432
E. A. FREUNDT BT AL.
(b) Solid medium
Bacto PPLO broth w/o CV (Difco)
Ionagar No. 2 (Code L 12, Oxoid)
Distilled water
Adjust pH to 7.8
Sterilise by autoclaving (121°C 20 min-1)
Eagle's essential vitamins ( x 100)
Glucose (50% (w/v) solution)
Swine serum (unheated)
p-NADH (1 yo (w/v) solution)
Cystein hydrochloride (1 % (w/v) solution)
Phenol red (0.06 (w/v) solution)
Penicillin (20 000 IU ml-1)
6 . Medium FF74: Friis medium (for M . jlocculare and M .
hy opneumoniae)
(a) Liquid medium
Brain heart infusion broth (Difco)
Double distilled water
Bacto PPLO broth w/o CV (Difco)
Hanks's balanced salt solution (modified, see (c) below)
(Sterilise by autoclaving 121 "C 2-5 min-l)
Fresh yeast extract (FG, see (d) below)
Horse serum (inactivated)
Swine serum (from SPF pigs) inactivated
Phenol red (0.06% (w/v) solution)
Bacitracin (2.5% (w/v) solution)
Meticillin (2.5 yo (w/v) solution)
(b) Solid medium
Agar-Agar (Merck: art. 1613)
D EAE-dextran
Hanks's balanced salt solution (modified, see (c) below)
(Sterilise by autoclaving 121 "C 2--5 min-l)
After cooling below 100°C this solution is added to one portion of
(c) Hanks's balanced salt solution (HBSS), modified
The solution is prepared from two stock solutions, A and B.
(A) NaCl, 80.0 g; KCl, 4.0 g; MgS04, 7Hz0, 1.0 g; MgC12,
6Hz0, 1.0 g; dissolve in 400 ml water, add CaClz (anhydrous)
1.4 g, and water to 500 ml.
(B) NazHP04.12Hz0, 1.5 g in 400 ml water; add KHzPO4,
0.6 g and waterto 500 ml.
HBSS: 25 ml of A is added to 400 ml of water, mixed with 25 ml
of B and 50 ml of water.
preheated FF74 broth.
(d) Yeast extract F G
Fleischmann pure dry yeast, type 2040
Double distilled water
2.25 g
1.4 g
90.0 ml
0.025 ml
2.0 ml
12.0 ml
1 .O ml
1 .O ml
5.0 ml
0.25 ml
0.82 g
75 .O ml
0.87 g
50.0 ml
6.0 ml
15.0 ml
15.0 ml
5.0 ml
1 *O ml
1.0 ml
1.3 g
0.017 g
16.0 ml
125 g
750 ml
E. A. FREUNDT BT AL.
(b) Solid medium
Bacto PPLO broth w/o CV (Difco)
Ionagar No. 2 (Code L 12, Oxoid)
Distilled water
Adjust pH to 7.8
Sterilise by autoclaving (121°C 20 min-1)
Eagle's essential vitamins ( x 100)
Glucose (50% (w/v) solution)
Swine serum (unheated)
p-NADH (1 yo (w/v) solution)
Cystein hydrochloride (1 % (w/v) solution)
Phenol red (0.06 (w/v) solution)
Penicillin (20 000 IU ml-1)
6 . Medium FF74: Friis medium (for M . jlocculare and M .
hy opneumoniae)
(a) Liquid medium
Brain heart infusion broth (Difco)
Double distilled water
Bacto PPLO broth w/o CV (Difco)
Hanks's balanced salt solution (modified, see (c) below)
(Sterilise by autoclaving 121 "C 2-5 min-l)
Fresh yeast extract (FG, see (d) below)
Horse serum (inactivated)
Swine serum (from SPF pigs) inactivated
Phenol red (0.06% (w/v) solution)
Bacitracin (2.5% (w/v) solution)
Meticillin (2.5 yo (w/v) solution)
(b) Solid medium
Agar-Agar (Merck: art. 1613)
D EAE-dextran
Hanks's balanced salt solution (modified, see (c) below)
(Sterilise by autoclaving 121 "C 2--5 min-l)
After cooling below 100°C this solution is added to one portion of
(c) Hanks's balanced salt solution (HBSS), modified
The solution is prepared from two stock solutions, A and B.
(A) NaCl, 80.0 g; KCl, 4.0 g; MgS04, 7Hz0, 1.0 g; MgC12,
6Hz0, 1.0 g; dissolve in 400 ml water, add CaClz (anhydrous)
1.4 g, and water to 500 ml.
(B) NazHP04.12Hz0, 1.5 g in 400 ml water; add KHzPO4,
0.6 g and waterto 500 ml.
HBSS: 25 ml of A is added to 400 ml of water, mixed with 25 ml
of B and 50 ml of water.
preheated FF74 broth.
(d) Yeast extract F G
Fleischmann pure dry yeast, type 2040
Double distilled water
2.25 g
1.4 g
90.0 ml
0.025 ml
2.0 ml
12.0 ml
1 .O ml
1 .O ml
5.0 ml
0.25 ml
0.82 g
75 .O ml
0.87 g
50.0 ml
6.0 ml
15.0 ml
15.0 ml
5.0 ml
1 *O ml
1.0 ml
1.3 g
0.017 g
16.0 ml
125 g
750 ml