220 A MANUAL OF BACTERIOLOGY 



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

 then made into a suspension by adding a few drops of sterile 0-1 per 

 cent, sodium chloride solution and well rubbing up with a sterile glass 

 or aluminium 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 some time, preferably in a shaking machine, 

 so as thoroughly to break up the masses of organisms. The con- 

 tents of the vessel, which should measure 5 c.c. or thereabouts, are 

 then centrifuged for some minutes, the suspension is poured off from 

 the deposit into a second sterile flask and is now 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 transferred 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 microscopical fields. Assum- 

 ing 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 1500 bacteria are admixed. Since equal volumes 

 of blood and suspension have been taken, one cubic millimetre of 



bacterial suspension will contain 5 ' 000>0 ^ Q X 15Q = 15,000,000 



>00 



bacteria. But one cubic centimetre contains 1000 cubic milli- 

 metres, therefore the suspension contains 15,000,000 x 1000 = 

 15,000,000,000 bacteria per cubic centimetre, and by appropriate 

 dilution any bacterial content of the suspension may be obtained. 

 Thus, if 1,000,000,000 organisms per cubic centimetre is desired, 

 1 c.c. of the suspension must be diluted with 14 c.c. of salt solution. 

 To the prepared dilution of the bacterial suspension 0-5 percent, of 

 carbolic acid, or 0-2 per cent, of trikresol, is added, and the flask is 

 placed in a water-bath at 56 to 60 C. for one or one and a half 



