Agar 20 grammes.

Emulsify in 200 c.c. distilled water.

2. Wash the agar emulsion into a tared 2-litre flask with 400 c.c.

distilled water.

3. Weigh out

Peptone 0.5 gramme.

Emulsify in 50 c.c. distilled water and add to the contents of the flask.

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

5. Weigh out and mix

Dextrose 10.0 grammes.

Pota.s.sium phosphate 0.5 gramme.

Magnesium sulphate 0.2 gramme.

Pota.s.sium nitrate 0.06 gramme.

and add to the contents of the flask.

6. Adjust the weight of the medium ma.s.s to the calculated figure for one litre (1025 grammes) by the addition of distilled water at 100C.

7. t.i.trate the medium ma.s.s and adjust the reaction to +5.

8. Cool to 60 C. Clarify with egg and filter.

9. Tube in quant.i.ties of 10 c.c. and sterilise as for nutrient agar.

~Beyrinck's Solution. I.~--(_For the cultivation of nitrogen fixing organisms._)

1. Weigh out and mix 1 gramme pota.s.sium hydrogen phosphate, 0.2 gramme magnesium sulphate, and 0.02 gramme sodium chloride.

2. Dissolve in water 1000 c.c., in a 2-litre flask.

3. Add 1 c.c. of a one per thousand aqueous solution of ferrous sulphate.

4. Add 1 c.c. of a one per thousand solution manganese sulphate.

5. Weigh out 20 grammes dextrose and add to the contents of the flask (dextrose up to 40 grammes may be used for the different organisms).

6. Steam for twenty minutes, filter.

7. Tube, and sterilise as for nutrient bouillon.

~Beyrinck's Solution. II.~--(_For growth of Azobacter._)

Proceed as in preparing solution No. I, subst.i.tuting mannite for dextrose in step 5.

~Winogradsky's Solution (for Nitric Organisms).~--

1. Weigh out and mix.

Pota.s.sium phosphate 1.0 gramme Magnesium sulphate 0.5 gramme Calcium chloride 0.01 gramme Sodium chloride 2.0 grammes

and dissolve in

Distilled water 1000 c.c.

2. Fill into flasks, in quant.i.ties of 20 c.c. and add to each a small quant.i.ty of freshly washed magnesium carbonate.

3. Sterilise in the steamer at 100 C. for twenty minutes on each of three consecutive days.

4. Add to each flask containing 20 c.c. solution, 2 c.c. of a sterile 2 per cent. solution of ammonium sulphate.

5. Incubate at 37 C. for forty-eight hours and eliminate any contaminated culture flasks. Store the remainder for future use.

~Winogradsky's Solution (for Nitrous Organisms).~--

1. Weigh out and mix

Ammonium sulphate 1 gramme Pota.s.sium sulphate 1 gramme

and dissolve in

Distilled water 1000 c.c.

2. Add 5 to 10 grammes basic magnesium carbonate, previously sterilised by boiling.

3. Fill into flasks and sterilise, etc., as for previous solution.

~Silicate Jelly (Winogradsky).~--

1. Weigh out and mix

Ammonium sulphate 0.40 gramme Magnesium sulphate 0.05 gramme Calcium chloride 0.01 gramme

and dissolve in

Distilled water 50 c.c.

Label--Solution A.