METHODS OF COUNTING BACTEEIA 131 



some of the heated vaccine to an agar tube and incubating for 

 twenty-four hours. Appropriate doses are then with all aseptic 

 precautions measured by means of a sterile graduated pipette, 

 and placed in little glass bulbs or ampoules drawn out to a 

 capillary tube at one end. It is usual to add sufficient \ per 

 cent, phenol in sterile saline to make the contents of the 

 ampoule up to about 1 c.c. The ampoules 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, the organism is 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 and kept for forty -two hours at 37° C. The 

 bacteria are then killed by the flask being put into a water bath 

 at 53° C. for twenty minutes ; - 5 per cent, lysol is added, and 

 the bacteria in the vaccine are counted. 



The dosage is adjusted to the standard described in 

 Chapter XV. 



Special methods are adopted in preparing the vaccines used 

 in connection with tuberculosis, cholera, plague, etc., and are 

 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, advisable s to 

 know roughly 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. The diluent ought also to contain some antiseptic, 

 especially when the organisms are motile. 



In MacAlister's method, the suspension of bacteria is diluted 

 with decinormal solution of hydrochloric acid, and a drop of the 

 mixture is placed in the counting chamber. The acid causes 

 the organisms to deposit on the two glass surfaces and they can 

 readily be counted in the two planes, this being carried out with 

 dark ground illumination. A dry 7 mm. lens is used along 



