Recommended media for the culture method: Mycoplasma
The following media are recommended. Other media may be used providing their ability to sustain the
growth of mycoplasmas has been demonstrated on each batch in the presence and absence of the
product to be examined.
Recommended media for the detection of Mycoplasma gallisepticum
(a) Liquid medium
Beef heart infusion broth (1) 90.0 ml
Horse serum (unheated) 20.0 ml
Yeast extract (250 g/l) 10.0 ml
Thallium acetate (10 g/l solution) 1.0 ml
Phenol red (0.6 g/l solution)
5.0 ml
Penicillin (20 000 IU/ml)
0.25 ml
Deoxyribonucleic acid (2 g/l solution)
1.2 ml
Adjust to pH 7.8.
(b) Solid medium
Prepare as described above replacing beef heart infusion broth by beef heart infusion agar containing 15
g/l of agar.
Recommended media for the detection of Mycoplasma synoviae
(a) Liquid medium
Beef heart infusion broth (1)
90.0 ml
Essential vitamins (2)
0.025 ml
Glucose monohydrate (500 g/l solution)
2.0 ml
Swine serum (inactivated at 56 °C for 30 min)
12.0 ml
-Nicotinamide adenine dinucleotide (10 g/l solution)
1.0 ml
Cysteine hydrochloride (10 g/l solution)
1.0 ml
Phenol red (0.6 g/l solution)
5.0 ml
Penicillin (20 000 IU/ml)
0.25 ml
Mix the solutions of -nicotinamide adenine dinucleotide and cysteine hydrochloride and after 10 min
add to the other ingredients. Adjust to pH 7.8.
(b) Solid medium
Beef heart infusion broth (1)
90.0 ml
Ionagar (3)
1.4 g
Adjust to pH 7.8, sterilise by autoclaving then add:
Essential vitamins (2)
0.025 ml
Glucose monohydrate (500 g/l solution)
2.0 ml
Swine serum (unheated)
12.0 ml
-Nicotinamide adenine dinucleotide (10 g/l solution)
1.0 ml
Cysteine hydrochloride (10 g/l solution)
1.0 ml
Phenol red (0.6 g/l solution)
5.0 ml
Penicillin (20 000 IU/ml)
0.25 ml
Recommended media for the detection of non-avian mycoplasmas
(a) Liquid medium
Hanks' balanced salt solution (modified) (4)
800 ml
Distilled water
67 ml
Brain heart infusion (5)
135 ml
PPLO Broth (6)
248 ml
Yeast extract (170 g/l)
60 ml
Bacitracin
250 mg
Meticillin
250 mg
Phenol red (5 g/l)
4.5 ml
Thallium acetate (56 g/l)
3 ml
Horse serum
165 ml
Swine serum
165 ml
Adjust to pH 7.4 - 7.45.
(b) Solid medium
Hanks' balanced salt solution (modified) (4)
200 ml
DEAE-dextran
200 mg
Ionagar (3)
15.65 mg
Mix well and sterilise by autoclaving. Cool to 100 °C. Add to 1740 ml of liquid medium as described
above.
(1) Beef heart infusion broth
Beef heart (for preparation of the infusion)
500 g
Peptone
10 g
Sodium chloride
5g
Distilled water to
1000 ml
Sterilise by autoclaving.
(2) Essential vitamins
Biotin
100 mg
Calcium pantothenate
100 mg
Choline chloride
100 mg
Folic acid
100 mg
i-Inositol
200 mg
Nicotinamide
100 mg
Pyridoxal hydrochloride
100 mg
Riboflavine
10 mg
Thiamine hydrochloride
100 mg
Distilled water to
1000 ml
(3) Ionagar
A highly refined agar for use in microbiology and immunology prepared by an ion-exchange procedure
which results in a product having superior purity, clarity and gel strength.
It contains about:
Water
12.2 per cent
Ash
1.5 per cent
Acid-insoluble ash
0.2 per cent
Chlorine
Phosphate (calculated as P2O5)
0.3 per cent
Total nitrogen
0.3 per cent
Copper
8 ppm
Iron
170 ppm
Calcium
0.28 per cent
Magnesium
0.32 per cent
(4) Hanks' balanced salt solution (modified)
Sodium chloride
6.4 g
Potassium chloride
0.32 g
Magnesium sulphate heptahydrate
0.08 g
Magnesium chloride hexahydrate
0.08 g
Calcium chloride, anhydrous
0.112 g
Disodium hydrogen phosphate dihydrate
0.0596 g
Potassium dihydrogen phosphate, anhydrous
0.048 g
Distilled water to
800 ml
(5) Brain heart infusion
Calf-brain infusion
200 g
Beef-heart infusion
250 g
Proteose peptone
10 g
Glucose monohydrate
2g
Sodium chloride
5g
Disodium hydrogen phosphate, anhydrous
25 g
Distilled water to
1000 ml
(6) PPLO broth
Beef-heart infusion
50 g
Peptone
10 g
Sodium chloride
5g
Distilled water to
1000 ml
Mannitol
Bacterial endotoxins (2.6.14)
If intended for use in the manufacture of parenteral preparations without a further appropriate
procedure for the removal of bacterial endotoxins:
— less than 4 IU/g for parenteral preparations having a concentration of 100 g/L or less of
mannitol;
— less than 2.5 IU/g for parenteral preparations having a concentration of
more than 100 g/L of mannitol.
