112 MICROSCOPIC METHODS 



emulsion of the bacterium to be tested having been prepared, a 

 quantity of this is also drawn up to the mark. The two fluids 

 are then thoroughly mixed by being first blown out on to a 

 sterile slide and then being drawn back into the pipette and 

 expelled, this being repeated several times. A cover-glass is 

 placed over the drop, and the slide is placed in the incubator at 

 37 C. for fifteen minutes. The cover-glass is then slipped off 

 so as to make a film preparation which in the case of ordinary 

 bacteria may be stained by Irishman's method. The number 

 of bacteria present in, say, 50 polymorphonuclear cells succes- 

 sively examined is determined and an average struck. The 

 method was first used for showing that in cases of staphylococcus 

 infection the average number of bacteria taken up was less than 

 in a control in which the same bacterial emulsion was exposed 

 to the blood of a healthy individual. In making such an 

 observation drops from the two mixtures are placed on the same 

 slide under separate cover-glasses and the preparation incubated. 

 One cover is then slipped to one end of the slide and the other to 

 the other, the two films being then stained as one. 



According to Wright's view the process of phagocytosis in 

 blood outside the body is not a simple one, and^, before a 

 leucocyte takes up a bacterium the latter must be acted on 

 in some way by substances present in the serum, which Wright 

 calls opsonins (see Immunity). The technique by which the 

 actions of these opsonins is studied has been elaborated by 

 Wright and his co-workers in connection with his work on 

 bacterial vaccines, especially in relation to infection by the 

 pyogenic cocci and the tubercle bacillus. This technique 

 involves (1) the preparation of the bacterial emulsion, (2) the 

 preparation of the leucocytes, (3) the preparation of samples of 

 (a) serum from a normal person, (6) serum from the infected 

 person. 



(1) Preparation of bacterial emulsion. In the case of 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. The mixture is placed in a tube and centri- 

 fugalised so as to deposit any masses of bacteria which may be 

 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 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 the case of 



