~English Proof Agar (Blaxall).~--Subst.i.tute Witte's peptone for that of Cha.s.saing, and proceed as for French proof agar.

~French Mannite Agar, Sabouraud.~--(_For cultivation of Favus._)

Proceed exactly as in preparing French Proof agar _vide supra_ subst.i.tuting Mannite (38 grammes) for maltose.

_Media for the Study of Milk Bacteria._

~Gelatine Agar.~--This medium is prepared by adding to nutrient gelatine sufficient agar to ensure the solidity of the medium when incubated at temperatures above 22 C. If it is intended to employ an incubating temperature of 30C., 10 per cent. gelatine and 0.5 per cent. agar must be dissolved in the meat extract before the addition of the peptone and salt; while for incubating at 37C., 12 per cent. gelatine and 0.75 per cent. agar must be used. Avoid the addition of more agar than is absolutely necessary, otherwise the action upon the medium of such organisms as elaborate a liquefying ferment may be r.e.t.a.r.ded or completely absent.

1. Measure out 400 c.c. double strength meat extract into a "tared"

2-litre flask, and add to it gelatine, 100 grammes.

2. Weigh out powdered agar, 5 grammes, emulsify with 100 c.c., cold distilled water and add to the contents of the flask.

3. Dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes.

4. Weigh out peptone, 10 grammes; salt, 5 grammes; emulsify with 100 c.c. double strength meat extract previously heated to 60C., and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes. Then adjust the weight to the calculated figure for one litre (in this instance 1120 grammes) by the addition of distilled water at 100C.

6. Estimate the reaction; control the result. Then add sufficient caustic soda solution to render the reaction +10.

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

8. Cool to 60 C. Clarify with egg as for nutrient agar.

9. Filter through papier Chardin, using the hot-water funnel.

10. Tube, and sterilise as for nutrient agar.

~Agar Gelatine (Guarniari).~--

1. Measure out double strength meat extract, 400 c.c., into a "tared"

2-litre flask, and add to it gelatine, 50 grammes.

2. Weigh out powdered agar, 3 grammes; emulsify with cold distilled water, 50 c.c., and add to the contents of the flask.

3. Dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes.

4. Weigh out Witte's peptone, 25 grammes; salt, 5 grammes, and emulsify with 100 c.c. double strength meat extract previously heated to 60C., and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes.

6. Weigh the flask and make up the medium ma.s.s to the calculated figure for one litre (1083 grammes) by the addition of distilled water at 100C.

7. Neutralise carefully to litmus paper by the successive additions of small quant.i.ties of normal soda solution.

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

9. Cool to 60 C. Clarify with egg as for nutrient agar.

10. Filter through papier Chardin, using the hot-water funnel.

11. Tube, and sterilise as for nutrient agar.

~Whey Gelatine.~--

1. Curdle fresh milk by warming to 60C., and adding rennet; filter off the whey into a sterile "tared" flask.

2. Estimate and note the reaction of the whey.

3. Weigh out gelatine, 10 per cent., and add it to the whey in the flask.

4. Bubble live steam through the mixture fifteen minutes to dissolve the gelatine; and weigh.

5. Estimate the reaction of the medium ma.s.s; then add sufficient caustic soda solution to restore the reaction of the medium ma.s.s (i. e., total weight minus weight of flask) to the equivalent of the original whey.

6. Cool to 60 C. and clarify with egg as for nutrient gelatine (_vide_ page 166).

7. Filter through papier Chardin.

8. Tube, and sterilise as for nutrient gelatine.

~Whey Agar.~--

1. Curdle fresh milk by warming to 60C., and adding rennet; filter off the whey into a sterile flask.

2. Weigh out agar, 1.5 or 2 per cent., and add it to the whey in the flask.

3. Bubble live steam through the mixture for twenty minutes, to dissolve the agar.

4. Cool to 60C.; clarify with egg as for nutrient agar (_vide_ page 168).

5. Filter through papier Chardin, using the hot-water funnel.

6. Tube, and sterilise as for nutrient agar.

~Litmus Whey.~--

1. Curdle fresh milk by warming to 60 C. and adding rennet.

2. Filter off the whey through b.u.t.ter muslin into a sterile flask.