AGGLUTINATION TESTS 143 



For obtaining an agglutinating or bacteriolytic serum for bacteria we inject 

 about i c.c. of the killed bacterial bouillon culture subcutaneously or into the peri- 

 toneal cavity of the rabbit. The easiest way to inject the rabbit is to hold the animal 

 head down and plunge the needle in the median line into the abdominal cavity, 

 forcing in the contents of the syringe. The intestines gravitate downward and by 

 entering the needle below the limits of the bladder we avoid injuring any vital part. 

 It may be more satisfactory to at first inject only about 1/2 c.c., and then if there is 

 very little reaction, as shown by the appetite and spirits of the rabbit, to inject 

 about four days later i c.c. About four or five injections at intervals of three to 

 five days will usually produce an immune serum. 



Injection of the antigenic material (blood cells, serum or bacterial emulsion) into 

 the marginal ear vein may be employed. With this method, however, I have had 

 several rabbits die in what was considered anaphylactic shock. (For the method 

 of immunizing rabbits to produce a hsemolytic serum see Wassermann test.) Some 

 animals do not seem to be capable of producing antibodies, so that it may be necessary 

 to use one or more rabbits before a satisfactory serum is obtained. The most con- 

 venient way of obtaining serum for a test is to cut across one of the marginal veins 

 of the rabbit's ear, and collect the blood in a Wright's U-tube. Centrifugalizing, 

 we have the serum ready for use. 



The vein can be made to stand out prominently by applying a compress dipped 

 into very hot water. When a large amount of serum is desired it is better to use a 

 test-tube with two pieces of glass tubing passing through a double perforated rub- 

 ber stopper. To one of the projecting pieces of glass tubing a stout hypodermic 

 needle is attached through the medium of 8 inches of rubber tubing and to the 

 second piece of glass tubing passing through the stopper of the large test-tube 

 another piece of rubber tubing is attached for suction. To obtain blood from the 

 rabbit find the ensiform cartilage and insert the needle in the notch to the left and 

 gently force it upward. Applying suction with the mouth the blood flows into the 

 test-tube so soon as the needle enters the heart. By placing the tube of blood in 

 the refrigerator the serum separates out from the clot. The removal of 20 to 30 c.c. 

 of blood does not seem to affect the animals in the least and they can be used in this 

 way time and time again. The immune body and agglutinin in serum remain 

 active for weeks when kept in the refrigerator. The complement and opsonin, 

 however, begin to deteriorate at once and have disappeared by the fifth day. Con- 

 sequently, for opsonic and bacteriolytic and haemolytic experiments, fresh serum 

 twelve to twenty-four hours must be used, or it may be activated. 



AGGLUTINATION TESTS. 



There are two methods of testing the aggultinating power of a 

 serum -the microscopical and the macroscopical or sedimentation 

 method. 



i. For the microscopical method draw up serum to the mark 0.5 of the white 

 pipette. Then draw up salt solution to the mark n. This when mixed gives a 

 dilution of i to 20. One loopful of the diluted serum and one loopful of a bouillon 



