METHODS OF COUNTING BACTERIA 135 



normal saline. A uniform emulsion is necessary, and if clumps 

 are present these must be disintegrated with a shaking- machine, 

 or deposited by centrifuging. A sample of the living emulsion 

 is withdrawn for the enumeration of the organisms (vide infra\ 

 and the vaccine is then sterilised by heating in a water bath at 

 60 C. for half an hour. With certain staphylococci a longer 

 exposure, say an hour, is advisable, and sometimes in such cases 

 a higher temperature must be employed. The efficiency of the 

 sterilisation must be tested by transferring some of the heated 

 vaccine to an agar tube and incubating for twenty-four hours. 

 Appropriate doses (see Chapter VII.) are then with all aseptic 

 precautions measured by means of a sterile graduated pipette, 

 and placed, along with an equal volume of *5 per cent, lysol, in 

 little glass bulbs drawn out to a capillary tube at one end. These 

 when charged are sealed, and for use the sealed end is broken 

 off, the contents are sucked up into a sterile hypodermic needle, 

 and injected fairly deeply into the skin, usually in the region of 

 the flank. 



In the case of the typhoid bacillus, organisms are used of such 

 virulence that a quarter of a twenty-four hours' old sloped agar 

 culture, when administered hypodermically, will kill a guinea- 

 pig of from 350 to 400 grams. Flasks of bouillon are inoculated 

 with such a culture for forty-two hours at 37 C. The bacteria 

 are then killed by the flask being put into a water bath at 55 C. 

 for twenty minutes ; '5 per cent, lysol is added, and the bacteria 

 in the vaccine are counted. By such methods, vaccines against 

 any of the pyogenic cocci and against any members of the coli- 

 typhoid group can be made. 



The vaccines used in tuberculosis, cholera, and plague will be 

 described in the chapters on these diseases. 



Methods of counting the Bacteria in Dead Cultures. In 

 the making of vaccines it is, as indicated above, necessary to 

 know the total number of bacterial cells, whether dead or living, 

 present in a culture, for the dead as well as the living contain 

 the toxins which may stimulate the therapeutic capacities of 

 the body. A sufficiently accurate enumeration of the bacteria 

 in a vaccine emulsion can usually be made by counting a suitably 

 diluted sample with a Thoma-Zeiss hsemocytometer. For this 

 purpose Zeiss supplies a special cover-glass, ground thin in the 

 middle so that an oil immersion lens can be used. This is an 

 advantage, but in many cases a dry lens is sufficient, especially 

 if a small quantity of stain, e.g., gentian violet, is added to the 

 diluent. 



Wright's method consists in making a mixture of blood (whose 



