94 PHARMACEUTICAL BACTERIOLOGY 



with Turck ruling 1 is used (No. 2 ocular with ^ in. objective) which can be 

 secured from any bacteriological supply house. The instructions for 

 using it can be obtained from the dealer, though the measuring values 

 indicated on the hemacytometer are sufficient to indicate the manner of 

 making the counts. The rulings generally used for bacterial countings are 

 ^00 sq. mm. X Mo mm - deep, making an area of Mooo cu. mm., or re- 

 duced to decimal fractions, 0.04 sq. mm! X o.i mm. deep = 0.004 cu. mm. 

 We will suppose that the average of 20 counts shows 5 bacilli, then i cu. 

 mm. would contain 20,000 bacilli or 20,000,000 in i cc. 



The direct count is, in many instances, very unsatisfactory for several 

 reasons . Particles other than micro-organisms may be mistaken for bacilli 

 or cocci and, furthermore, it cannot be known for a certainty that the organ- 

 isms are dead or alive. If they are present in great abundance (10,000,000 

 to 100,000,000 and more per cc.), ordinary smear preparations may be 

 stained, using methyl blue or Hoffmann's violet. Dead bacilli, that is 

 those which have been dead for some time, do not take the stain well, 

 due to the fact that the cellplasm is disintegrated. 



Tomato pastes, anchovy pastes, catsups, some mineral waters and 

 imilar preparations, may contain bacteria in such numbers that dilutions 

 are desirable or necessary to make counting possible. Dilutions of 

 i-io, i-ioo will, as a rule, be sufficient. Weigh or measure one part 

 (i gm. or i cc.) of the substance, add it to 9 or 99 parts filtered distilled 

 water and mix thoroughly by shaking. 



If the direct count shows bacilli in great numbers or if for any reason 

 sewage contamination is suspected, and also to determine the number of 

 living bacilli and spores suspected, proceed as follows: 



II. Plate Culture Counts. Make one set of plate cultures, using lactose 

 litmus agar, 2 and incubate at 20 C. Make a second set of plate cultures, 

 also upon lactose litmus agar, and incubate at 38 C. The usual dilution 

 methods are followed when necessary, using preferably o.i cc. quantities 

 for the plates. This temperature differential test is considered of great 

 importance. Colon bacilli and other micro-organisms, whose natural 

 habitat is the intestinal canal, will develop actively at the higher tempera- 

 ture (38 C.), whereas the usual air, soil and water bacteria develop best 

 at the lower temperature (20 C.). If the high temperature colonies ap- 

 proximate the low temperature colonies, sewage contamination may be 

 suspected. If in addition many of the high temperature lactose litmus 

 agar colonies show pink or light vermilion, the sewage contamination is 

 practically proven. The colon bacillus, as well as sewage streptococci, 



1 To render the ruled lines visible rub a very soft pencil over the ruled area. 

 2 Add i per cent, of lactose to the usual agar medium and enough tincture of litmus 

 to give it a lilac tinge. 



