by hand or in a shaking machine)
for ten minutes.

4. Liquefy and measure out into a flask

Nutrient agar 750 c.c.

then cool to 55 deg. C.

5. Mix the oleic acid emulsion with the agar.

6. Add 10 c.c. sterile neutral red, 1 per cent. aqueous solution.

7. Tube in quantities of 10 c.c., slant, and allow to set.

8. Incubate for forty-eight hours at 37 deg. C. and reject any
contaminated tubes. Store the sterile tubes for future use.

_Coli-typhoid Group._

~Parietti's Bouillon.~--

1. Measure out pure hydrochloric acid, 4 c.c., and add to it carbolic
acid solution (5 per cent.), 100 c.c. Allow the solution to stand at
least a few days before use.

2. This solution is added in quantities of 0.1, 0.2. and 0.3 c.c.
(delivered by means of a sterile graduated pipette) to tubes each
containing 10 c.c. of previously sterilised nutrient bouillon (_vide_
page 163).

3. Incubate at 37 deg. C. for forty-eight hours to eliminate contaminated
tubes. Store the remainder for future use.

~Carbolised Bouillon.~--

1. Prepare nutrient bouillon (_vide_ page 163, sections 1 to 6). Measure
out 1000 c.c.

2. Weigh out carbolic acid, 1 gramme (2.5 or 5 grammes may be needed for
special purposes), and dissolve it in the medium.

3. Tube, and sterilise as for bouillon.

~Carbolised Gelatine.~--

1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). Measure
out 1000 c.c.

2. Weigh out carbolic acid, 5 grammes (= 0.5 per cent.), and dissolve it
in the gelatine.

3. Filter if necessary through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

One or 2.5 grammes of carbolic acid (= 0.1 per cent. or 0.25 per cent.)
are occasionally used in place of the 5 grammes to meet special
requirements.

~Carbolised Agar.~--

1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out
1000 c.c.

2. Weigh out 1 gramme pure phenol and dissolve in the medium.

3. Filter if necessary through papier Chardin.

4. Tube, and sterilise as for nutrient agar.

~Litmus Gelatine.~--

1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 8).

2. Add sterile litmus solution, sufficient to tint the medium a deep
lavender colour.

3. Tube, and sterilise as for nutrient gelatine.

~Lactose Litmus Bouillon (Lakmus Molke).~--

1. Weigh out peptone, 4 grammes, and emulsify it with 200 c.c. meat
extract (_vide_ page 148), previously heated to 60 deg. C.

2. Weigh