METHODS OF TESTING FOR AGGLUTINATION 117 



the mixture is placed in an upright thin glass tube ; sedimenta- 

 tion is shown by the formation within a given time (say from two 

 hours at 55° C. to twenty-four hours "at room temperature) of a 

 somewhat flocculent layer at the bottom, the fluid above being 

 clear. Two points should be attended to : (a) controls should 

 always be made with normal serum and with the bacterial 

 emulsion alone, and (b) the serum to be tested should never 

 be brought in the undiluted condition into contact with the 

 bacteria. The stages of procedure are the following : — 



1. Blood is conveniently obtained by pricking the lobe of the ear, 

 which should previously have been washed with a mixture of alcohol 

 and ether, and allowed to dry. The blood is drawn up into a Wright's 

 blood-capsule (Fig. 41) or into the hollow bulbous portion of a capillary 

 pipette, such as in Fig. 40, a. (These pipettes can be readily made by 

 drawing out quill glass-tubing in a flame. It is convenient always to 

 have several ready for use.) The pipette is kept in the upright position, 

 one end being closed. For purposes of transit, break off the bulb at 

 the constriction and seal the ends. After the serum has separated from 

 the coagulum the bulb is broken through near its upper end, and the 

 serum removed by means of another capillary pipette. The serum is then 

 to be diluted. 



2. The serum may be diluted (a) by means of a graduated pipette — 

 either a leucocytometer pipette (Fig. 40, b) or some corresponding form. 

 In this way successive dilutions of 1 : 10, 1 : 20, 1 : 100, etc., can be 

 rapidly made. This is the best method, (b) By means of a capillary 

 pipette with a mark on the tube, the serum is drawn up to the mark 

 and then blown out into a glass capsule ; equal quantities of bouillon 

 are successively measured in the same way, and added till the requisite 

 dilution is obtained, (c) By means of a platinum needle with a loop 

 at the end (Delepine's method). A loopful of serum is placed on a slide, 

 and the desired number of similar loopfuls of bouillon are separately 

 placed around on the slide. The drops are then mixed. 



A very convenient and rapid method of combining the steps 1 and 2 

 is to draw a drop of blood up to the mark 1 or '5 on a leucocytometer 

 pipette, and draw the bouillon after it till the bulb is filled. A dilution 

 of 10 or 20 times is thus obtained. Then blow the mixture into a 

 U-shaped tube (Fig. 40, c), and centrifugalise or simply allow the red 

 corpuscles to separate by standing. (In this method, of course, the 

 dilution is really greater than if pure serum were used, and allowance 

 must therefore be made in comparing results.) The presence of red 

 corpuscles is no drawback in the case of the microscopic method, but when 

 sedimentation tubes are used the corpuscles should be separated first. 



3. The bacteria to be tested should be taken from young cultures, 

 preferably not more than twenty-four hours old, incubated at 37° C. 

 They may be used either as a bouillon culture or as an emulsion made 

 by adding a small portion of an agar culture to bouillon or '8 per cent, 

 solution of sodium chloride. In the latter case the mass of bacteria on a 

 platinum loop should be gently broken down at the margin of the fluid in 

 a watch-glass. When a thick turbidity is thus obtained, any remaining 

 fragments should first be removed, and then the organisms should be uni- 

 formly mixed with the rest of the fluid. The bacterial emulsion ought to 

 have a faint but distinct turbidity. (When the exact degree of sediment- 



