PREPARATION OF LEUCOCYTES 123 



present. 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 con- 

 tain 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 the 

 case of the tubercle bacillus, as short a variety of the organism 

 as possible should be selected, and a mass of growth off a solid 

 medium is taken (bacilli in mass can be obtained in the market 

 from wholesale chemists) and is well washed with changes of 

 distilled water, drained on filter paper in a Petri dish, and 

 thoroughly rubbed up with a little 1'5 per cent, saline in an 

 agate mortar, so as to disintegrate the bacterial masses and get 

 an emulsion composed as far as possible of individual bacilli. It 

 is extremely difficult to obtain a good emulsion of tubercle bacilli, 

 i.e. one that shall consist as far as possible of separate bacilli 

 on the one hand without clumps, and on the other without 

 portions of disintegrated bacilli. The rubbing-up in the mortar, 

 which usually occupies many hours, must be done very slowly 

 and gently with a very light pestle, and the manipulation must 

 be frequently controlled by microscopic observation. A thick 

 cream should be obtained, and this should be sterilised by 

 steaming for half an hour on three successive days. Before 

 sterilisation it is convenient to seal up the stock emulsion in 

 small quantities in a number of pieces of quill tubing, so that 

 in the subsequent procedures only small portions of the emulsion 

 are exposed to aerial contamination at one time. For actual 

 use, one of those tubes is opened, a little is withdrawn with a 

 sterile pipette, and a weak emulsion made in the same way as 

 with the staphylococcus, except that 1 '5 per cent, saline is used. 

 The stock tube may be sealed with wax and kept for use again. 

 A fresh emulsion ought to be made up for each day's work. 



(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 bringc'the blood in contact with the citrate and^'prevent 

 coagulation. The equivalent of about ten to twenty drops of 



