BACTERIOLOGICAL TECHNIC. 67 



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 c.c.), ordinary smear preparations maybe stained, 

 using methyl blue or fuchsin. Dead bacilli, that is those which have been 

 dead for some time, do not take the stain well, due" to the fact that the cell- 

 plasm is disintegrated. 



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

 similar preparations, may contain bacteria in such numbers that dilutions 

 are desirable or necessary to make counting possible. A dilution of one in 

 ten will, as a rule, be sufficient. Weigh or measure one part (i gm or r 

 c.c.) of the substance, add it to nine 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, 1 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 c.c. quantities for 

 the plates. This temperature differential test is considered of great impor- 

 tance. Colon bacilli and other micro-organisms, whose natural habitat is 

 the intestinal canal, will develop actively at the higher temperature (38 C.), 

 whereas the usual air, soil and water bacteria develop best at the lower tem- 

 perature (20 C.). If the high temperature colonies approximate the low 

 temperature colonies, sewage contamination may be suspected. If in addi- 

 tion 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, give pink colonies, the latter being the 

 brighter, more vermilion in coloration, due to the formation of acid (in the 

 fermenting lactose) . Examine the pink colonies under the microscope. The 

 colon microbe is rod-shaped, rather thick, non-sporing. and shows motility in 

 recent broth cultures, whereas the streptococci are smaller and are not rod- 

 shaped. High temperature colonies as compared with low temperature col- 

 onies should not exceed 1:100 or 1:25. If the proportion is 1:4 or less, 

 sewage contamination is very likely. After 36 hours the pink colonies may 

 turn blue, due to the development of ammonia and amines. 



Naturally the high temperature colonies must be studied within twenty- 

 four to thirty hours whereas the low temperature cultures require much more 

 time, two to four days. 



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

 to give it a lilac tinge. 



