128 PRACTICAL METHODS IN IMMUNITY. 



H^MOLYTIC EXPERIMENTS. 



Take the blood of the animal that has been used to immunize the 

 rabbit and receive it in a graduated centrifuge tube containing salt 

 solution which has had i% of sodium citrate added to it. This 

 prevents the coagulation of the blood. After mixing, centrifuge and 

 pipette off supernatant fluid. Note the graduation reached by the 

 sediment of red cells and make up with salt solution to 20 times its 

 volume. If the cells reach the 1/2 c.c. mark, add 10 c.c. of salt solu- 

 tion. This gives a 5% emulsion of red cells the percentage usually 

 used in hemolytic experiments. To carry out the test, simply add i c.c. 

 of this 5% mixture of red cells to each of the series of tubes containing 

 diluted serum, as with the macroscopic agglutination tests. Place in 

 the incubator for 2-5 hours. The red cells settle to the bottom, and 

 tubes showing haemolysis have a light reddish to rich haemoglobin 

 color. Tubes not showing haemolysis remain white. 



BACTERIOLYTIC EXPERIMENTS. 



These may be carried out in the peritoneal cavity of a guinea-pig, 

 injecting mixtures of immune sera and the bacterial culture. 



Upon withdrawing, after 15-60 minutes, the bacteria are granular 

 and disintegrated. This is the well-known Pfeiffer's phenomenon, 

 and was once considered the most important test for cholera. See 

 Cholera. There have been several accidents (death of Orgel) with 

 this test, and it is not practicable except in a well- equipped laboratory. 

 Instead of a guinea-pig, we may simply take a fresh serum of known 

 dilution, and mix it with an equal quantity of the bacterial emulsion 

 in a capillary pipette; sealing off the end of the pipette, we incubate for 

 15 minutes. Then filing off the end we mix the culture thoroughly on a 



test-tubes. Then take a loopful (2 mg.) of culture from an 18 to 24 hour old agar 

 culture and emulsify it thoroughly in the dilution in the first test-tube repeat the 

 process in the second tube and so on. This procedure is much bafer than when 

 live cultures are added with a pipelte. Again, the dilution is unchanged by this 

 addition whereas it is doubled when an equal volume of culture is added to the 

 diluted serum. A control should always be made in normal salt solution. After 

 incubating, observe flocculent precipitates (agglutination) by tilting the fluid in the 

 tubes to form a thin layer and to obtain the most advantageous light and look for 

 a fine curdy precipitate (agglutination) or a uniformly turbid emulsion (negative 

 reaction). 



