Witte's peptone 10 grammes Sodium chloride 5 grammes Nutrose 10 grammes

and dissolve in 200 c.c. of serum water heated to 80 C.

4. Mix the agar emulsion and the peptone-nutrose solution in a "tared"

flask of 2-litre capacity and add a further 100 c.c. serum water.

5. Complete the solution of the various ingredients by bubbling live steam through the flask as in making nutrient agar.

6. Add further 250 c.c. serum water.

7. Weigh the flask and its contents: then (1045 grammes + weight of flask) minus (weight of flask and its present contents) = weight of fluid required to make up the bulk of the medium to 1 litre. Add the requisite amount of sterile distilled water.

8. t.i.trate and estimate the reaction of the medium ma.s.s. Then standardise to reaction of +2.5.

9. Clarify with egg, and filter as for nutrient agar. (In clarifying, after the addition of the egg white the mixture should be in the steamer for full two hours.)

10. After filtration is complete measure the filtrate, and to every 150 c.c. of the medium add:

Litmus solution (Kahlbaum) 20 c.c.

Krystal violet aqueous solution (1:1000) (B. Hoechst) 1.5 c.c.

Lactose 1.5 grammes

11. Tube in quant.i.ties of 15 c.c.

12. Sterilise in the steamer at 100 C. for thirty minutes on each of three successive days--i. e., by the discontinuous method for three days.

~Egg Medium (Dorset).~--

1. Prepare 1000 c.c. of a 0.85 per cent. solution of sodium chloride in a stout 2-litre flask.

2. Sterilise in the autoclave at 120 C. for twenty minutes. Cool to 20 C.

3. Take 12 fresh eggs; wash the sh.e.l.ls first with water then with undiluted formalin: allow the sh.e.l.ls to dry.

4. Break the eggs into a sterile graduated cylinder and measure the total volume of the mixed whites and yolks. Add one part sterile saline solution to three parts mixed eggs.

5. Transfer this mixture to a large wide-mouthed stoppered bottle previously sterilised. Add sterile gla.s.s beads and shake thoroughly in a mechanical shaker for about thirty minutes, or whip with an egg-whisk.

6. Filter through coa.r.s.e b.u.t.ter muslin into a sterile flask.

NOTE.--A few drops of alcoholic solution of basic fuchsin (sufficient to give a definite pink colour), or a few drops of waterproof Chinese ink added to the medium at this stage facilitates the subsequent "fishing" of colonies.

7. Tube in quant.i.ties of 10 c.c.

8. Solidify in the sloping position in the insp.i.s.sator at 75 C. for one hour.

9. Place the tubes for forty-eight hours in the incubator at 37 C., and eliminate any contaminated tubes.

To prevent drying, 0.5 c.c. glycerine bouillon (see page 209) may be added to each tube between steps 8 and 9.

10. Cap those tubes of media which remain sterile with india-rubber caps and store for future use.

~Potato.~--

1. Choose fairly large potatoes, wash them well, and scrub the peel with a stiff nail-brush.

2. Peel and take out the eyes.

3. Remove cylinders from the longest diameter of each potato by means of an apple-corer or a large cork-borer (i. e., one of about 1.4 cm.

diameter).

The reaction of the fresh potato is strongly acid to phenolphthalein.

If, therefore, the potatoes are required to approximate +10, as for the cultivation of some of the vibrios, the cylinders should be soaked in a 1 per cent. solution of sodium carbonate for thirty minutes.

4. Cut each cylinder obliquely from end to end, forming two wedge-shaped portions.

5. Place a small piece of sterilised cotton-wool, moistened with sterile water, at the bottom of a sterile test-tube; insert the potato wedge into the tube so that its base rests upon the cotton-wool. Now plug the tube with cotton-wool (Fig. 111).

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

[Ill.u.s.tration: FIG. 111.--Potato tube.]

NOTE.--The cork borer reserved for cutting the potato cylinders should be silver electro-plated both inside and out, and the knife used for dividing the cylinders should be of silver or silver plated. When these precautions are adopted the potato wedges will retain their white color and will not show the discoloration so often observed when steel instruments are employed.

~Beer Wort.~--Wort is chiefly used as a medium for the cultivation of yeasts, moulds, etc., both in its fluid form and also when made solid by the addition of gelatine or agar. The wort is prepared as follows:

1. Weigh out 250 grammes crushed malt and place in a 2-litre flask.

2. Add 1000 c.c. distilled water, heated to 70 C., and close the flask with a rubber stopper.

3. Place the flask in a water-bath regulated to 60C. and allow the maceration to continue for one hour.

4. Strain through b.u.t.ter muslin into a clean flask and heat in the steamer for thirty minutes.

5. Filter through Swedish filter paper.

6. Tube in quant.i.ties of 10 c.c. or store in flasks.

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

The natural reaction of the wort should _not_ be interfered with.

NOTE.--It is sometimes more convenient to obtain "_unhopped_"[6] beer wort direct from the brewery. In this case it is diluted with an equal quant.i.ty of distilled water, steamed for an hour, filtered, filled into sterile flasks or tubes, and sterilised by the discontinuous method.

~Wort Gelatine.~--