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 quant.i.ties 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 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 60C.

2. Weigh out salt, 2 grammes, and lactose, 20 grammes, and mix with the emulsion.

3. Wash the mixture into a sterile litre flask with 200 c.c. meat extract and add 600 c.c. distilled water.

4. Heat in the steamer at 100 C. for thirty minutes, to completely dissolve the peptone, etc.

5. _Neutralise carefully to litmus paper_ by the successive additions of small quant.i.ties of decinormal soda solution.

6. Replace in the steamer for twenty minutes to precipitate phosphates, etc.

7. Filter through two thicknesses of Swedish filter paper.

8. Add sterile litmus solution, sufficient to colour the medium a deep purple.

9. Tube, and sterilise as for bouillon.

~Lactose Litmus Gelatine (Wurtz).~--

1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 4).

2. Render the reaction of the medium ma.s.s -5.

3. Replace in the steamer at 100 C. for twenty minutes.

4. Clarify with egg as for gelatine.

5. Weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the medium.

6. Filter through papier Chardin.

7. Add sufficient sterile litmus solution to colour the medium pale lavender.

8. Tube, and sterilise as for nutrient gelatine.

~Lactose Litmus Agar (Wurtz).~--

1. Prepare nutrient agar (_vide_ page 167, sections 1 to 4).

2. Render the reaction of the medium ma.s.s -5.

3. Replace in the steamer at 100 C. for twenty minutes.

4. Cool to 60 C. and clarify with egg as for nutrient agar.

5. Weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the medium.