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 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 pota.s.sium 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 pota.s.sium phosphate (KH_{2}PO_{4}) 2.0 grammes Pota.s.sium 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 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.

NOTE.--In certain cases a corresponding percentage of lactose, maltose, or saccharose is subst.i.tuted 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.