MEASUREMENT OF GROUP AGGLUTININS 121 



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. 44, 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- 

 ing 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. 44, 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 

 kl. The fluids are then drawn several times up into the bulb, and returned 

 to the capillary tube so as to mix, arid finally blown carefully down close 

 to the lower end, which is then sealed off. The sediment collects at the 

 lower extremity. 



It is often important to observe not merely the fact that agglutination 

 occurs, but also the weakest concentration of the serum with which the 

 reaction can be obtained. 



Measurement of Group Agglutinins. In the case of certain 

 groups of allied organisms, notably the b. coli and its allies, 

 it has been found that when a serum clumps one member of the 

 group it frequently also clumps the allied forms. If the greatest 

 dilution with which agglutination is obtained be estimated, the 

 end-points for the different strains affected will be found to 

 differ. The determination of the end-point is important, as the 

 disease condition from which the serum is derived is generally 

 caused by the organism which is clumped in highest dilution. 



