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 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 princ.i.p.al 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 60C.

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 quant.i.ties 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 quant.i.ties 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 quant.i.ties or percentages will necessitate corresponding alterations in the calculated medium figure.