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The Bile Salt Media.
These media were prepared exactly as recommended by MacConkey (1908), Neutral Red being used as the indicator throughout. To the stock Bile Salt Peptone water solu- tion, the various sugar media were made by adding respectively Glucose 1%, Lactose 1%, Cane Sugar 1%, Dulcit 0.5%, Adonit 0.5%, Inulin 1%, Inosit 0.5%, Mannite 1%.
Great care was taken not to overheat the media in the sterilisation and Durham's tubes for gas fermation were always used.
For the water samples concentrated solution was used for the larger quantities as required.
The Bile Salt Agar was made by dissolving 2% of Agar and 1% Lactose in the stock fluid clearing with white egg and using: Neutral Red as the indicator.
The Litmus milk, Gelatine and the ordinary Agar Media were all carefully brought to a +10 (Eyre Scale) Reaction to Phenolphthalein; 12% Gelatine was used for the Gelatine Media and extract of beef was used both in the case of the Gelatine and Agar Media.
Acidity in Litmus Whey.
The cultures were grown for 3 days at 37° C. in a known volume of "medium and then tested by the addition of N A ŎH.
N
10
The results can only be considered approximate and it is very doubtful if the results obtained were worth the trouble involved.
The Reduction of Nitrates.
The medium used was as follows:-2 grammes of Nitrite free Potassium Nitrate and 10 grammes of Peptone were added to a litre of Am monia free distilled water.
The reduction was tested for by means of Ilvosay's solution made up as described by Thresh (1904). The reaction was applied cualitatively only, there was unfortunately no time to carry out the quantitative method described by Erich Pelz (1911). Using the qualitative test only, the results given proved to be of little value. The first 120 Lactose fermenters taken from the stock of pure cultures isolated from the water all gave the reaction and further tests also gave positive results.
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These results agree with those of MacConkey (1909), Savage (1906), etc. Houston (1902-3) states that possibly the test is neglected without sufficient reason and that a negative result with a coli-like microbe isolated from water would be a point against considering such a microbe as of intestinal origin".
Indol Reaction.
The method used for this test was Ehrlich's as described by Bohme (1906) and Marshall (1907). No case was considered negative unless the organism had first been grown for six days before applying the test. In doubtful cases the culture after addition of the reagents was shaken up with Amyl Alcohol to extract any colouring matter which might be there. As the test was thus used with a considerable amount of care a good deal of importance was attached to a negative result.
Vosges and Proskauer's Reaction.
A Glucose tube was inoculated and grown at 37° C. for four days, a strong solution of alkali was then added and the tube stood at Room Temperature for twenty-four hours. It was then examined for the reaction.
The Neutral Red Reaction.
This reaction was tested for either in Neutral Red Broth or in Neutral Red Glucose Agar shakes, made up according to Savage (1901). In my experience this is an unsatis- factory test and its use was continued only so as to be able to classify the organism by the "Flaginac" and "Excretal" methods.