THE TECHNIQUE OF SERUM REACTIONS 251 



In obtaining blood from larger animals, horses, sheep, etc., a cannula 

 be introduced into the jugular or internal saphenous veins. The 

 skin is shaved and sterilized and a rubber tourniquet placed about the 

 neck or thigh, as the case may be, in order to cause the vein to stand 

 out. A small incision may be made through the skin over the vein, but 

 Is not necessary. A cannula, with rubber tubing attached, is then 

 plunged into the vein and the blood caught in sterile high cylindrical 

 jars, allowed to clot, and placed in the refrigerator. The serum is taken 

 off after twenty-four to forty-eight hours with sterile pipettes. 



Agglutination Tests. For the determination of the agglutinating 

 power of serum it is necessary to make suitable dilutions of the serum, 

 and to prepare an even emulsion of the microorganisms to be tested. 

 The test may be made microscopically or macroscopically. The micro- 

 scopic test is the one in general use in the diagnosis of typhoid fever, 

 and is occasionally applied to some other diseases. In its application 

 to typhoid fever it is usually spoken of as the Gruber-Widal reaction. 



Twelve- to eighteen-hour broth cultures of the typhoid bacillus, 

 grown at incubator temperature, may be used. It is preferable, how- 

 ever, to use an emulsion of a twelve to twenty-four hour old agar culture 

 in physiological salt solution (0.85 per cent). The salt-soluti'on emulsion 

 is made by adding about 10 c.c. of normal salt solution to the fresh agar 

 slant culture, carefully detaching the culture from the surface of the 

 agar with a flexible platinum wire, and pipetting off the emulsion thus 

 made. With some microorganisms it is sufficient simply to allow the 

 larger clumps to settle and to pipette off the supernatant turbid emulsion. 

 With other microorganisms, the tendency to form clumps makes it 

 necessary to resort to further methods of securing an even distribution 

 of the bacteria. This may be done either by sucking the emulsion in and 

 out through a narrow pipette held perpendicularly against the bottom of 

 a watch glass, as in Wright's technique for the opsonic test (see section 

 on Opsonins, p. 285), or by carefully rubbing the clumps against the 

 watch glass with a stiff platinum wire. In the case of the tubercle ba- 

 cillus not even this suffices, but it becomes necessary to grind the moist 

 bacillary masses in a mortar before emulsifying. With the tubercle bacil- 

 lus, too, it is preferable to use salt solution at 1.5 per cent concentration. 

 In preparing cultures of streptococcus and pneumococcus for ag- 

 glutination tests, it has been found convenient by Hiss to grow for 

 about four days in flasks of a 1% glucose, 2% pepton meat-infusion 

 broth, to which has been added 1% of calcium carbonate (p. 126). 

 The calcium neutralizes the inhibiting acid formed in the broth 



