22O PHARMACEUTICAL BACTERIOLOGY. 



centrifugalized and the serum used. The spinal fluid is obtained in the 

 usual manner by lumbar puncture. 



3. Complement. The normal blood serum of a guinea-pig. The blood 

 from one guinea-pig is required, thus making it necessary to sacrifice one 

 animal for each test. The blood must be used fresh, as the serum loses its 

 complementing value if kept over twenty-four hours. The blood is defibri- 

 nated, centrifugalized, and the serum used. If stored, it should be frozen. 



4. Hemolytic Serum. The blood serum of a rabbit that has been injected 

 with washed lamb's blood-corpuscles. The rabbit is immunized as follows: 

 The lamb's blood is first obtained, best by cutting its ear and allowing 10 c.c. 

 of blood to run into 30 c.c. of a i per cent, sodium citrate solution in normal 

 salt solution. (This will prevent the blood from clotting.) It is then 

 centrifugalized, the supernatant fluid pipetted off, and the blood-corpuscles 

 washed with normal salt solution by repeated centrifugalization and dejec- 

 tion of the supernatant fluid. Five c.c. of the washed blood-corpuscles are 

 injected into the rabbit five (5) or six (6) times at repeated intervals of five (5) 

 days. On about the tenth day after the last injection, blood is taken from 

 the rabbit, centrifugalized, and the serum used. Before using this serum, 

 it is necessary to test its power after being inactivated (heated for three- 

 quarters of an hour at 56 C. to destroy complement). The test is made 

 to determine the minimum quantity of the serum that will hemolyze i c.c. 

 of the 5 per cent, suspension of lamb's blood-corpuscles, with o.i c.c. of 

 complement (normal guinea-pig serum). Various quantities of the serum 

 to be tested are put in a series of test-tubes with i c.c. of the suspension of 

 lamb's blood corpuscles and o.i c.c. of the complement in each tube. The 

 tubes are put in the incubator at 37 C., for an hour and then examined to 

 determine the smallest quantity of serum that produced hemolysis. (The 

 proper quantity is usually i c.c. of a i in 2000 dilution, in normal salt solution. 

 The quantity necessary for the reaction is two minimal units, thus i c.c. of a 

 1000 dilution is used for the reaction.) The dilution used should never be 

 lower than i :iooo. If it happens to be lower it will be necessary to give the 

 rabbit a few more injections of blood-corpuscles, before using its serum. 



5. Lamb's Blood Corpuscles. Five c.c. of defibrinated lamb's blood are 

 collected and washed with normal salt solution in the same way as the rab- 

 bit's blood. Then a 5 per cent, suspension in normal salt solution is made. 



The antigen, the patient's serum and the hemolytic serum must be in- 

 activated (to destroy complement) before using, by heating them for three- 

 quarters of an hour at 56 C. The two sera should be inactivated as soon 

 as made. 



The antigen, antibody (patient's serum) complement, and hemolytic 

 serum should each be so diluted with normal salt solution that i c.c. of the 

 dilution will contain the necessary quantities needed for the reaction. 



