5. Tube, and stiffen at 100 C. in the serum insp.i.s.sator.

6. Incubate at 37 C. for forty-eight hours and eliminate any contaminated tubes; store the remainder for future use.

~Egg-alb.u.men (Tarchanoff and Kolesnikoff).~--

1. Place unbroken hens' eggs in dekanormal caustic soda solution for ten days. (After this time the white becomes firm like gelatine.)

2. Carefully remove the sh.e.l.l and cut the egg into fine slices.

3. Wash for two hours in running water.

4. Place the egg slices in a large beaker and sterilise in the steamer at 100 C. for one hour.

5. Transfer each slice of egg by means of a pair of sterilised forceps to a Petri dish or large capsule.

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

~Egg Alb.u.min Broth (Lipschuetz).~--

1. Weigh out

Egg alb.u.min (extra fine powder, Merck). 4 grammes

and place in a 2-litre flask with a number of sterile gla.s.s beads.

2. Measure out distilled water 200 c.c. into a half-litre flask and warm to 37 C. in the incubator.

3. Add the water to the flask containing the alb.u.min and beads and dissolve by shaking.

4. Add n/10-NaOH, 40 c.c. Allow the mixture to stand for thirty minutes with frequent shaking.

5. Filter through Swedish filter paper.

6. Sterilise by boiling two or three times at intervals of two hours.

7. Add ordinary nutrient bouillon 600 c.c.

8. Fill into small Erlenmeyer flasks in quant.i.ties of 50 c.c.

9. Incubate for forty-eight hours at 37C.--discard any contaminated flasks and store the remainder for future use.

~Egg Alb.u.min Agar.~--

1. Prepare egg alb.u.min solution as above 1-6.

2. Liquefy and measure out ordinary nutrient agar 600 c.c. and add to the egg alb.u.min solution (in place of the nutrient broth).

3. Complete as above 8-9.

_Diplococcus Meningitidis Intracellularis._

~Ascitic Fluid Agar (Wa.s.sermann)~ _Synonym_ ~N-as-gar (Mervyn Gordon).~

1. Liquefy and measure out into a sterile flask:

Nutrient agar 600 c.c.

2. Measure out into a half litre flask

Distilled water 210 c.c.

and add to it

Ascitic fluid 90 c.c.

Nutrose 6 grammes

3. Heat over a bunsen flame, shaking constantly until the fluid boils, and the nutrose is dissolved.

4. Add the nutrose ascitic solution to the fluid agar.

5. Heat in the steamer for thirty minutes, then filter.

6. Tube and sterilise as for nutrient agar.

NOTE.--The finished medium in this case measures 900 c.c.

only since inconvenient fractions would be introduced in making up to one litre exactly.

_Diplococcus Pneumoniae._

~Blood Agar (Washbourn).~--

1. Melt up several tubes of nutrient agar (_vide_ page 167) and allow them to solidify in the oblique position.

2. Place the tubes, in the horizontal position, in the "hot" incubator for forty-eight hours, to evaporate off some of the condensation water.

3. Kill a small rabbit with chloroform and nail it out on a board (as for a necropsy). Moisten the hair thoroughly with 2 per cent. solution of lysol.

4. Sterilise several pairs of forceps, scissors, etc., by boiling.

5. Reflect the skin over the thorax with sterile instruments.

6. Open the thoracic cavity by the aid of a fresh set of sterile instruments.