252 INFECTION AND IMMUNITY 



by the microorganisms and permits the development of a mass culture. 

 The flasks should be shaken at least once a day. The broth may be 

 pipetted off and clumps removed by centrifugation. Without this tech- 

 nique it is sometimes difficult to get sufficient growths of these bacteria 

 for any quantity of emulsion unless large surfaces of agar are employed 

 in special receptacles or by making many slant cultuies. 



The serum dilutions are obtained by first making a one to ten dilu- 

 tion of serum with normal salt solution. The serum used for this pur- 

 pose is of red blood corpuscles by centrifugation. From the 1 to 10 

 dilution any number of higher dilutions may be made, by mixing given 

 parts of the 1 to 10 dilution with normal salt solution; thus one part 

 of a 1 to 10 dilution plus an equal quantity of salt solution gives a dilu- 

 tion of 1 to 20. One part of one to ten dilution plus two parts of normal 

 salt solution gives one to thirty, etc. It must not be forgotten that, 

 when equal parts of the serum and bacillary emulsion have, been mixed, 

 each one of these dilutions is doubled. 



In making the microscopic agglutination test, equal quantities of 

 serum dilution and bacterial emulsion are mixed upon a cover-slip. 

 The mixture may be made either by measuring out a drop of each sub- 

 stance with a standard platinum loop, depositing them close together 

 on the cover-slip, and mixing; or equal quantities may be sucked up, 

 each to a given mark, in a capillary pipette, mixed by suction in and 

 out, and deposited upon the cover-slip. The cover-slip is inverted over 

 a hollow glass slide, the rim of which has been greased with vaseline. 

 The drop is then observed through a (Leitz) No. 7 lens, ocular No. 3. 

 Macroscopic agglutination, preferable for exact laboratory research, 

 is made in narrow test tubes measuring about 0.5 cm. in diameter and 

 about 5 cm. in length (Fig. 60). 



Equal quantities, usually 1 c.c. each, of serum dilution and emulsion 

 are mixed. A series of tubes is prepared, in each subsequent one ot 

 which the dilution is higher. These mixtures may be placed in the in- 

 cubator for a few hours and then kept at room temperature. After re- 

 moval from the incubator agglutination is in some instances hastened 

 by transference to the ice chest. When agglutination takes place in 

 these tubes, clumps of bacteria may be seen to form, which settle to 

 the bottom of the tube, very much like snow-flakes. The surface of 

 the sediment is heaped up and irregular. The supernatant fluid becomes 

 entirely clear. When the reaction does not occur the sediment is an even, 

 granular one with a flat surface, and the emulsion remains turbid. 



Instead of using tet tubes as described above, Wright has sug- 



