BACTERIOLOGICAL TECHNIC ' 95 



give pink colonies, the latter being the brighter, more vermilion in colora- 

 tion, 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 tem- 

 perature colonies as compared with low temperature colonies should not 

 exceed i :ioo or 1:25. If the proportion is i -.4 or less, sewage contamina- 

 tion 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. 



If the temperature and color differential tests indicate sewage con- 

 tamination, then the following additional tests should be carried out. 



III. Indol Reaction and Gas Formula. — The indol reaction has already 

 been explained. The gas formula is determined as follows: To sets of 

 four graduated fermentation tubes containing glucose bouillon and lac- 

 tose bouillon, add o.i, 0.2, 0.5, and 10 cc. of the suspected liquid and 

 incubate at 38° for 48 hours. If gas formation is observed the presence 

 of colon bacilH may be suspected. If the o.i cc. tubes show gas forma- 

 tion then the presence of colon bacilli may be assumed. Fill the bulb 

 of a tube, showing gas formation, with a 2-per cent, solution of sodic 

 hydrate, hold thumb tightly over the opening and mix contents by tilting 

 back and forth carefully. The portion of gas absorbed is CO2 whereas 

 the unabsorbed portion is supposedly hydrogen. The colon bacillus 

 shows a gas formation of ^i hydrogen. Of course the total volume 

 of gas is recorded before the sodic hydrate is added. 



The gas formula with a positive indol reaction is practically conclusive 

 as far as the presence of the colon bacillus is concerned. Add to this the 

 other tests and we have conclusive evidence of sewage .contamination. 



The colon bacillus, the bacilli of the hog cholera group aJid others, 

 have the power of reducing neutral red; producing a greenish-yellow 

 fluorescence. For this reaction use glucose bouillon to which has been 

 added i per cent, of a 0.5 per cent, solution of neutral red. In examining 

 milk, the pus cell and leucocyte count is considered important; centrifu- 

 galize 10 cc. of milk for five minutes, pour off supernatant milk and 

 mix sediment with 0.5 cc. normal salt solution and make counts of pus 

 cells and leucocytes per cc. from the amount (0.5CC.). Abundant pus cells 

 and leucocytes indicate abscess or other pathological condition of milk 

 ducts or glands. This test is, however, of little significance excepting in 

 the hands of authorities on diseases of cows. It is stated that as many as 

 100,000 leucocytes per cc. may occur in apparently healthy animals . 



