264 A MANUAL OF BACTERIOLOGY 



The vaccine is prepared by growing the organism under appro- 

 priate conditions, the staphylococcus on agar, the streptococcus, 

 pneumococcus, and gonococcus on blood-agar, etc. The growth 

 is then made into a suspension by adding a few drops of sterile 

 saline solution and well rubbing up with a sterile glass or alu- 

 minium rod. Two or three tubes are treated in this way ; the 

 suspension is poured into a sterile tube or small flask of stout 

 glass, the culture tubes are rinsed out with a little more of the 

 salt solution, and the washings added to the suspension, two or 

 three sterile glass beads are added, and the vessel, sealed or 

 corked, is shaken vigorously for half an hour in a shaking machine, 

 so as thoroughly to break up the masses of organisms. If a 

 shaking machine is not available, the tube may be shaken by 

 hand for a few minutes and the suspension centrifuged for five 

 minutes to remove masses. The suspension, which should 

 measure not less than 5 c.c., after either of these operations is 

 ready for standardisation. 



Standardisation is carried out by Wright's method. Two or 

 three volumes of citrate solution are sucked up into a pipette such 

 as that used for opsonic determinations, the finger is pricked and 

 one volume of blood is taken up in the pipette, separated from the 

 citrate solution by an air-bubble, and finally one volume of the 

 bacterial suspension, also separated from the blood by an air- 

 bubble, is taken up. The whole contents of the pipette are then 

 well mixed by expelling on to a clean slide and sucking up three 

 or four times. About one-third of the mixture is then trans- 

 ferred to each of three clean slides, and the drops are spread with 

 the edge of a slide so as to obtain thin uniform smears. These are 

 allowed to dry, stained with Leishman's stain, and the number of 

 red corpuscles and bacteria is counted in a number of micro- 

 scopical fields. Assuming that there are 5,000,000 red cells in a 

 cubic millimetre of blood, it is easy to calculate approximately 

 the number of bacteria contained in the suspension. Suppose 

 that 500 red cells have been counted, and with these 1,500 bacteria 

 are admixed. Since equal volumes of blood and suspension 

 have been taken, one cubic millimetre of bacterial suspension will 



contain 5>000>0 ^ A X 1>50 = 15,000,000 bacteria. But one cubic 

 500 



centimetre contains 1,000 cubic millimetres, therefore the suspen- 

 sion contains 15,000,000 x 1,000 = 15,000,000,000 bacteria per 



