THE OPSONTC TECHNIQUE 111 



is to draw a drop of Hood 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. 46, 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 w r hen 

 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, inciibated 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 microscope. The form of glass slide used for hang-drop cultures 

 (Fig. 27) 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. 46, d. 

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

 is sucked up after it to separate it from the bacterial emulsion, which 

 is then drawn up in the same quantity ; the diluted serum will then 

 occupy the position Jcl. The fluids are then drawn several times up 

 into the bulb and returned to the capillary tube so as to mix, and finally 

 blown carefully down close to the lower end, which is then sealed off'. 

 The sediment collects at the lower extremity. 



It may be said that.it is often important to observe not only the 

 strongest concentration of a serum which will produce agglutination but 

 also the weakest. 



Method of measuring the Phagocytic Capacity of the 

 Leucocytes the Opsonic Technique. This was first done 

 by Leishman by a very simple method as follows : A piece 

 of quill tubing is drawn out to a capillary diameter so as 

 to make a pipette about six inches long. The point is 

 broken off and a rubber nipple adjusted to the wide end, a 

 mark is made with an oil pencil about three-quarters of an 

 inch above the orifice. Blood is drawn from the finger up 

 to the mark, then an air-bubble is allowed to pass in. A thin 



