i68 



PRACTICAL METHODS IN IMMUNITY 



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

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

 convenient 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. Centrifugaliz- 

 ing, 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 as 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. Such sera may also be dried on paper 

 as for amboceptor paper (Noguchi). The complement and opsonin, however, begin 

 to deteriorate at once and have disappeared by the fifth day. Consequently, 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 agglutinating power of a 

 serum the microscopical and the macroscopical or sedimentation 

 method. 



The Widal Reaction. 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. (It is more convenient to make the serum 

 dilutions with a graduated rubber bulb capillary pipette.) One loopful of the diluted 

 serujn and one loopful of a bouillon culture or salt solution suspension of the organism 

 to be testeo" gives a dilution of i to 40. One loopful of the i to 20 diluted serum and 

 3 loopfuls of the bacterial suspension give a dilution of i to 80. These two dilutions 

 answer in ordinary diagnostic tests. The red pipette with a i to 100 to i to 200 

 dilution may be used where dilutions approaching i to 1000 are desired. Having 

 mixed the diluted serum and the bacterial suspension on a cover-glass, we invert it 

 over a vaselined concave slide and examine with a high power, a dry objective (}/ 

 i nch) . It is neater to press down the vaselined periphery of the concavity on the cover- 

 glass. This sticks to the borders of the cover-glass and the preparation is easily 

 handled. It is simpler to make a ring of vaseline to fit the cover-glass and make 

 the mixture of diluted serum and culture in the center of this ring or square. Then 

 apply the cover-glass, press it down on the vaseline ring and examine as with the 

 ordinary hanging drop. In making dilutions it is preferable to use salt solution, as 



