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.
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:
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.
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 glass 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.
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 assumed the shape of a wedge, as in figure 100, b.
When opened out the filter assumes the shape represented in figure 100, c.
The folded filter is next placed inside a glass 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.
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 glass funnel, in which the filtration of the agar is carried on, by a hot-water jacket. This is done by placing the glass funnel inside a double-walled copper funnel—the space 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.
This plan is not absolutely satisfactory, although very generally employed on occasion, and it is preferable to fill the medium into long-necked flint glass 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 quantities, 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-cock 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 glass 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 quantities 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.
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 partition 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 brass guide rod which transfixes the partition (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.
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 Witté'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 masses 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 100°C. (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:
Lemco Broth.—
1. Measure out 250 c.c. distilled water into a litre flask.
2. Weigh out 10 grammes Liebig's Lemco Meat Extract on a piece of clean filter paper and add to the water in the flask. Shake the flask well to make an even emulsion of the meat extract.
3. Weigh out Witté's peptone (10 grammes), salt (5 grammes). Mix into smooth paste with 100 c.c. distilled water previously heated to 60°C.
4. Add the peptone salt emulsion to the meat extract emulsion in the flask and add 650 c.c. distilled water. Heat in the steamer for forty-five minutes.
5. Standardise the medium and complete as for nutrient bouillon.
Nutrient Gelatine.—
1. Weigh a 2-litre flask on a trip balance (Fig. 106) and note the weight, or counterpoise carefully.
An extremely useful counterpoise is a small sheet-brass cylinder about 38 mm. high and 38 mm. in diameter, with a funnel-shaped top and provided with a side tube by which its contents, fine "dust" shot, may be emptied out (Fig. 107).
2. Measure out double strength meat extract, 500 c.c., into the "tared" flask.
3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and make into a thick paste with 150 c.c. distilled water; then add the emulsion to the meat extract in the flask; also add 100 grammes sheet gelatine cut into small pieces; place the flask in the water-bath and raise to the boil.
4. Arrange a 5-litre tin can (with copper bottom, such as is used in the preparation of distilled water) by the side of the water bath, fill the can with boiling water and place a lighted Bunsen burner under it. Fit a long safety tube to the neck of the can and also a delivery tube, bent twice at right angles; adjust the tube to reach to the bottom of the interior of the flask containing the gelatine, etc. (Fig. 108).
5. Keep the water in the steam can vigourously boiling, and so steam at 100°C, bubbling through the medium mass, for ten minutes, by which time complete solution of the gelatine is effected. A certain amount of steam will condense as water in the medium flask during this process—hence the necessity for the use of double strength meat extract—but if the water bath is kept boiling this condensation will not exceed 100 c.c.
6. Weigh the flask and its contents; then (1115[4] grammes + weight of the flask) minus (weight of the flask and its contents) equals the weight of water required to make up the bulk to 1 litre. The addition of the requisite quantity of water is carried out as follows:
In one pan of the trip balance place the counterpoise of the tared flask (or its equivalent in weights) together with the weights making up the calculated medium weight. In the opposite pan place the flask containing the medium mass. Now add boiling distilled water from a wash bottle until the two pans are exactly balanced.
7. Titrate and estimate the reaction of the medium mass; control the result. Calculate the amount of soda solution required to make the reaction of the medium mass +10 (i. e., calculate for 1000 c.c., less the quantity used for the titrations).
8. Add the necessary amount of soda solution and heat in the steamer at 100° C. for twenty minutes, to precipitate the phosphates, etc.
9. Allow the medium mass to cool to 60° C. Well whip the whites of two eggs, add to the contents of the flask and replace in the steamer at 100° C. for about half an hour (until the egg-albumen has coagulated and formed large, firm masses floating on and in clear gelatine).
10. Filter through papier Chardin into a sterile flask.
11. Tube in quantities of 10 c.c.
12. Sterilise in the steamer at 100° C. for twenty minutes on each of three consecutive days—i. e., by the discontinuous method.
Nutrient Agar-agar.—
1. Weigh a 2-litre flask and note the weight—or counterpoise exactly.
2. Measure out double strength meat extract, 500 c.c., into the "tared" flask.
3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and 20 grammes of powdered agar, and make into a thick paste with 150 c.c. distilled water, and add to the meat extract in the flask; place the flask in a water-bath.
4. Arrange the steam can and water-bath as already directed (for the preparation of gelatine) and figured.
5. Bubble live steam (at 100° C.) through the medium mass, for twenty-five minutes, by which time complete solution of the agar is effected.
6. Now weigh the flask and its contents; then (1035[5] grammes + weight of flask) minus (weight of flask and its contents) equals the weight of water required to make up the bulk of the medium to 1 litre. Add the requisite amount (see preparation of gelatine, page 166, step 6).
7. Titrate, and estimate the reaction of the medium mass; control the result. Calculate the amount of soda solution required to make the reaction of the medium mass + 10 (i. e., calculated for 1000 c.c., less the quantity used for the titrations).
8. Add the necessary amount of soda solution and replace in the steamer for twenty minutes (to complete the precipitation of the phosphates, etc.).
9. Allow the medium mass to cool to 60° C. Well whip the whites of two eggs, add to the contents of the flask, and replace in the steamer at 100° C. for about one hour (until the egg-albumen has coagulated and formed large, firm masses floating on and in clear agar.)
10. Filter through papier Chardin, by the aid of a hot-water funnel, if necessary (Fig. 101), into a sterile flask.
11. Tube in quantities of 10 c.c. or 15 c.c.
12. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days—i. e., by the discontinuous method.
Blood-serum (Inspissated).—
1. Sterilise cylindrical glass jar (Fig. 109) and its cover by dry heat, or by washing first with ether and then with alcohol and drying.
2. Collect blood at the slaughter house from ox or sheep in the sterile cylinder.
3. Allow the vessel to stand for fifteen minutes for the blood to coagulate. (This must be done before leaving the slaughterhouse, otherwise the serum will be stained with hæmoglobin.)
4. Separate the clot from the sides of the vessel by means of a sterile glass rod (the yield of serum is much smaller when this is not done), and place the cylinder in the ice-chest for twenty-four hours.
5. Remove the serum with sterile pipettes, or syphon it off, and fill into sterile tubes (5 c.c. in each) or flasks.
6. Heat tubes containing serum to 56° C. in a water-bath for half an hour on each of two successive days.
7. On the third day, heat the tubes, in a sloping position, in a serum inspissator to about 72° C. (A coagulum is formed at this temperature which is fairly transparent; above 72° C., a thick turbid coagulum is formed.)
The serum inspissator (Fig. 110) in its simplest form is a double-walled rectangular copper box, closed in by a loose glass lid, and cased in felt or asbestos—the space between the walls is filled with water. The inspissator is supported on adjustable legs so that the serum may be solidified at any desired "slant," and is heated from below by a Bunsen burner controlled by a thermo-regulator. The more elaborate forms resemble the hot-air oven (Fig. 26) in shape and are provided with adjustable shelves so that any desired obliquity of the serum slope can be obtained.
8. Place the tubes in the incubator at 37° C. for forty-eight hours in order to eliminate those that have been contaminated. Store the remainder in a cool place for future use.
Alternative Method.
Steps 1-5 as above.
6. Sterilise the serum by the fractional method—that is, by exposure in a water-bath to a temperature of 56° C. for half an hour on each of six consecutive days; store in the fluid condition.
7. Coagulate in the inspissator when needed.
Serum Water.—
This forms the basis of many useful media, and is prepared as follows:
1. Collect blood in the slaughterhouse (see page 168) and when firmly clotted collect all the expressed serum and measure in a graduated cylinder.
2. For every 100 c.c. of serum add 300 c.c. distilled water and mix in a flask.
3. Heat the mixture in the steamer at 100° C. for thirty minutes. (This destroys any diastatic ferment present in the serum and partially sterilises the fluid.)
4. Filter if turbid.
5. If not needed at once complete the sterilisation of the serum water by two subsequent steamings at 100° C. for twenty minutes at twenty-four hour intervals.
Citrated Blood Agar. Guy's.—
1. Kill a small rabbit with chloroform vapour, and nail it out on a board (as for a necropsy); moisten the hair thoroughly with 2 per cent. solution of lysol.
2. Sterilise several pairs of forceps, scissors, etc. by boiling.
3. Reflect the skin over the thorax with sterile instruments.
4. Open the thoracic cavity by the aid of a fresh set of sterile instruments.
5. Open the pericardium with another set of sterile instruments.
6. Sear the surface of the left ventricle with a red-hot iron.
7. Take a sterile capillary pipette (Fig. 13, c); break off the sealed extremity with a pair of sterile forceps.
8. Steady the heart in a pair of forceps and thrust the point of the pipette through the wall of the ventricle and through the seared area, apply suction to the plugged end of the pipette and fill it with blood.
9. Transfer the entire quantity of blood collected from the rabbit's heart to a small Erlenmeyer flask containing a number of sterile glass beads and 5 c.c. concentrated sod. citrate solution. (See page 378.)
10. Agitate thoroughly and set aside for a couple of hours.
11. Melt up several tubes of nutrient agar (see page 167) and cool to 42° C.
12. With a sterile 10 c.c. graduated pipette transfer 1 c.c. citrated blood from the Erlenmeyer flask to each tube of liquefied agar. Rotate the tube between the hands in order to diffuse the citrated blood evenly throughout the agar.
13. Place the tubes in a sloping position and allow the medium to set.
14. Place tubes of blood agar for forty-eight hours in the incubator at 37° C. and at the end of that time eliminate any contaminated tubes.
15. Store such tubes as remain sterile for future use.
Milk.—
1. Pour 1 litre of fresh cow's or goat's milk into a large separating funnel, and heat in the steamer at 100° C. for one hour.
2. Remove from the steamer and estimate the reaction of the milk (normal cows' milk averages +17). If of higher acidity than +20, or lower than +10, reject this sample of milk and proceed with another supply of milk from a different source.
Reject milk to which antiseptics have been added as preservatives.
3. Allow the milk to cool, when the fat or cream will rise to the surface and form a thick layer.
4. Draw off the subnatant fat-free milk into sterile tubes (10 c.c. in each).
5. Sterilise in the steamer at 100° C. for twenty minutes on each of five successive days.
6. Incubate at 37° C. for forty-eight hours and eliminate any contaminated tubes. Store the remainder for future use.
Litmus Milk.—
1. Prepare milk as described above, sections 1 to 3.
2. Draw off the subnatant fat-free milk into a flask.
3. Add sterile litmus solution, sufficient to colour the milk a deep lavender.
4. Tube, sterilise, etc., as for milk.
Nutrose Agar (Eyre).—
(This is a modification of the well known Drigalski-Conradi medium originally introduced for the isolation of B. typhosus).
1. Collect 250 c.c. perfectly fresh ox serum (vide Blood Serum, page 168, steps 1 to 5) and add to it 450 c.c. sterile distilled water.
2. Weigh out agar powder, 20 grammes, and emulsify it with 250 c.c. of the cold serum water.
3. Weigh out
| Witté's peptone | 10 grammes |
| Sodium chloride | 5 grammes |
| Nutrose | 10 grammes |
and dissolve in 200 c.c. of serum water heated to 80° C.
4. Mix the agar emulsion and the peptone-nutrose solution in a "tared" flask of 2-litre capacity and add a further 100 c.c. serum water.
5. Complete the solution of the various ingredients by bubbling live steam through the flask as in making nutrient agar.
6. Add further 250 c.c. serum water.
7. Weigh the flask and its contents: then (1045 grammes + weight of flask) minus (weight of flask and its present contents) = weight of fluid required to make up the bulk of the medium to 1 litre. Add the requisite amount of sterile distilled water.
8. Titrate and estimate the reaction of the medium mass. Then standardise to reaction of +2.5.
9. Clarify with egg, and filter as for nutrient agar. (In clarifying, after the addition of the egg white the mixture should be in the steamer for full two hours.)
10. After filtration is complete measure the filtrate, and to every 150 c.c. of the medium add:
| Litmus solution (Kahlbaum) | 20 c.c. |
| Krystal violet aqueous solution (1:1000) (B. Hoechst) | 1.5 c.c. |
| Lactose | 1.5 grammes |
11. Tube in quantities of 15 c.c.
12. Sterilise in the steamer at 100° C. for thirty minutes on each of three successive days—i. e., by the discontinuous method for three days.
Egg Medium (Dorset).—
1. Prepare 1000 c.c. of a 0.85 per cent. solution of sodium chloride in a stout 2-litre flask.
2. Sterilise in the autoclave at 120° C. for twenty minutes. Cool to 20° C.
3. Take 12 fresh eggs; wash the shells first with water then with undiluted formalin: allow the shells to dry.
4. Break the eggs into a sterile graduated cylinder and measure the total volume of the mixed whites and yolks. Add one part sterile saline solution to three parts mixed eggs.
5. Transfer this mixture to a large wide-mouthed stoppered bottle previously sterilised. Add sterile glass beads and shake thoroughly in a mechanical shaker for about thirty minutes, or whip with an egg-whisk.
6. Filter through coarse butter muslin into a sterile flask.
Note.—A few drops of alcoholic solution of basic fuchsin (sufficient to give a definite pink colour), or a few drops of waterproof Chinese ink added to the medium at this stage facilitates the subsequent "fishing" of colonies.
7. Tube in quantities of 10 c.c.
8. Solidify in the sloping position in the inspissator at 75° C. for one hour.
9. Place the tubes for forty-eight hours in the incubator at 37° C., and eliminate any contaminated tubes.
To prevent drying, 0.5 c.c. glycerine bouillon (see page 209) may be added to each tube between steps 8 and 9.
10. Cap those tubes of media which remain sterile with india-rubber caps and store for future use.
Potato.—
1. Choose fairly large potatoes, wash them well, and scrub the peel with a stiff nail-brush.
2. Peel and take out the eyes.
3. Remove cylinders from the longest diameter of each potato by means of an apple-corer or a large cork-borer (i. e., one of about 1.4 cm. diameter).
The reaction of the fresh potato is strongly acid to phenolphthalein. If, therefore, the potatoes are required to approximate +10, as for the cultivation of some of the vibrios, the cylinders should be soaked in a 1 per cent. solution of sodium carbonate for thirty minutes.
4. Cut each cylinder obliquely from end to end, forming two wedge-shaped portions.
5. Place a small piece of sterilised cotton-wool, moistened with sterile water, at the bottom of a sterile test-tube; insert the potato wedge into the tube so that its base rests upon the cotton-wool. Now plug the tube with cotton-wool (Fig. 111).
6. Sterilise in the steamer at 100° C. for twenty minutes on each of five consecutive days.
Note.—The cork borer reserved for cutting the potato cylinders should be silver electro-plated both inside and out, and the knife used for dividing the cylinders should be of silver or silver plated. When these precautions are adopted the potato wedges will retain their white color and will not show the discoloration so often observed when steel instruments are employed.
Beer Wort.—Wort is chiefly used as a medium for the cultivation of yeasts, moulds, etc., both in its fluid form and also when made solid by the addition of gelatine or agar. The wort is prepared as follows:
1. Weigh out 250 grammes crushed malt and place in a 2-litre flask.
2. Add 1000 c.c. distilled water, heated to 70° C., and close the flask with a rubber stopper.
3. Place the flask in a water-bath regulated to 60°C. and allow the maceration to continue for one hour.
4. Strain through butter muslin into a clean flask and heat in the steamer for thirty minutes.
5. Filter through Swedish filter paper.
6. Tube in quantities of 10 c.c. or store in flasks.
7. Sterilise in the steamer at 100° C. for twenty minutes on each of three consecutive days.
The natural reaction of the wort should not be interfered with.
Note.—It is sometimes more convenient to obtain "unhopped"[6] beer wort direct from the brewery. In this case it is diluted with an equal quantity of distilled water, steamed for an hour, filtered, filled into sterile flasks or tubes, and sterilised by the discontinuous method.
Wort Gelatine.—
1. Measure out wort (prepared as above), 900 c.c., into a sterile flask.
2. Weigh out gelatine, 100 grammes (= 10 per cent.), and add it to the wort in the flask.
3. Bubble live steam through the mixture for ten minutes, to dissolve the gelatine.
4. Cool to 60°C.; clarify with egg as for nutrient gelatine (vide page 164).
5. Filter through papier Chardin.
6. Tube, and sterilise as for nutrient gelatine.
Wort Agar.—
1. Measure out wort (as above), 700 c.c., into a sterile flask.
2. Weigh out powdered agar, 20 grammes; mix into a smooth paste with 200 c.c. of cold wort and add to the wort in the flask.
3. Bubble live steam through the mixture for twenty minutes, to dissolve the agar.
4. Cool to 60° C.; clarify with egg as for nutrient agar (vide page 167).
5. Filter through papier Chardin, using the hot-water funnel.
6. Tube, and sterilise as for nutrient agar.
Peptone Water (Dunham).—
1. Weigh out Witté's peptone, 10 grammes, and salt, 5 grammes, and emulsify with about 250 c.c. of distilled water previously heated to 60° C.
2. Pour the emulsion into a litre flask and make up to 1000 c.c. by the addition of distilled water.
3. Heat in the steamer at 100° C. for thirty minutes.
4. Filter through Swedish filter paper.
5. Tube in quantities of 10 c.c. each.
6. Sterilise in the steamer at 100° C. for twenty minutes on each of three consecutive days.
"Sugar" or "Carbohydrate" Media.—
Formerly the ability of bacteria to induce hydrolytic changes in carbohydrate substances was observed only in connection with a few well-defined sugars, but of recent years it has been shown that when using litmus as an indicator these so-called "fermentation reactions" facilitate the differentiation of closely allied species, and the list of substances employed in this connection has been considerably extended. The media prepared with them are now no longer regarded as special, but are comprised in the "stock media" of the laboratory. The chief of these substances are the following, arranged in accordance with their chemical constitution:
These substances should be obtained from Kahlbaum (of Berlin); in the pure form, and when possible as large crystals, and the method of preparing a medium containing either of them may be exemplified by describing Dextrose Solution.
Dextrose Solution.—
1. Weigh out