[ 2 ] ; 
(4) Addition to Peptone water of 5 cc. and sometimes of other 
amounts of the water to be examined and testing for Indol at the end 
of 48 hours. 
As determined by plating the number of organisms in all natural waters 
is very high as compared with European standard and the number when 
cultivated at blood heat is as great as at room temperatures and hi«-her a^ 
a rule than in gelatine plates at 22° C. 
For purposes of comparison the plates used were of Agar-Agar made 
with Liebig’s Extract and neutralised so as to be of an alkalinity + 10 as 
tested by Phenolphthalein. The plates were kept at a temperature of 37 0 C. 
and counts were made up to the 5th day. Few new colonies are found 
after the third day whether the plate be kept at 37° C., in the cold incubator 
at 22 C. or at room temperature. 
MacConkey’s medium is, in my opinion, of particular value, and the 
absence of the formation of acid and gas within 24 hours is a certain proof of 
the purity of the water. It is not a test of sewage contamination as many j 
natural waters will give the reaction even with .4 cc. or less. Amongst 
these waters is that found in the stems of upright growing bamboos 
\\ Inch ha\ e been perforated by a borer and in which water is found. 
In such a stem with an aperture from 4- to ^ of an inch any contamination 
l>\ animals except insects and other small mvertibrates is impossible. The 
\ame of the medium is that it is a crucial test of the purity of w r ater. 
Plating from the growth in this medium is of considerable value. 
For this purpose it is essential that young cultures preferably 24 hours 
old should be used as the more delicate organisms, those most closely related 
to the human intestinal organisms, pathogenic or non-pathogenic, are 
destroyed in the acid medium into which this medium is converted by the 
growth of organisms. If plated from older cultures, these organisms may 
not be found. 
Plating from this bile salt broth in Gelatine is best as it is the non¬ 
liquefying organisms that are of importance. 
In this manner from nearly all such w'aters short bacilli can be isolated 
which decompose glucose or lactose or both with the formation of gas or 
wdth the formation of acid only. They are non-spore forming are usually ! 
motile but vary in motility. They do not retain their stain when treated 
by Gram’s method. Only a few of them curdle milk and that not for three 
or four days as a rule. 
Few of them form indol and none indol and nitrites. Some are not 
pathogenic to rats or guinea pigs whether injected subcutaneously or into 
the peritoneum. Others are fatal w'hen injected intraperitoneally but not 
wTen injected subcutaneously. An extensive local slough or pus may be 
caused or no lesion at all may result from the subcutaneous injection. 
In appearance of growth they are often indistinguishable, and in any 
case from the non-pathogenic to the most virulent there is no more 
difference in the growth on Agar, Gelatine, Potato, etc., than in various 
laboratory strains ot B. Coli communis to which organism they appear 
to be related. 
The presence of these classes of organisms in minute samples of the 
wrater is indicated by the use of MacConkey’s medium. Though not of the 
same importance as that class of organisms would be in a temperate climate 
I consider that such w’ater should be viewed with suspicion. 
