STREPTOCOCCI IN WATER 297 
to forty-eight hours, and, at the end of that period a loopful from the 
surface of each tube should be examined as a hanging drop under the 
microscope. The broth should then be carcfully poured off so as to 
leave at the bottom any precipitate which has been produced. Add a 
few drops of dilute sodium carbonate solution, to dissolve the bile salts 
and examine again in the hanging drop. If distinct chains are not found, 
streptococci may be regarded as absent.’’ This method has been found 
by Thresh to yield the best results. 
Savage’s Method for Isolation of Streptococcus from Water, etc. 
Add 0.1 and 1 c.c. of the sample (water) to tubes of glucose neutral 
red broth and 10 cc of the sample to a tube of double strength 
neutral red broth. Incubate the tubes at 37° C. for forty-eight hours 
and then examine microscopically in hanging drop for chains of cocci. 
The broth tube containing the quantity of water next below the one 
giving a positive result is examined several times in fresh preparations. 
Where it is doubtful whether chains are present, a little of the fluid is 
centrifugalized and stained with methylene blue. The streptococci are 
best isolated from glucose broth tubes by adding a platinum loopful to 
a litmus lactose plate and distributing over its surface. All suspicious 
strains must be subcultured and worked out. 
Method for Separation of B. coli and Streptococci in Water. ‘“ Inoc- 
ulate the desired quantity of water, preferably 1 c.c. into dextrose broth 
fermentation tube. Incubate at 37° C. Examine after six to twelve 
hours. Within two to three hours after gas formation has appeared, 
plate the broth into litmus lactose agar, incubating for twelve to eigh- 
teen hours at 37° C. If, at the end of this time, no acid-producing 
colonies are present, it is probably safe to assume that there were no 
colon bacilli present.” 
After the first plating from dextrose broth, replace the fermentation 
tube for thirty-six hours and plate in litmus lactose agar. This culture 
should give a nearly pure culture of streptococci, if these organisms 
were originally present in the water. 
Bacillus Enteritidis Sporogenes. This organism was discovered by 
Klein in 1895 in diarrhoea stools. Its use as an indicator of pollution 
rests on the fact that it is present in feces and, therefore, in sewage. 
This organism should be regarded as one of a group of bacteria the 
members of which are very closely related. Table XX VIIT quoted from 
Thresh will indicate the essential differences between certain members of 
this group. Bacillus welchit (Bacillus aerogenes capsulatus) 1s probably 
identical with Klein’s Bacillus enteritidis sporogenes. Welch describes 
his bacillus as non-motile but otherwise the descriptions and the changes 
