120 MICROSCOPIC METHODS. 



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

 either a leucocytometer pipette (Fig. 41, b) or some corresponding 

 form. In this way successive dilutions of I : 10, I : 20, I : 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 I and 

 2 is to draw a drop of blood up to the mark i or . 5 on a leucocytometer 

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

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

 U-shaped tube (Fig. 41, 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. 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 uniformly mixed with the 

 rest of the fluid. The bacterial emulsion ought to have a faint but 

 distinct turbidity. (When the exact degree of sedimenting power of a 

 serum is to be tested expressed as the highest dilution in which it 

 produces complete sedimentation within twenty-four hours a standard 

 quantity (by weight) of bacteria must be added to a given quantity of 

 bouillon. This is not necessary for clinical diagnosis.) 



4. To test microscopically, mix equal quantities (measured by a 

 marked capillary pipette) of the diluted serum and the bacterial emulsion 

 on a glass slide, cover with a cover-glass and examine under the micro- 

 scope. The form of glass slide used for hang-drop cultures (Fig. 26) 

 will be found very suitable. The ultimate dilution of the serum will, 

 of course, be double the original dilution. 



To observe sedimentation mix equal parts of diluted serum and of 

 bacterial emulsion and place in a thin glass tube a simple tube with 

 closed end or a U-tube. Keep in upright position for twenty-four 

 hours. One of Wright's sedimentation tubes is shown in Fig. 41, d. 

 Diluted serum is drawn up to fill the space mn, a small quantity of air 



