The Elements of Bacteriological Technique Part 39

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 60C.; 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 Witte'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 quant.i.ties 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 const.i.tution:

_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 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.