ilisation, 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