OPSONIC METHODS 121 



(1) Preparation of Bacterial Emulsion. — In the case of ordinary organ- 

 isms, e.g., the pyogenic cocci, a little of a twenty-four-hour living culture 

 on a sloped agar tube is taken and rubbed up in a watch-glass with "85 per 

 cent, saline so as to obtain an emulsion consisting of single bacterial delist 

 With certain' organisms, e.g., streptococci in chains, a good deal of tritura- 

 tion may be necessary, and often centrifuging must be practised, for the 

 removal of clumps. Only by experience can a knowledge be gained of 

 the amount of culture to be used in the first instance, but the resultant 

 emulsion usually should exhibit only the merest trace of cloudiness to the 

 naked eye. If too strong an emulsion be used, the leucocytes may take 

 up so many organisms that these cannot be accurately enumerated. 

 When intensely pathogenic organisms are used, e.g., b. pestis, m. meli- 

 tensis, Wright recommends that the culture should be first killed by 

 emulsifying in 40 per cent, formalin. The latter is then removed by 

 centrifuging and the deposit washed with saline. In the case of the 

 tubercle bacillus, Wright directs that a 7-10 day culture in glycerin broth 

 should be sterilised by heat, collected on a 

 filter, washed with salt solution, and dried. 

 Ten milligrams of the dry culture should 

 be powdered in a small agate mortar, a 

 drop of 1 per cent, saline added, and 

 the sticky paste triturated for about five 

 minutes ; further saline is added drop by 

 drop till a thick emulsion is obtained of 

 the bulk of about 1 c. c. This is centrifuged 

 and the supernatant suspension pipetted 

 off and diluted to the necessary degree. 



(2) Preparation of Leucocytes. — Here the 

 observer uses his own blood cells. A 

 1 '5 per cent, solution of sodium citrate in 

 •85 per cent, sodium chloride is prepared. 

 This is placed in a glass tube 3 inches 

 long, made by drawing out a piece of half- *'m- 41.— Wright's blood-cap- 

 inch tubing to a point, the tube being sule > and method of filling 

 filled nearly to the brim. A handkerchief same - 



being bound round the finger, this is now 



pricked, and the- blood allowed to flow directly into the fluid, to the 

 bottom of which it sinks. The tube ought to be inverted between the 

 addition of every few drops of blood, so as to bring the blood in contact 

 with the citrate and prevent coagulation. The equivalent of about ten to 

 twenty drops of blood should be obtained. The diluted blood is then 

 centrifuged, and when the corpuscles are separated the supernatant 

 fluid is removed, fresh saline is siibstituted, and the centrifugalisation 

 repeated. A second washing with saline is practised, the supernatant 

 fluid removed, and the greyish surface layer of blood, which is rich in 

 leucocytes, removed by a fine pipette. The leucocytes may be thoroughly 

 mixed by drawing up in a fine pipette and blowing out again, this being 

 repeated several times. 



(3) Preparation of the Sera. — Each sample of serum is prepared by the 

 methods described in the case of agglutination (p. 117). If a Wright's 

 capsule is used the blood is taken as shown in Fig. 41. The ends are 

 sealed and the capsule is now hung by the bend on the edge of a centri- 

 fuge tube, and the serum separated by spinning the instrument. In each 

 case serum from a normal individual should be prepared as a control. 



