4. Incubate at 37 C. for forty-eight hours to eliminate any contaminated tubes. Store the remainder for future use.

~Blood Serum (Councilman and Mallory).~--

1. Collect blood serum in slaughterhouse, coagulate, remove serum and tube (_vide_ page 168).

Great care must be taken to avoid the inclusion of air bubbles--indeed if only a few tubes are filled at one time, it is a good plan to stand them upright in the receiver of an air pump and to exhaust as completely as possible before transferring to the serum insp.i.s.sator.

2. Heat the tubes in a slanting position in hot-air steriliser at 90 C.

till firmly coagulated, say half an hour.

3. Sterilise in steam steriliser at 100 C. for 20 minutes on each of three successive days.

Resulting medium not translucent, but opaque and firm.

_B. Tuberculosis._

~Egg Medium (Lubenau).~--

This modification of Dorset's egg medium (_quod vide_ page 174) is preferred by some for the growth of the tubercle bacillus of the human type. It consists in the addition of one part of 6 per cent. glycerine in normal saline solution, to the egg mixture between steps 4 and 5.

~Glycerine Bouillon.~--

1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1 to 6).

2. Measure out glycerine, 60 c.c. (= 6 per cent.), and add to the bouillon.

3. Tube, and sterilise as for bouillon.

~Glycerine Agar.~--

1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out 1000 c.c.

2. Measure out pure glycerine, 60 c.c. (= 6 per cent.), and add to the agar.

3. Tube, and sterilise as for nutrient agar.

~Glycerine Blood-serum.~--

1. Prepare blood-serum as described, page 168, sections 1 to 4.

2. Add 5 per cent. pure glycerine.

3. Complete as described above for ordinary blood-serum, sections 5 to 7.

NOTE.--Different percentages of glycerine--from 4 per cent.

to 8 per cent.--are used for special purposes. Five per cent. is that usually employed.

~Glycerinated Potato.~--

1. Prepare ordinary potato wedges (_vide_ page 174, sections 1 to 4).

2. Soak the wedges in 25 per cent. solution of glycerine for fifteen minutes.

3. Moisten the cotton-wool pads at the bottom of the potato tubes with a 25 per cent. solution of glycerine.

4. Insert a wedge of potato in each tube and replug the tubes.

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

~Animal Tissue Media (Frugoni).~--

1. Take a number of sterile test-tubes 16 3 or 4 cm., plugged with cotton wool, and into each insert a 2 cm. length of stout gla.s.s tubing (about 1 cm. diameter); fill in glycerine (6 per cent.) bouillon to the upper level of the piece of gla.s.s tubing. Sterilise in the steamer at 100 C. for twenty minutes on each of three successive days.

2. Kill a small rabbit by means of chloroform vapour.

3. Under strictly aseptic precautions remove the lungs, liver and other solid organs and transfer them to a sterile double gla.s.s dish.

4. With the help of sterile scissors and forceps divide the organs into roughly rectangular blocks 3 1.5 1 cm.

5. Pour into the dish a sufficient quant.i.ty of sterile glycerine solution (6 per cent. in normal saline), cover, and allow to stand for one hour.

6. Introduce a block of tissue into each tube so that it rests upon the upper end of the piece of gla.s.s tubing. (The surface of the tissue will now be kept moist by capillary attraction and condensation).

7. Sterilise in the autoclave at 120 C. for thirty minutes.

8. Cap the tubes and store them in the ice chest for future use.

Tissues obtained at postmortems can also be used after preliminary sterilisation by boiling or autoclaving.

_Media for the Study of Special Cocci._

_Diplococcus Gonorrhoeae._

~Ascitic Bouillon (Serum Bouillon).~--

1. Collect ascitic fluid (pleuritic fluid, hydrocele fluid, etc.), by aspiration directly into sterile flasks, under strictly aseptic precautions.

2. Mix the serum with twice its bulk of sterile nutrient bouillon (_vide_ page 163).