The Elements of Bacteriological Technique Part 34

2. The estimation be performed at the boiling-point.

3. Phenolphthalein be used as the indicator.

The estimation is carried out by means of t.i.tration experiments against standard solutions of caustic soda, in the following manner:

_Method of Estimating the Reaction._--

_Apparatus Required_: _Solutions Required_:

1. 25 c.c. burette graduated 1. 10N NaOH, accurately in tenths of a centimetre. standardised.

2. 1 c.c. pipette graduated in 2. n/1 NaOH, accurately hundredths, and provided standardised with rubber tube, pinch-c.o.c.k, and delivery nozzle.

3. 25 c.c. measure (cylinder or 3. n/10 NaOH, accurately pipette, calibrated for standardised.

98C.--_not_ 15C).

4. Several 60 c.c. conical 4. 0.5 per cent. solution of beakers or Erlenmeyer phenolphthalein in 50 per flasks. cent. alcohol.

5. White porcelain evaporating basin, filled with boiling water and arranged over a gas flame as a water-bath.

6. Bohemian gla.s.s flask, fitted as a wash-bottle, and filled with distilled water, which is kept boiling on a tripod stand.

METHOD.--Arrange the apparatus as indicated in figure 97.

(A) 1. Fill the burette with n/10 NaOH.

2. Fill the pipette with n/1 NaOH.

[Ill.u.s.tration: FIG. 97.--Arrangement of apparatus for t.i.trating media.]

3. Measure 25 c.c. of the meat extract (previously heated in the steamer at 100 C. for forty-five minutes) into one of the beakers by means of the measure; rinse out the measure with a very small quant.i.ty of boiling distilled water from the wash-bottle, and then add this rinse water to the meat extract already in the beaker.

4. Run in about 0.5 c.c. of the phenolphthalein solution and immerse the beaker in the water-bath, and raise to the boil.

5. To the medium in the beaker run in n/10 NaOH cautiously from the burette until the end-point is reached, as indicated by the development of a pinkish tinge, shown in figure 98 (b). Note the amount of decinormal soda solution used in the process.

NOTE.--Just before the end-point is reached, a very slight opalescence may be noted in the fluid, due to the precipitation of dibasic phosphates. After the true end-point is reached, the further addition of about 0.5 c.c.

of the decinormal soda solution will produce a deep magenta colour (Fig. 98, c), which is the so-called "end-point" of the American Committee of Bacteriologists.

[Ill.u.s.tration: FIG. 98.--a, Sample of filtered meat extract or nutrient gelatine to which phenolphthalein has been added. The medium is acid, as evidenced by the unaltered colour of the sample. b, The same neutralised by the addition of n/10 NaOH. The production of this faint rose-pink colour indicates that the "end-point," or neutral point to phenolphthalein, has been reached. If such a sample is cooled down to say 30 or 20 C., the colour will be found to become more distinct and decidedly deeper and brighter, resembling that shown in c. c, Also if, after the end-point is reached, a further 0.5 c.c. or 1.0 c.c. n/10 NaOH be added to the sample, the marked alkalinity is evidenced by the deep colour here shown.]

(B) Perform a "control" t.i.tration (occasionally two controls may be necessary), as follows:

1. Measure 25 c.c. of the meat extract into one of the beakers, wash out the measure with boiling water, and add the phenolphthalein as in the first estimation.

2. Run in n/1 NaOH from the pipette, just short of the equivalent of the amount of _deci_-normal soda solution required to neutralise the 25 c.c.

of medium. (For example, if in the first estimation 5 c.c. of n/10 NaOH were required to render 25 c.c. of medium neutral to phenolphthalein, only add 0.48 c.c. of n/1 NaOH.) Immerse the beaker in the water-bath.

3. Complete the t.i.tration by the aid of the n/10 NaOH.

4. Note the amount of n/10 NaOH solution required to complete the t.i.tration, and add it to the equivalent of the n/1 NaOH solution previously run in. Take the total as the correct estimation.

_Method of Expressing the Reaction._--

The reaction or _t.i.tre_ of meat extract, medium, or any solution estimated in the foregoing manner, is most conveniently expressed by indicating the number of cubic centimetres of normal alkali (or normal acid) that would be required to render _one litre_ of the solution exactly neutral to phenolphthalein.

[Ill.u.s.tration: FIG. 99.--Stock bottle for dekanormal soda solution.]

The sign + (plus) is prefixed to this number if the original solution reacts acid, and the sign - (minus) if it reacts alkaline.

For example, "meat extract + 10," indicates a sample of meat extract which reacts acid to phenolphthalein, and would require the addition of 10 c.c. of _normal_ NaOH per litre, to neutralise it.

NOTE.--Such a solution would probably react alkaline to litmus.

Conversely, if as the result of our t.i.tration experiments we find that 25 c.c. of meat extract require the addition of 5 c.c. n/10 NaOH to neutralise, then 1000 c.c. of meat extract will require the addition of 200 c.c. n/10 NaOH = 20 c.c. n/1 NaOH.

And this last figure, 20, preceded by the sign + (i. e., +20), to signify that it is acid, indicates the reaction of the meat extract.

NOTE.--The standard soda solutions should be prepared by accurate measuring operations, controlled by t.i.trations, from a stock solution of 10N NaOH, which should be very carefully standardised. If a large supply is made or the consumption is small this stock solution must be kept in an aspirator bottle to which air can only gain access after it has been dried and rendered free from CO_{2}. This may be done by first leading it over H_{2}SO_{4} and soda lime, or soda lime alone, by some such arrangement as is shown in figure 99, which also shows a constant burette arrangement for the delivery of small measured quant.i.ties of the dekanormal soda solution.

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 _t.i.tre_ of the medium ma.s.s; 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 t.i.tration 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 _t.i.tre_ 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 t.i.trations, 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 quant.i.ty 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.