TITRATING H^EMOLYSIN 329 



Sterile test-tubes, 16X2 cm. 

 Small sterile test-tubes, 9X1 cm. 

 Small test-tube rack, or roll of plasticine. 

 Capillary teat pipettes. 



Stout rubber band or length of small rubber tubing. 

 Reagents Required and Method of Preparation: 



1. Normal saline solution. 



2. Haemolytic serum inactivated by preliminary heating to 

 56 C. for 15 minutes (vide supra) in test-tube labelled H. S. 



3. Complement. Fresh guinea-pig serum in test-tube labelled C. 



Kill a normal guinea-pig with chloroform vapour. 



Open the thorax with all aseptic precautions, and collect 



as much blood as possible from the heart with a sterile 



Pasteur pipette. 

 Transfer it to a sterile centrifuge tube and place the tube 



in the incubator at 37 C. Two hours later separate 



the clot from the sides of the tube, and centrifugalise 



thoroughly. 

 Pipette off the clear serum to a clean sterilised test-tube. 



4. Erythrocyte solution, in test-tube labelled E. 



Collect and wash human red blood cells (see page 388, 1-8). 

 Measure the volume of red cells available and prepare a 2 

 per cent, suspension in normal saline solution. 



METHOD. 



1. Take two test-tubes and number them i and 2, 

 and pipette into each 9 c.c. of normal saline solution. 



2. Add i c.c. of haemolytic rabbit serum to tube No. i 

 and mix thoroughly: take up i c.c. of the mixture and 

 add it to tube No. 2 ; mix thoroughly. 



3 . Set up ten small test-tubes in test-tube rack or in 

 roll of plasticine, and number i to 10. 



4. Pipette into tube No. i 0.5 c.c. =0.5 c.c. 1 



haemolytic serum [ From tube 



Pipette into tube No. 2 o.i c.c. = 0.1 c.c. { H. S. 

 haemolytic serum 



Pipette into tube No. 3 0.5 c.c. = 0.05 c.c. 



hasmolytic serum 

 Pipette into tube No. 4 0.3 c.c. = 0.03 c.c. 



haemolytic serum 

 Pipette into tube No. 5 0.2 c.c. =0.02 c.c. 



haemolytic serum 

 Pipette into tube No. 6 o.i c.c. = 0.01 c.c. 



haemolytic serum 



From 

 tube i. 



