Standardisation of Media

Differences in the reaction of the medium in which it is grown will provoke not only differences in the rate of growth of any given bacterium, but also well-marked differences in its cultural and morphological characters; and nearly every organism will be found to affect a definite "optimum reaction"—a point to be carefully determined for each. For most bacteria, however, the "optimum" usually approximates fairly closely to +10; and as experiment has shown that this reaction is the most generally useful for routine laboratory work, it is the one which may be adopted as the standard for all nutrient media derived from meat extract.

Briefly, the method of standardising a litre of media to +10 consists in subtracting 10 from the initial titre of the medium mass; the remainder indicates the number of cubic centimetres of normal soda solution that must be added to the medium, per litre, to render the reaction +10.

Standardising Nutrient Bouillon.—For example, 1000 c.c. bouillon are prepared; at the first titration it is found

1. 25 c.c. require the addition of 5.50 c.c. n/10 NaOH to neutralise.

Two controls give the following results:

2. 25 c.c. require the addition of 5.70 c.c. n/10 NaOH to neutralise.

3. 25 c.c. require the addition of 5.60 c.c. n/10 NaOH to neutralise.

Averaging these two controls, 25 c.c. require the addition of 5.65 c.c. n/10 NaOH to neutralise, and therefore 1000 c.c. require the addition of 226 c.c. n/10 NaOH, or 22.60 c.c. n/1 NaOH, or 2.26 c.c. n/10 NaOH.

Initial titre of the bouillon = +22.6, and as such requires the addition of (22.6 c.c. - 10 c.c.) = 12.6 c.c. of n/1 NaOH per litre to leave its finished reaction +10.

But the three titrations, each on 25 c.c. of medium, have reduced the original bulk of bouillon to (1000 - 75 c.c.) = 925 c.c. The amount of n/1 NaOH required to render the reaction of this quantity of medium +10 may be deduced thus:

1000 c.c.:925 c.c.::12.6 c.c.:x.

Then x = 11.65 c.c. n/1 NaOH.

Whenever possible, however, the required reaction is produced by the addition of dekanormal soda solution, on account of the minute increase it causes in the bulk, and the consequent insignificant disturbance of the percentage composition of the medium. By means of a pipette graduated to 0.01 c.c. it is possible to deliver very small quantities; but if the calculated amount runs into thousandth parts of a cubic centimetre, these are replaced by corresponding quantities of normal or even decinormal soda.

In the above example it is necessary to add 11.65 c.c. normal NaOH or its equivalent, 1.165 c.c. dekanormal NaOH. The first being too bulky a quantity, and the second inconveniently small for exact measurement, the total weight of soda is obtained by substituting 1.16 c.c. dekanormal soda solution, and either 0.05 c.c. of normal soda solution or 0.5 c.c. of decinormal soda solution.

Standardising Nutrient Agar and Gelatine.—The method of standardising agar and gelatine is precisely similar to that described under bouillon.