124 METHODS OF EXAMINING SERUM 



ordinary organisms, e.g., the pyogenic cocci, a little of a twenty- 

 four hour living culture off 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 cells. With 

 certain organisms, e.g., streptococci in chains, a good deal of 

 trituration 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. 

 Wright states it will then contain from 7000 to 10,000 million 

 bacteria per c.cm. If too strong an emulsion be used, the 

 leucocytes may take up so many organisms that these cannot be 

 accurately enumerated. In all cases the aim should be to obtain 

 an emulsion of such strength that about an average of two 

 bacteria per leucocyte will be taken up. When intensely 

 pathogenic organisms are used, e.g., b. pestis, m. melitensis, 

 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 on 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-inch tubing to a point, the tube being filled nearly to the 

 brim. A handkerchief 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 centri- 

 fugalised, and when the corpuscles are separated the supernatant 

 fluid is removed, 1 per cent, saline is substituted, and the 

 centrifugaiisation repeated. A second washing with saline is 



