Chapter VII: Appendix: 492 (5)

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 deg. C. for twenty minutes, to precipitate the phosphates, etc.

9. Allow the medium mass to cool to 60 deg. C. Well whip the whites of two eggs, add to the contents of the flask and replace in the steamer at 100 deg. 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 deg. 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 deg. 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 deg. C. Well whip the whites of two eggs, add to the contents of the flask, and replace in the steamer at 100 deg. 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 deg. 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 haemoglobin.)

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 deg. 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 deg. C. (A coagulum is formed at this temperature which is fairly transparent; above 72 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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 deg. C. for twenty minutes on each of five successive days.

6. Incubate at 37 deg. 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

Witte's peptone 10 grammes
Sodium chloride 5 grammes
Nutrose 10 grammes

and dissolve in 200 c.c. of serum water heated to 80 deg. 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 deg. 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 deg. C. for twenty minutes. Cool to 20 deg. 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 deg. C. for one hour.

9. Place the tubes for forty-eight hours in the incubator at 37 deg. 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 deg. 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 deg. C., and close the flask with a rubber stopper.

3. Place the flask in a water-bath regulated to 60 deg. 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 deg. 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 deg. 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 deg. 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 Witte's peptone, 10 grammes, and salt, 5 grammes, and emulsify with about 250 c.c. of distilled water previously heated to 60 deg. 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 deg. 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 deg. 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), laevulose, 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 deg. C.

2. Place in a flask and add

Distilled water 850 c.c.

3. Steam in the steamer at 100 deg. 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 deg. 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 deg. 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 CO_{2} through the solution until reaction is definitely acid.

11. Sterilise in the steamer at 100 deg. 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 Witte's peptone, 20 grammes (= 2 per cent.),
and emulsify with 200 c.c. distilled water previously warmed
to 60 deg. 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 deg. 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 deg. 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.

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 deg. 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
Asparagin 3.4 grammes
Ammonium lactate 10.0 grammes
Sodium chloride 5.0 grammes
Magnesium sulphate 0.2 gramme
Calcium chloride 0.1 gramme
Acid potassium phosphate (KH_{2}PO_{4}) 1.0 gramme

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
Asparagin 4 grammes
Sodium phosphate, (Na_{2}HPO_{4}) 12OH 2 grammes
Ammonium lactate 6 grammes
Sodium chloride 5 grammes
and dissolve in
Distilled water 1000 c.c.

2. Tube, and sterilise as for nutrient bouillon.

NOTE.--Either of the above asparagin media, after the
addition of 10 per cent. gelatine or 1.5 per cent. agar, may
be advantageously employed in the solid condition.

~Proteid Free Broth (Uschinsky).~--

1. Weigh out and mix
Calcium chloride 0.1 gramme
Magnesium sulphate 0.2 gramme
Acid potassium phosphate (KH_{2}PO_{4}) 2.0 grammes
Potassium aspartate 3.0 grammes
Sodium chloride 5.0 grammes
Ammonium lactate 6.0 grammes

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 deg. C. for forty-eight hours.

4. Heat in the steamer at 100 deg. 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 deg. C. for forty-eight hours.

7. Heat in the steamer at 100 deg. 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.

NOTE.--In certain cases a corresponding percentage of
lactose, maltose, or saccharose is substituted for glucose.

~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.

NOTE.--Other "sugar" media are prepared by substituting a
corresponding percentage of lactose, maltose (or any other
of the substances referred to under "Sugar Media," page 177)
for the glucose.

~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.

NOTE.--The lactate of iron may be substituted for the
tartrate.

~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.

NOTE.--The nitrate of sodium or ammonium may be substituted
for that of potassium, or the salt may be added in the
proportion of from 0.1 to 1 per cent. to meet special
requirements.

~Iron Peptone Solution (Pakes).~--

1. Weigh out peptone, 30 grammes, and emulsify it with 200 c.c. tap water, previously heated to about 60 deg. 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 deg. 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 Witte's peptone, 10 grammes, and emulsify it with 200 c.c. ammonia-free distilled water previously heated to 60 deg. 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 deg. 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 deg. 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

Sodium chloride 2.0 grammes
Magnesium sulphate 0.1 gramme
Calcium chloride 0.2 gramme
Monopotassium phosphate 2.0 grammes

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

3. Weigh out and mix

Asparagin 2 grammes
Mannite 2 grammes

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

Peptone 20 grammes
Mannite 1 gramme

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 deg. 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 deg. 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 deg. 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 Witte's peptone, 1 per cent.; salt, 0.5 per cent.; gelatine, 10 per cent.

5. Heat in the steamer at 100 deg. 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 deg. 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 deg. C. for ninety minutes to dissolve the agar.

5. Cool to 60 deg. 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

Serum water (See page 170) 1000 c.c.

2. Weigh out

Dextrose 10 grammes

and dissolve in the serum water.

3. Filter through Swedish filter paper.

4. Measure out and add to the medium

Litmus solution (Kahlbaum) 50 c.c.

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

Laevulose, 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

Potassium phosphate 4.0 grammes
Magnesium sulphate 2.0 grammes
Ammonium sulphate 4.0 grammes
Sodium chloride 0.25 gramme

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 deg. 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 deg. C. until the mixture solidifies.

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

(A solid medium of the colour of _cafe 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 deg. 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 deg. 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 deg. 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 deg. C. (_vide_ page 166).

5. Cool to below 60 deg. 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 deg. C. (_vide_ page 166).

6. Cool to below 60 deg. 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 deg. 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

Acid potassium phosphate (KH_{2}PO_{4}) 5.0 grammes
Calcium phosphate 0.5 gramme
Magnesium sulphate 5.0 grammes
Ammonium tartrate 10.0 grammes

and dissolve in

Distilled water 1000 c.c.

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

~Naegeli's Solution.~--

1. Weigh out and mix

Dibasic potassium phosphate (K_{2}HPO_{4}) 1.0 gramme
Magnesium sulphate 0.2 gramme
Calcium chloride 0.1 gramme
Ammonium tartrate 10.0 grammes

and dissolve in

Distilled water 1000 c.c.

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 deg. 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 deg. 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 x 7 cm. high).

4. Sterilise block in its dish for one hour in the hot-air oven at 115 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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 Witte'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 deg. C. If it is intended to employ an incubating temperature of 30 deg. 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 deg. 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 deg. 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 deg. 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 deg. C. for twenty minutes.

8. Cool to 60 deg. 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 Witte's peptone, 25 grammes; salt, 5 grammes, and emulsify with 100 c.c. double strength meat extract previously heated to 60 deg. 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 deg. 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 deg. C. for twenty minutes.

9. Cool to 60 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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 deg. 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

Fresh milk 1000 c.c.

2. Add

Hydrochloric acid (or glacial acetic acid) 1.5 c.c.

and boil.

3. Filter off coagulated casein.

4. Neutralise to litmus by the addition of n/1 caustic soda solution and boil. Whey now cloudy and acid again.

5. Again neutralise to litmus by addition of n/10 caustic soda solution.

6. Filter.

7. Tint the whey with neutral litmus solution to a deep purple colour.

8. Tube and sterilise as for milk.

~Litmus Whey Gelatine.~--

1. Measure out milk 1000 c.c. into a tared 2-litre flask.

2. Add hydrochloric acid (or glacial acetic acid) 1.5 c.c. and boil for five minutes.

3. Filter off the casein, and make the whey faintly alkaline to litmus.

4. Weigh out

Peptone 10 grammes

and emulsify in a few cubic centimeters of the whey and return to the flask.

5. Weigh out

Gelatine 50 grammes

add it to the whey in the flask and incorporate the mixture by bubbling through live steam.

6. Clear with egg and filter.

7. Make the weight of the medium mass to the calculated figure for one litre (1060 grammes) by the addition of distilled water.

8. Weigh out

Dextrose 15 grammes

and dissolve in the fluid whey gelatine.

9. Add sterile litmus solution to the required tint.

10. Tube and sterilise for twenty minutes in steamer at 100 deg. C. on each of five successive days.

This medium will remain semi-fluid at the room temperature, and may be used for cultures in the cool or hot incubator.

~Litmus Whey Agar~ is prepared in a similar manner to Whey Gelatine, with the substitution of 15 grammes of agar for the gelatine.

~Malt Extract Solution (Herschell).~--

1. Measure into a flask distilled water 1000 c.c.