s

and dissolve in 200 c.c. of serum water heated to 80 deg. 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. Titrate and estimate the reaction of the medium mass. 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 quantities of 15 c.c.

12. Sterilise in the steamer at 100 deg. 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 deg. C. for twenty minutes. Cool
to 20 deg. C.

3. Take 12 fresh eggs; wash the shells first with water then with
undiluted formalin: allow the shells 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 glass beads and shake thoroughly in a
mechanical shaker for about thirty minutes, or whip with an egg-whisk.

6. Filter through coarse butter 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 quantities of 10 c.c.

8. Solidify in the slop