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 deg. C. for twenty minutes on each of
_five_ consecutive days.

~Animal Tissue Media (Frugoni).~--

1. Take a number of sterile test-tubes 16 x 3 or 4 cm., plugged with
cotton wool, and into each insert a 2 cm. length of stout glass tubing
(about 1 cm. diameter); fill in glycerine (6 per cent.) bouillon to the
upper level of the piece of glass tubing. Sterilise in the steamer at
100 deg. 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 glass dish.

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

5. Pour into the dish a sufficient quantity 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 glass tubing. (The surface of the tissue will
now be kept moist by capillary attraction and condensation).

7. Sterilise in the autoclave at 120 deg. 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).~-