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 (11154 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 (10355 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:
| Monosaccharides | Dextrose (glucose), lævulose, galactose, mannose, arabinose, xylose. |
| Disaccharides | Maltose, lactose, saccharose. |
| Trisaccharides | Raffinose (mellitose). |
| Polysaccharides | Dextrin, inulin, starch, glycogen, amidon. |
| Glucosides | Amygdalin, coniferin, salicin, helicin, phlorrhizin. |
| Polyatomic alcohols | Trihydric, Glycerin. |
| Tetrahydric, Erythrite. | |
| Pentahydric, Adonite. | |
| Hexahydric, Dulcite, (dulcitol or melampirite), isodulcite (rhamnose), mannite (mannitol), sorbite (sorbitol), inosite. |
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
| Peptone | 20 grammes |
| Glucose | 10 grammes |
and grind together in a mortar; then emulsify in 100 c.c. of distilled water heated to 60° C.
2. Place in a flask and add
Distilled water 850 c.c.
3. Steam in the steamer at 100° C. for twenty minutes to dissolve the peptone and glucose.
4. Add
Kubel-Tiemann litmus solution (Kahlbaum) 50 c.c.
(The substances enumerated above react acid to phenolphthalein, but variously toward the neutral litmus solution. To such as react acid, add very cautiously n/1 sodium hydrate solution to the medium in bulk until the neutral tint has returned).
5. Fill into tubes in which have previously been placed the inverted Durham’s gas tubes.
6. Sterilise in the steamer at 100° C. for twenty minutes on each of three successive days.
Note.—On no account should these media be sterilised in the autoclave, as temperatures above 100° C. themselves induce hydrolytic changes in the substances in question. It is equally important that the twenty minutes should not be exceeded in sterilisation, as neglect of this precaution may discolour the litmus or lead to the production of yellowish tints when the tubes are subsequently inoculated with acid-forming bacteria.
Neutral Litmus Solution.
The most satisfactory is the Kubel-Tiemann, prepared by Kahlbaum. It can however be made in the laboratory as follows:
1. Weigh out
Commercial litmus 50 grammes,
and place in a well stoppered 500 c.c. bottle; measure out and add 300 c.c. alcohol 95 per cent.
2. Shake well at least once a day for seven days—the alcohol acquires a green colour.
3. Decant off the green alcohol and fill a further 300 c.c. 95 per cent. alcohol into the bottle and repeat the shaking.
4. Repeat this process until on adding fresh alcohol the fluid only becomes tinged with violet.
5. Pour off the alcohol, leaving the litmus as dry as possible. Connect up the bottle to an air pump and evaporate off the last traces of alcohol.
6. Transfer the dry litmus to a litre flask, measure in 600 c.c. distilled water and allow to remain in contact 24 hours with frequent shakings.
7. Filter the solution into a clean flask and add one or two drops of pure concentrated sulphuric acid until the litmus solution is distinctly wine-red in colour.
8. Add excess of pure solid baryta and allow to stand until the reaction is again alkaline.
9. Filter.
10. Bubble CO2 through the solution until reaction is definitely acid.
11. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days. This sterilises the solution and also drives off the carbon dioxide, leaving the solution neutral.
Media for anaerobic cultures. In addition to the foregoing media, all of which can be, and are employed in the cultivation of anaerobic bacteria, certain special media containing readily oxidised substances are commonly used for this purpose. The principal of these are as follows:
Bile Salt Broth (MacConkey).—
1. Weigh out Witté’s peptone, 20 grammes (= 2 per cent.), and emulsify with 200 c.c. distilled water previously warmed to 60°C.
2. Weigh out sodium taurocholate (commercial), 5 grammes (= 0.5 per cent.), and glucose, 5 grammes (= 0.5 per cent.), and dissolve in the peptone emulsion.
3. Wash the peptone emulsion into a flask with 800 c.c. distilled water, and heat in the steamer at 100° C. for twenty minutes.
4. Filter through Swedish filter paper into a sterile flask.
5. Add sterile litmus solution sufficient to colour the medium to a deep purple, usually 13 per cent. required.
6. Fill, in quantities of 10 c.c., into tubes containing small gas tubes (vide Fig. 104, page 161). Sterilise in the steamer at 100° C. for twenty minutes on each of three consecutive days.
Glucose Formate Bouillon (Kitasato).—
1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6).
2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the fluid.
3. Tube, and sterilise as for bouillon.
Glucose Formate Gelatine (Kitasato).—
1. Prepare nutrient gelatine (vide page 164, sections 1 to 7) and measure out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the hot gelatine.
3. Filter through papier Chardin.
4. Tube, and sterilise as for nutrient gelatine.
Glucose Formate Agar (Kitasato).—
1. Prepare nutrient agar (vide page 167, sections 1 to 8). Measure out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the agar.
3. Tube, and sterilise as for nutrient agar.
Sulphindigotate Bouillon (Weyl).—
1. Measure out nutrient bouillon (vide page 163, sections 1 to 6 1000 c.c.).
2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the fluid.
3. Tube, and sterilise as for bouillon.
Sulphindigotate Gelatine (Weyl).—
1. Prepare nutrient gelatine (vide page 164, sections 1 to 7). Measure out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the hot gelatine.
3. Filter through papier Chardin.
4. Tube, and sterilise as for nutrient gelatine.
Sulphindigotate Agar.—
1. Prepare nutrient agar (vide page 167, sections 1 to 8). Measure out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the hot agar.
3. Tube, and sterilise as for nutrient agar.
Note.—The Sulphindigotate media are of a blue colour, which during the growth of anaerobic bacteria is oxidised and decolourised to a light yellow.
Footnotes
4 This figure is obtained by adding together 1 litre water, 1000 grammes; 10 per cent. gelatine, 100 grammes; 1 per cent. peptone, 10 grammes; 0.5 per cent. salt, 5 grammes; total, 1115 grammes. Modifications of the above process, as to quantities and percentages, will require corresponding alterations of the figures. The average weight of a measured litre of 10 per cent. nutrient gelatine when prepared in this way after filtration is 1080 grammes.
5 This figure is obtained by adding together 1 litre of water (meat extract), 1000 grammes; 2 per cent. agar, 20 grammes; 1 per cent. peptone, 10 grammes; 0.5 per cent. salt, 5 grammes—total 1035 grammes. Modifications of the process as to quantities or percentages will necessitate corresponding alterations in the calculated medium figure. The average weight of a measured litre of 2 per cent. agar when prepared in this way, after filtration, is 1010.5 grammes.
6 “Hopped” wort exerts a toxic effect upon many bacteria, including the lactic acid bacteria.