Meat Extract

A watery solution of the extractives, etc., of lean meat (usually beef) forms the basis of several nutrient media. This solution is termed "meat extract" and it has been determined empirically that its preparation shall be carried out by extracting half a kilo of moist meat with one litre of water. For many purposes, however, it is more convenient to have a more concentrated extract; one kilo of meat should therefore be extracted with one litre of water, to form "Double Strength" meat extract.

It was customary at one time, and is even now in some laboratories to use either "shin of beef" or "beef-steak"—both contain muscle sugar which often needs to be removed before the nutrient medium can be completed. Heart muscle (bullock's heart or sheep's heart) is much to be preferred and from the point of economy, ease and cleanliness of manipulation, and extractive value, the imported frozen bullock's hearts provide the best extract.

Meat extract (Fleischwasser) is prepared as follows:

1. Measure 1000 c.c. of distilled water into a large flask (or glass beaker, or enamelled iron pot) and add 1000 grammes (roughly, 2-1/2 pounds) of fresh lean meat—e. g., bullock's heart—finely minced in a mincing machine.

2. Heat the mixture gently in a water-bath, taking care that the temperature of the contents of the flask does not exceed 40° C. for the first twenty minutes. (This dissolves out the soluble proteids, extractives, salts, etc.)

3. Now raise the temperature of the mixture to the boiling-point, and maintain at this temperature for ten minutes. (This precipitates some of the albumins, the hæmoglobin, etc., from the solution.)

4. Strain the mixture through sterile butter muslin or a perforated porcelain funnel, then filter the liquid through Swedish filter paper into a sterile "normal" litre flask, and when cold make up to 1000 c.c. by the addition of distilled water—to replace the loss from evaporation.

5. If not needed at once, sterilise the meat extract in bulk in the steam steriliser for twenty minutes on each of three consecutive days.

Calf, sheep, or chicken flesh is occasionally substituted for the beef; or the meat extract may be prepared from animal viscera, such as brain, spleen, liver, or kidneys.

Note.—As an alternative method, 5 c.c. of Brand's meat juice or 3 grammes of Wyeth's beef juice, or 10 grammes Liebig's extract of meat (Lemco) may be dissolved in 1000 c.c. distilled water, and heated and filtered as above to form ordinary or single strength meat extract.

Media, prepared from such meat extracts are, however, eminently unsatisfactory when used for the cultivation of the more highly parasitic bacteria; although when working in tropical and subtropical regions their use is well-nigh compulsory.

Reaction of Meat Extract.—Meat extract thus prepared is acid in its reaction, owing to the presence of acid phosphates of potassium and sodium, weak acids of the glycolic series, and organic compounds in which the acid character predominates. Owing to the nature of the substances from which it derives its reaction, the total acidity of meat extract can only be estimated accurately when the solution is at the boiling-point.

Moreover, it has been observed that prolonged boiling (such as is involved in the preparation of nutrient media) causes it to undergo hydrolytic changes which increase its acidity, and the meat extract only becomes stable in this respect after it has been maintained at the boiling-point for forty-five minutes.

Although meat extract always reacts acid to phenolphthalein, it occasionally reacts neutral or even alkaline to litmus; and again, meat extract that has been rendered exactly neutral to litmus still reacts acid to phenolphthalein. This peculiar behaviour depends upon two factors:

1. Litmus is insensitive to many weak organic acids the presence of which is readily indicated by phenolphthalein.

2. Dibasic sodium phosphate which is formed during the process of neutralisation is a salt which reacts alkaline to litmus, but neutral to phenolphthalein. In order, therefore, to obtain an accurate estimation of the reaction of any given sample of meat extract, it is essential that—

1. The meat extract be previously exposed to a temperature of 100° C. for forty-five minutes.

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

3. Phenolphthalein be used as the indicator.

The estimation is carried out by means of titration 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 in tenths of a centimetre. 1. 10N NaOH, accurately standardised.

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

3. 25 c.c. measure (cylinder or pipette, calibrated for 98°C.—not 15°C). 3. n/10 NaOH, accurately standardised

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

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

6. Bohemian glass 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.

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 quantity 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.

(B) Perform a "control" titration (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 titration by the aid of the n/10 NaOH.

4. Note the amount of n/10 NaOH solution required to complete the titration, 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 titre 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.

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 titration 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 titrations, 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 CO2. This may be done by first leading it over H2SO4 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 quantities of the dekanormal soda solution.