5. Place in the steamer at 100 C. for thirty minutes.

6. Filter.

7. Weigh out 20 grammes saccharose and add to the filtrate.

8. Tube, and sterilise as for nutrient bouillon.

~Haricot Agar.~--

1. Measure out 400 c.c. distilled water into a "tared" 2-litre flask.

2. Weigh out 15 grammes agar and mix into a thick paste with 100 c.c.

cold distilled water, and add to the flask.

3. Dissolve the agar by bubbling live steam through the mixture as in making nutrient agar.

4. Weigh out 250 grammes haricot beans, place in the flask with the agar mixture.

5. Add 1 c.c. of 1 per cent. aqueous solution sodium bicarbonate.

6. Weigh out 10 grammes sodium chloride and add to the contents of the flask.

7. Place in the steamer at 100 C. for thirty minutes.

8. Adjust the weight of the medium ma.s.s to 1030 grammes (the figure per litre obtained experimentally) by the addition of distilled water at 100 C.

9. Cool to 60C., clarify with egg and filter.

10. Weigh out 20 grammes saccharose and add to the contents of the flask.

11. Tube, and sterilise as for nutrient agar.

~Wood Ash Agar.~--

1. Measure 400 c.c. distilled water into a tared 2-litre flask.

2. Weigh out 10 grammes agar and make into a thick paste with 100 c.c.

cold distilled water.

3. Add this agar paste to the distilled water in the flask.

4. Dissolve the agar by pa.s.sing live steam through it, as in preparing nutrient agar.

5. Weigh out 5 grammes clean wood ash and place in a second flask containing 200 c.c. distilled water with some sterile gla.s.s beads: shake thoroughly in a mechanical shaker for ten minutes.

6. Heat in steamer at 100C., for thirty minutes.

7. After removal from the steamer dry the outside of the flask thoroughly, place it over a Bunsen flame and boil for one minute.

8. Filter directly into the flask containing the melted agar mixture.

9. Weigh out 4 grammes maltose. Add to the contents of the flask.

10. Adjust the weight of the medium ma.s.s to the calculated figure for one litre (1019 grammes) by the addition of distilled water at 100C.

11. Replace the flask in the steamer for twenty minutes, cool to 60C., and clarify with egg and filter.

12. Tube, and sterilise as for nutrient agar.

_Media for the Study of Special Bacilli._

_B. Acnes._

~Oleic Acid Agar (Fleming).~--

1. Measure out into a sterile stout gla.s.s bottle which already contains about 10 sterile gla.s.s beads

Ascitic fluid 250 c.c.

2. Weigh out

Oleic acid 25 grammes

and add it to the ascitic fluid in the bottle.

3. Emulsify evenly by shaking (either 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 55C.

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 quant.i.ties of 10 c.c., slant, and allow to set.

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

_Coli-typhoid Group._

~Parietti's Bouillon.~--