The Elements of Bacteriological Technique / A Laboratory Guide for Medical, Dental, and Technical Students. Second Edition Rewritten and Enlarged.

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

3. Steam in the steamer at 100° C. for twenty minutes to dissolve the peptone and glucose.

4. Add

(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.

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

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:

XII. SPECIAL MEDIA.

In this chapter are collected a number of media which have been elaborated by various workers for special purposes, grouped together under headings which indicate their chief utility. In many instances the name of the originator of the medium is given, but without reference to his original instructions, since these are in many cases inadequate to the requirements of the isolated worker, who would probably fail to reproduce the medium in a form giving the results attributed to it by its author. Such modifications have therefore been introduced as make for uniformity between the different batches of media.

A considerable number of coloured media, chiefly intended for work with intestinal bacteria, have been included; but beyond the fact that the author's modification of the Drigalski-Conradi medium has been included amongst the routine media of the laboratory, no comment has been made upon their relative values, since only by observation and practice can the skill necessary to utilise their full value be acquired.

The instructions as to sterilisation are rarely given in full; the routine method of exposure in the steam steriliser at 100° C. (without pressure) for twenty minutes on each of three successive days for all fluid media, and thirty minutes on each of three successive days for all liquefiable or solid media must be carried out; and only when these general rules are to be departed from are further details given.

Media for the Study of the Chemical Composition of Bacteria.

Asparagin Medium (Uschinsky).—

1. Weigh out and mix

2. Dissolve the mixture in distilled water 1000 c.c.

3. Add glycerine, 40 c.c.

4. Tube, and sterilise as for nutrient bouillon.

Asparagin Medium (Frankel and Voges).—

1. Weigh out and mix

and dissolve in

2. Tube, and sterilise as for nutrient bouillon.

Proteid Free Broth (Uschinsky).—

1. Weigh out and mix

2. Dissolve the mixture in distilled water 1000 c.c.

3. Add glycerine 30 c.c.

4. Tube and sterilise as for nutrient broth.

Media for the Study of Biochemical Reaction.

Inosite-free Media—Bouillon (Durham).—

1. Prepare meat extract, 1000 c.c. (vide page 148), from bullock's heart which has been "hung" for a couple of days.

2. Prepare nutrient bouillon (+10), 1000 c.c. (vide, page 161), from the meat extract, and store in 1-litre flask.

3. Inoculate the bouillon from a pure cultivation of the B. lactis aerogenes, and incubate at 37° C. for forty-eight hours.

4. Heat in the steamer at 100° C. for twenty minutes to destroy the bacilli and some of their products.

5. Estimate the reaction of the medium and if necessary restore to +10.

6. Inoculate the bouillon from a pure cultivation of the B. coli communis and incubate at 37° C. for forty-eight hours.

7. Heat in the steamer at 100° C. for twenty minutes.

Now fill two fermentation tubes with the bouillon, tint with litmus solution, and sterilise; inoculate with B. lactis aerogenes. If no acid or gas is formed, the bouillon is in a sugar-free condition; but if acid or gas is present, again make the bouillon in the flask +10, reinoculate with one or other of the above-mentioned bacteria, and incubate; then test again. Repeat this till neither acid nor gas appears in the medium when used for the cultivation of either of the bacilli referred to above.

8. After the final heating, stand the flask in a cool place and allow the growth to sediment. Filter the supernatant broth through Swedish filter paper. If the filtrate is cloudy, filter through a porcelain filter candle.

9. Tube, and sterilise as for bouillon.

Bouillon prepared in the above-described manner will prove to be absolutely sugar-free; and from it may be prepared nutrient sugar-free gelatine or agar, by dissolving in it the required percentage of gelatine or agar respectively and completing the medium according to directions given on pages 166 and 167. The most important application of inosite-free bouillon is its use in the preparation of sugar bouillons, whether glucose, maltose, lactose, or saccharose, of exact percentage composition.

Sugar (Dextrose) Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6) or sugar-free bouillon (vide supra).

2. Weigh out glucose (anhydrous), 20 grammes (= 2 per cent.), and dissolve in the fluid.

3. Tube, and sterilise as for bouillon.

Ordinary commercial glucose serves the purpose equally well, but is not recommended, as during the process of sterilisation it causes the medium to gradually deepen in colour.

Sugar Gelatine.—

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 dissolve in the hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

Sugar 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.), and dissolve in the clear agar.

3. Tube, and sterilise as for nutrient agar.

Iron Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 141, sections 1 to 6).

2. Weigh out ferric tartrate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

Lead Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6).

2. Weigh out lead acetate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

Nitrate Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6).

2. Weigh out potassium nitrate, 5 grammes (= 0.5 per cent.), and dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

Iron Peptone Solution (Pakes).—

1. Weigh out peptone, 30 grammes, and emulsify it with 200 c.c. tap water, previously heated to about 60°C.

2. Wash the emulsion into a litre flask with 800 c.c. tap water.

3. Weigh out salt, 5 grammes, and sodium phosphate, 3 grammes, and dissolve in the mixture in the flask.

4. Heat the mixture in the steamer at 100° C. for thirty minutes, to complete the solution of the peptone, and filter into a clean flask.

5. Fill into tubes in quantities of 10 c.c. each.

6. Add to each tube 0.1 c.c. of a 2 per cent. neutral solution of ferric tartrate. (A yellowish-white precipitate forms.)

7. Sterilise as for nutrient bouillon.

Lead Peptone Solution.—

Prepare as for iron peptone solution but in step 6 substitute 0.1 c.c. of a 1 per cent. neutral aqueous solution of lead acetate.

Nitrate Peptone Solution (Pakes).—

1. Weigh out Witté's peptone, 10 grammes, and emulsify it with 200 c.c. ammonia-free distilled water previously heated to 60°C.

2. Wash the emulsion into a flask and make up to 1000 c.c., with ammonia-free distilled water.

3. Heat in the steamer at 100° C. for twenty minutes.

4. Weigh out sodium nitrate, 1 gramme, and dissolve in the contents of the flask.

5. Filter through Swedish filter paper.

6. Tube, and sterilise as for nutrient bouillon.

Litmus Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6).

2. Add sufficient sterile litmus solution to tint the medium a dark lavender colour. (Media rendered +10 will usually react very faintly alkaline or occasionally neutral to litmus.)

3. Tube, and sterilise as for bouillon.

Rosolic Acid Peptone Solution.—

1. Weigh out rosolic acid (corallin), 0.5 gramme, and dissolve it in 80 per cent. alcohol, 100 c.c. Keep this as a stock solution.

2. Measure out peptone water (Dunham), 100 c.c., and rosolic acid solution, 2 c.c., and mix.

3. Heat in the steamer at 100° C. for thirty minutes.

4. Filter through Swedish filter paper.

5. Tube, and sterilise as for nutrient bouillon.

Capaldi-Proskauer Medium, No. I.—

1. Weigh out and mix

2. Dissolve in water 1000 c.c. in a 2-litre flask

3. Weigh out and mix

and add to contents of flask.

4. Measure out 25 c.c. of the solution and titrate it against decinormal sodic hydrate, using litmus as the indicator. Control the result and estimate the amount of sodic hydrate necessary to be added to render the remainder of the solution neutral to litmus. Add this quantity of sodic hydrate.

5. Filter.

6. Add litmus solution 47.5 c.c. (= 5 per cent.).

7. Tube, and sterilise as for nutrient bouillon.

Capaldi-Proskauer Medium No. II.—

1. Weigh out and mix

2. Dissolve in water 1000 c.c. in a 2-litre flask.

3. Neutralise to litmus as in No. I (vide supra, Step 4).

4. Filter.

5. Add litmus solution 47.5 c.c. (= 5 per cent.).

6. Tube, and sterilise as for nutrient bouillon.

Urine Media. Bouillon.—

1. Collect freshly passed urine in sterile flask.

2. Place the flask in the steamer at 100° C. for thirty minutes.

3. Filter through two thicknesses of Swedish filter paper.

4. Tube, and sterilise as for nutrient bouillon. (Leave the reaction unaltered.)

Urine Gelatine.—

1. Collect freshly passed urine in sterile flask.

2. Take the specific gravity, and, if above 1010, dilute with sterile water until that gravity is reached.

3. Estimate (with control) at the boiling-point, and note the reaction of the urine.

4. Weigh out gelatine, 10 per cent., and add to the urine in the flask.

5. Heat in the steamer at 100° C. for one hour to dissolve the gelatine.

6. Estimate the reaction and add sufficient caustic soda solution to restore the reaction of the medium mass to the equivalent of the original urine.

7. Cool to 60° C. and clarify with egg as for nutrient gelatine (vide page 166).

8. Filter through papier Chardin.

9. Tube, and sterilise as for nutrient gelatine.

Urine Gelatine (Heller).—

1. Collect freshly passed urine in sterile flask.

2. Filter through animal charcoal to remove part of the colouring matter.

3. Take the specific gravity, and if above 1010, dilute with sterile water till this gravity is reached.

4. Add Witté's peptone, 1 per cent.; salt, 0.5 per cent.; gelatine, 10 per cent.

5. Heat in the steamer at 100° C. for one hour, to dissolve the gelatine, etc.

6. Add normal caustic soda solution in successive small quantities, and test the reaction from time to time with litmus paper, until the fluid reacts faintly alkaline.

7. Cool to 60° C. and clarify with egg as for nutrient gelatine (vide page 166).

8. Filter through papier Chardin.

9. Tube, and sterilise as for nutrient gelatine.

Urine Agar.—

1. Collect freshly passed urine in sterile flask.

2. Take the specific gravity and if above 1010, dilute with sterile water till this gravity is reached.

3. Weigh out 1.5 per cent. or 2 per cent. powdered agar, and add it to the urine.

4. Heat in the steamer at 100° C. for ninety minutes to dissolve the agar.

5. Cool to 60° C. and clarify with egg as for nutrient agar (vide page 168).

6. Filter through papier Chardin, using the hot-water funnel.

7. Tube, and sterilise as for nutrient agar.

(Leave the reaction unaltered.)

Serum Sugar Media (Hiss).—

In these media the fermentation of carbohydrate substance by bacterial action is indicated by the coagulation of the serum proteids in addition to the production of an acid reaction.

Serum Dextrose Water (Hiss).—

1. Measure out into a litre flask

2. Weigh out

and dissolve in the serum water.

3. Filter through Swedish filter paper.

4. Measure out and add to the medium

5. Tube in quantities of 10 c.c. and sterilise in the steamer at 100° C. for twenty minutes on each of three successive days.

Lævulose, galactose, maltose, lactose, etc., can be substituted in similar amounts for dextrose and the medium completed as above.

Omeliansky's Nutrient Fluid (For Cellulose Fermenters).—

1. Weigh out and mix

2. Dissolve in distilled water 4000 c.c.

3. Flask in quantities of 250 c.c.

4. Weigh out and add 5 grammes precipitated chalk to each flask.

5. Sterilise in the steamer at 100° C. for twenty minutes on each of three successive days.

Media for the Study of Chromogenic Bacteria.

Milk Rice (Eisenberg).—

1. Measure out nutrient bouillon, 70 c.c., and milk, 210 c.c., and mix thoroughly.

2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with the milk and broth mixture.

3. Fill the paste into sterile capsules, spreading it out so as to form a layer about 0.5 cm. thick, over the bottom of each.

4. Heat over a water-bath at 100° C. until the mixture solidifies.

5. Replace the lids of the capsules. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days.

(A solid medium of the colour of café au lait is thus produced.)

Milk Rice (Soyka).—

1. Measure out nutrient bouillon, 50 c.c., and milk, 150 c.c., and mix thoroughly.

2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with the milk and broth mixture.

3. Fill the paste into sterile capsules, to form a layer over the bottom of each.

4. Replace the lids of the capsules.

5. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days.

(A pure white, opaque medium is thus formed.)

Media for the Study of Phosphorescent and Photogenic Bacteria.

Fish Bouillon.—

1. Weigh out herring, mackerel, or cod, 500 grammes, and place in a large porcelain beaker (or enamelled iron pot).

2. Weigh out sodium chloride, 26.5 grammes; potassium chloride, 0.75 gramme; magnesium chloride, 3.25 grammes; and dissolve in 500 c.c. distilled water. Add the solution to the fish in the beaker.

3. Place the beaker in a water-bath and proceed as in preparing meat extract—i. e., heat gently at 40° C. for twenty minutes, then rapidly raise the temperature to, and maintain at, the boiling-point for ten minutes.

4. Strain the mixture through butter muslin into a clean flask.

5. Weigh out peptone, 5 grammes, and emulsify with about 200 c.c. of the hot fish water; incorporate thoroughly with the remainder of the fish water in the flask.

6. Heat in the steamer at 100° C. for twenty minutes to complete the solution of the peptone.

7. Filter through Swedish filter paper.

8. When the fish bouillon is cold, if it is to be used as fluid medium, make up to 1000 c.c. by the addition of distilled water. If, however, it is to be used as the basis for agar or gelatine media store it in the "Double Strength" condition.

9. Tube and sterilise as for nutrient bouillon.

As an alternative method "Marvis" fish food (16 grammes) may be substituted for the 500 grammes of fresh fish.

Fish Gelatine.—

1. Measure out double strength fish bouillon, 500 c.c., into a "tared" 2-litre flask.

2. Add sheet gelatine, 100 grammes, cut into small pieces.

3. Bubble live steam through the mixture for fifteen minutes to dissolve the gelatine.

4. Weigh the flask and its contents; adjust the weight to the calculated figure for one litre of medium (1135.5 grammes) by the addition of distilled water at 100° C. (vide page 166).

5. Cool to below 60°C., and clarify with egg.

6. Filter through papier Chardin.

7. Tube, and sterilise as for nutrient gelatine.

Shake well after the final sterilisation, to aerate the medium.

Fish Gelatine-Agar.—

1. Weigh out powdered agar, 5 grammes, and emulsify it with 200 c.c. double strength fish bouillon.

2. Wash the emulsion into a "tared" 2-litre flask with 300 c.c. fish bouillon.

3. Weigh out sheet gelatine, 70 grammes, cut it into small pieces and add it to the contents of the flask.

4. Bubble live steam through the mixture to dissolve the gelatine and agar.

5. Weigh the flask and contents. Adjust the weight to the calculated figure for one litre of medium (1110.5 grammes) by the addition of distilled water at 100° C. (vide page 166).

6. Cool to below 60° C. and clarify with egg.

7. Filter through papier Chardin.

8. Tube, and sterilise as for nutrient gelatine.

Shake well after the final sterilisation, to aerate the medium.

Media for the Study of Yeasts and Moulds.

Pasteur's Solution.—

(Reaction alkaline).

1. Weigh out and mix the ash from 10 grammes of yeast; ammonium tartrate, 10 grammes; cane sugar, 100 grammes.

2. Dissolve the mixture in distilled water, 1000 c.c.

3. Tube or flask, and sterilise as for nutrient bouillon.

Yeast Water (Pasteur).—

1. Weigh out pressed yeast, 75 grammes; place in a 2-litre flask and add 1000 c.c. distilled water.

2. Heat in the steamer at 100° C. for thirty minutes.

3. Filter through papier Chardin.

4. Tube or flask, and sterilise as for nutrient bouillon.

Cohn's Solution.—

1. Weigh out and mix

and dissolve in

2. Tube, or flask and sterilise as for nutrient bouillon.

Naegeli's Solution.—

1. Weigh out and mix

and dissolve in

2. Tube or flask; sterilise as for nutrient bouillon.

Plaster-of-Paris Discs.—

1. Take large corks, 2.5 cm. diameter, and roll a piece of stiff note-paper round each, so that about a centimetre projects as a ridge above the upper surface of the cork, and secure in position with a pin (Fig. 112).

2. Mix plaster-of-Paris into a stiff paste with distilled water, and fill each of the cork moulds with the paste.

3. When the plaster has set, remove the paper from the corks, and raise the plaster discs.

4. Place the plaster discs on a piece of asbestos board and sterilise by exposing in the hot-air oven to 150° C. for half an hour.

5. Remove the sterile discs from the oven by means of sterile forceps, place each inside a sterile capsule, and moisten with a little sterile water.

6. Sterilise in the steamer at 100° C. for thirty minutes on each of three consecutive days.

Gypsum Blocks (Engel and Hansen).—

These are in the form of truncated cones and for their preparation small tin moulds are required, each having a diameter of 5.5 cm. at the base and 4 cm. at the truncated apex. The height (or depth) of a mould is 4.5 to 5 cm.

1. Mix powdered calcined gypsum into a stiff paste with distilled water.

2. Fill the paste into the moulds and allow it to set and dry by exposure to air.

3. Remove the block from the mould and transfer it to a double glass dish of adequate size (7 cm. diameter × 7 cm. high).

4. Sterilise block in its dish for one hour in the hot-air oven at 115°C.

5. Carefully open the dish and add sterile distilled water to moisten the block and form a layer in the bottom of the dish 1 cm. deep.

Wine Must.—(Wine must is obtained from Sicily, in hermetically sealed tins, in a highly concentrated form—as a thick syrup—but not sterilised.)

1. Weigh out "wine must," 200 grammes, place in a 2-litre flask and add distilled water, 800 c.c.

2. Weigh out ammonium tartrate, 5 grammes, and add to the dilute must.

3. Place the flask in a water-bath regulated to 60° C. for one hour and incorporate the mixture thoroughly by frequent shaking.

4. Filter through papier Chardin.

5. Tube, and sterilise as for nutrient bouillon.

Wheat Bouillon (Gasperini).—

1. Weigh out and mix wheat flour, 150 grammes; magnesium sulphate, 0.5 gramme; potassium nitrate, 1 gramme; glucose, 15 grammes.

2. Dissolve the mixture in 1000 c.c. of water heated to 100°C.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient bouillon.

Bread Paste.—

1. Grate stale bread finely on a bread-grater.

2. Distribute the crumbs in sterile Erlenmeyer flasks, sufficient to form a layer about one centimetre thick over the bottom of each.

3. Add as much distilled water as the crumbs will soak up, but not enough to cover the bread.

4. Plug the flasks and sterilise in the steamer at 100° C. for thirty minutes on each of four consecutive days.

Media for the Study of Parasitic Moulds.

French Proof Agar (Sabouraud).—

1. Weigh out Chassaing's peptone, 10 grammes, and emulsify it with 200 c.c. distilled water previously heated to 60°C.

2. Weigh out powdered agar, 13 grammes, and emulsify with 200 c.c. cold distilled water.

3. Mix the two emulsions and wash into a tared 2-litre flask with 600 c.c. distilled water.

4. Bubble live steam through the mixture for twenty minutes, to dissolve the agar.

5. Cool to 60° C. and clarify with egg as for nutrient agar (vide page 168).

6. Filter through Papier Chardin, using the hot-water funnel.

7. Weigh out French maltose, 40 grammes, and dissolve in the agar.

8. Tube, and sterilise as for nutrient agar.

English Proof Agar (Blaxall).—Substitute Witté's peptone for that of Chassaing, and proceed as for French proof agar.

French Mannite Agar, Sabouraud.—(For cultivation of Favus.)

Proceed exactly as in preparing French Proof agar vide supra substituting Mannite (38 grammes) for maltose.

Media for the Study of Milk Bacteria.

Gelatine Agar.—This medium is prepared by adding to nutrient gelatine sufficient agar to ensure the solidity of the medium when incubated at temperatures above 22° C. If it is intended to employ an incubating temperature of 30°C., 10 per cent. gelatine and 0.5 per cent. agar must be dissolved in the meat extract before the addition of the peptone and salt; while for incubating at 37°C., 12 per cent. gelatine and 0.75 per cent. agar must be used. Avoid the addition of more agar than is absolutely necessary, otherwise the action upon the medium of such organisms as elaborate a liquefying ferment may be retarded or completely absent.

1. Measure out 400 c.c. double strength meat extract into a "tared" 2-litre flask, and add to it gelatine, 100 grammes.

2. Weigh out powdered agar, 5 grammes, emulsify with 100 c.c., cold distilled water and add to the contents of the flask.

3. Dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes.

4. Weigh out peptone, 10 grammes; salt, 5 grammes; emulsify with 100 c.c. double strength meat extract previously heated to 60°C., and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes. Then adjust the weight to the calculated figure for one litre (in this instance 1120 grammes) by the addition of distilled water at 100°C.

6. Estimate the reaction; control the result. Then add sufficient caustic soda solution to render the reaction +10.

7. Replace in the steamer at 100° C. for twenty minutes.

8. Cool to 60° C. Clarify with egg as for nutrient agar.

9. Filter through papier Chardin, using the hot-water funnel.

10. Tube, and sterilise as for nutrient agar.

Agar Gelatine (Guarniari).—

1. Measure out double strength meat extract, 400 c.c., into a "tared" 2-litre flask, and add to it gelatine, 50 grammes.

2. Weigh out powdered agar, 3 grammes; emulsify with cold distilled water, 50 c.c., and add to the contents of the flask.

3. Dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes.

4. Weigh out Witté's peptone, 25 grammes; salt, 5 grammes, and emulsify with 100 c.c. double strength meat extract previously heated to 60°C., and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes.

6. Weigh the flask and make up the medium mass to the calculated figure for one litre (1083 grammes) by the addition of distilled water at 100°C.

7. Neutralise carefully to litmus paper by the successive additions of small quantities of normal soda solution.

8. Replace in the steamer at 100° C. for twenty minutes.

9. Cool to 60° C. Clarify with egg as for nutrient agar.

10. Filter through papier Chardin, using the hot-water funnel.

11. Tube, and sterilise as for nutrient agar.

Whey Gelatine.—

1. Curdle fresh milk by warming to 60°C., and adding rennet; filter off the whey into a sterile "tared" flask.

2. Estimate and note the reaction of the whey.

3. Weigh out gelatine, 10 per cent., and add it to the whey in the flask.

4. Bubble live steam through the mixture fifteen minutes to dissolve the gelatine; and weigh.

5. Estimate the reaction of the medium mass; then add sufficient caustic soda solution to restore the reaction of the medium mass (i. e., total weight minus weight of flask) to the equivalent of the original whey.

6. Cool to 60° C. and clarify with egg as for nutrient gelatine (vide page 166).

7. Filter through papier Chardin.

8. Tube, and sterilise as for nutrient gelatine.

Whey Agar.—

1. Curdle fresh milk by warming to 60°C., and adding rennet; filter off the whey into a sterile flask.

2. Weigh out agar, 1.5 or 2 per cent., and add it to the whey 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 168).

5. Filter through papier Chardin, using the hot-water funnel.

6. Tube, and sterilise as for nutrient agar.

Litmus Whey.—

1. Curdle fresh milk by warming to 60° C. and adding rennet.

2. Filter off the whey through butter muslin into a sterile flask.

3. Neutralise to litmus by the cautious addition of citric acid solution 4 per cent. (Do not neutralise with mineral acid.)

4. Heat in the steamer at 100° C. for one hour to coagulate all the proteid.

(If the whey is cloudy when removed from the steamer allow it to stand for forty-eight hours in the ice chest and then decant off the clear fluid—or filter through a Berkefeld filter candle.)

5. Filter into a sterile flask.

6. Tint the whey with litmus solution to a deep purple red.

7. Tube, and sterilise as for milk.

Litmus Whey (Petruschky).—

1. Measure out into a flask