-immune serum + 0-1 c.c. complement. 



These are well shaken to mix their contents, and are kept for 

 half to one hour at 37 C. At the end of this time 0-1 c.c. of the 

 following mixture is added to tubes 1 and 3 : two volumes of heated 

 (to 56 C. for half an hour) serum hsemolysing sheep's red corpuscles 

 + one volume of washed sheep's corpuscles. To tube 2 is added 

 0-1 c.c. of a mixture of two volumes of physiological salt solution + 

 one volume of washed sheep's corpuscles. The tubes are kept for 

 a further hour or so at 37 C., and at the end of that time the 

 occurrence of haemolysis is noted. If the organism is homologous 

 with the immune serum, the immune body will fix the complement 

 in tube 1 and no haemolysis will occur ; in tube 3 haemolysis will 

 occur because the complement remains free. Tube 2 serves as a 

 control, and should show no haemolysis in three hours (though if 

 kept for eighteen to twenty-four hours haemolysis will occur if the 

 organism produces hcemolysins, apart from any action of comple- 

 ment). If the organism is not homologous with the immune serum, 

 haemolysis will occur in tube 1, because the complement docs not 

 become fixed, tubes 2 and 3 being the same as before. 



It is not even necessary to use the living organism ; the dead 

 organism or extracts thereof, and, in cases where the organism 

 cannot be cultivated, a dried and pulverised organ or an extract 

 thereof, has been employed. Certain non-specific substances may 

 sometimes be used as in the Wassermann reaction for syphilis (see 

 " Syphilis "). 



The haemolytic serum may be obtained by injecting rabbits with 

 a 10 per cent, suspension of well-washed sheep's red corpuscles. 

 The sheep's blood should be obtained as ascptically as possible 

 from the slaughterhouse ; the blood, as it runs, is caught in a 

 sterile wide-mouthed bottle containing a coil of fine wire witli which 

 it is defibrinated by shaking. The iiuid blood is then mixed with 



