The Elements of Bacteriological Technique Part 35

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 quant.i.ties; but if the calculated amount runs into thousandth parts of a cubic centimetre, these are replaced by corresponding quant.i.ties 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 quant.i.ty, and the second inconveniently small for exact measurement, the total weight of soda is obtained by subst.i.tuting 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.

THE FILTRATION OF MEDIA.

~Fluid media~ are usually filtered through stout Swedish filter paper (occasionally through a porcelain filter candle), and in order to accelerate the rate of filtration the filter paper should be folded in that form which is known as the "physiological filter," not in the ordinary "quadrant" shape, as by this means a large surface is available for filtration and a smaller area in contact with the gla.s.s funnel supporting it.

To fold the filter proceed thus:

1. Take a circular piece of filter paper and fold it exactly through its centre to form a semicircle (Fig. 100, a).

2. Fold the semicircle exactly in half to form a quadrant; make the crease 2, distinct by running the thumbnail along it, then open the filter out to a semicircle again.

3. Fold each end of the semicircle in to the centre and so form another quadrant; smooth down the two new creases 3 and 3a, thus formed and again open out to a semicircle.

4. The semicircle now appears as in figure 100, a, the dark lines indicating the creases already formed.

5. Fold the point 1 over to the point 3, and 1a to 3a, to form the creases 4 and 4a, indicated in the diagram by the light lines. Fold point 1 over to 3a, and 1a to 3, to form the creases 5 and 5a.

[Ill.u.s.tration: FIG. 100.--Filter folding: a, Filter folded in half, showing creases; b, appearance of filter on completion of folding; c, filter opened out ready for use.]

6. Thus far the creases have all been made on the same side of the paper. Now subdivide each of the eight sectors by a crease through its centre on the opposite side of the paper, indicated by the faint broken lines in the diagram. Fold up the filter gradually as each crease is made, and when finished the filter has a.s.sumed the shape of a wedge, as in figure 100, b.

When opened out the filter a.s.sumes the shape represented in figure 100, c.

The folded filter is next placed inside a gla.s.s funnel supported on a retort stand, and moistened with hot distilled water before the filtration of the medium is commenced.

~Liquefiable solid media~ are filtered through a specially made filter paper--"papier Chardin"--which is sold in boxes of twenty-five ready-folded filters.

[Ill.u.s.tration: FIG. 101.--Hot-water filter funnel and ring burner.]

Gelatine, when properly made, filters through this paper as quickly as bouillon does through the Swedish filter paper, and does _not_ require the use of the hot-water funnel.

Agar, likewise, if properly made, filters readily, although not at so rapid a rate as gelatine. If badly "egged," and also during the winter months, it is necessary to surround the gla.s.s funnel, in which the filtration of the agar is carried on, by a hot-water jacket. This is done by placing the gla.s.s funnel inside a double-walled copper funnel--the s.p.a.ce between the walls being filled with water at about 90 C.--and supporting the latter on a ring gas burner fixed to a retort stand (Fig. 101). The gas is lighted and the water jacket maintained at a high temperature until filtration is completed. If the steam steriliser of the laboratory is sufficiently large, it is sometimes more convenient to place the flask and filtering funnel bodily inside, close the steriliser and allow filtration to proceed in an atmosphere of live steam, than to use the gas ring and hot-water funnel.

STORING MEDIA IN BULK.

After filtration fill the medium into sterile litre flasks with cotton-wool plugs and sterilise in the steamer for twenty minutes on each of three consecutive days. After the third sterilisation, and when the flasks and contents are cool, cut off the top of the cotton-wool plug square with the mouth of the flask; push the plug a short distance down into the neck of the flask and fill in with melted paraffin wax to the level of the mouth. When the wax has set the flasks are stored in a cool dark cupboard for future use.

[Ill.u.s.tration: FIG. 102.--Rubber cap closing store bottle. a, before, and b, after sterilizing.]

This plan is not absolutely satisfactory, although very generally employed on occasion, and it is preferable to fill the medium into long-necked flint gla.s.s bottles (the quart size, holding nearly 1000 c.c., such as those in which Pasteurised milk is retailed) and to close the neck of the bottle by a special rubber cap.[3] This cap is made of soft rubber, the lower part, dome-shaped with thin walls, being slipped over the neck of the bottle (Fig. 102, a). The upper part is solid, but with a sharp clean-cut (made with a cataract or tenotomy knife) running completely through its axis from the centre of the disc to the top of the dome. During sterilisation the air in the neck of the bottle, expanded by the heat, is driven out through the valvular aperture in the solid portion of the stopper. On removing the bottle from the steam chamber, the liquid contracts as it cools, and the pressure of the external air drives the solid piece of rubber down into the neck of the bottle, and forces together the lips of the slit (Fig. 102, b). Thus sealed, the bottle will preserve its contents sterile for an indefinite period without loss from evaporation.

TUBING NUTRIENT MEDIA.

After the final filtration, the nutrient medium is usually "tubed"--_i.

e._, filled into sterile tubes in definite measured quant.i.ties, usually 10 c.c. This process is sometimes carried out by means of a large separator funnel fitted with a "three-way" tap which communicates with a small graduated tube (capacity 20 c.c. and graduated in cubic centimetres) attached to the side. The shape of this piece of apparatus, known as Treskow's funnel, renders it particularly liable to damage. It is better, therefore, to arrange a less expensive piece of apparatus which will serve the purpose equally well (Fig. 103).

A Geissler's three-way stop-c.o.c.k has the tube on one side of the tap ground obliquely at its extremity, and the tube on the opposite side cut off within 3 cm. of the tap. The short tube is connected by means of a perforated rubber cork with a 10 cm. length of stout gla.s.s tubing (1.5 cm. bore). The third channel of the three-way tap is connected, by means of rubber tubing, with the nozzle of an ordinary separator funnel.

Finally, the receiving cylinder above the three-way tap is graduated in cubic centimetres up to 20, by pouring into it measured quant.i.ties of water and marking the various levels on the outside with a writing diamond.

Fluid media containing carbohydrates are filled into fermentation tubes (_vide_ Fig. 21); or into ordinary media tubes which already have smaller tubes, inverted, inside them (Fig. 104), to collect the products of growth of gas-forming bacteria. When first filled, the small tubes float on the surface of the medium after the first sterilisation nearly all the air is replaced by the medium, and after the final sterilisation the gas tubes will be submerged and completely filled with the medium.

[Ill.u.s.tration: FIG. 103.--Separatory funnel and three-way tap arranged for tubing media.]

[Ill.u.s.tration: FIG. 104.--Gas tube (Durham).]

~Storing "Tubed" Media.~--Media after being tubed are best stored by packing, in the vertical position, in oblong boxes having an internal measurement of 37 cm. long by 12 cm. wide by 10 cm. deep. Each box (Fig.

105) has a movable part.i.tion formed by the vertical face of a weighted triangular block of wood, sliding free on the bottom (Fig. 105, A); or by a flat piece of wood sliding in a metal groove in the bottom of the box, which can be fixed at any spot by tightening the thumbscrew of a bra.s.s guide rod which transfixes the part.i.tion (Fig. 105, B). The front of the box is provided with a handle and a celluloid label for the name of the contained medium. These boxes are arranged upon shelves in a dark cupboard--or preferably an iron safe--which should be rendered as nearly air-tight as possible, and should have the words "media stores" painted on its doors.

[Ill.u.s.tration: FIG. 105.--Medium box, showing alternative part.i.tions A and B.]

FOOTNOTES:

[3] This rubber cap has been made for me by the Holborn Surgical Instrument Co., Thavies Inn, London, W. C.

XI. CULTURE MEDIA.

ORDINARY OR STOCK MEDIA.

~Nutrient Bouillon.~--

1. Measure out double strength meat extract, 500 c.c., into a litre flask and add 300 c.c. distilled water.

2. Weigh out Witte's peptone, 10 grammes (= 1 per cent.), salt, 5 grammes (= 0.5 per cent.), and mix into a smooth paste with 200 c.c. of distilled water previously heated to 60 C. (Be careful to leave no unbroken globular ma.s.ses of peptone.)

3. Add the peptone emulsion to the meat extract in the flask and heat in the steamer for forty-five minutes (to completely dissolve the peptone, and to render the acidity of the meat extract stable).

4. Estimate the reaction of the medium; control the result; render the reaction of the finished medium +10 (_vide_ page 155).

5. Heat for half an hour in the steamer at 100C. (to complete the precipitation of the phosphates, etc.).

6. Filter through Swedish filter paper into a sterile flask.

7. Fill into sterile tubes (10 c.c. in each tube).

8. Sterilise in the steamer for twenty minutes on each of three consecutive days--i. e., by the discontinuous method (_vide_ page 35).

NOTE.--As an alternative method when neither fresh nor frozen meat is available nutrient bouillon may be prepared from a commercial meat extract, as follows: