Solution A is boiled for a few minutes, cooled to room temperature, gassed
with 80% N2 + 20% CO2 gas mixture to reach a pH
below 6, then autoclaved anaerobically under the same gas mixture.
Solutions B, D, E and F are autoclaved separately under nitrogen. Solution
C is filter-sterilized and flushed with 80% N2 + 20% CO2
to remove dissolved oxygen. Solution B to F are added to the sterile, cooled
solution A in the sequence as indicated. The complete medium is
distributed anaerobically under 80% N2 + 20% CO2
into appropriate vessels. Final pH of the medium is 7.1 - 7.4. Addition of
10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution,
freshly prepared under N2 and filter-sterilized) may stimulate
growth at the beginning. For transfers use 5 - 10% inoculum.
Solution A
Distilled water
870.0
ml
Resazurin
1.0
mg
CaCl2 x 2 H2O
0.15
g
KCl
0.5
g
MgCl2 x 6 H2O
1.3
g
NaCl
7.0
g
NH4Cl
0.3
g
KH2PO4
0.2
g
Na2SO4
3.0
g
Solution B
Trace element solution SL-10
1.0
ml
Solution C
Distilled water
100.0
ml
NaHCO3
5.0
g
Solution D
Distilled water
10.0
ml
Na-acetate x 3 H2O
2.5
g
Solution E
Vitamin solution
10.0
ml
Solution F
Distilled water
10.0
ml
Na2S x 9 H2O
0.4
g
Trace element solution SL-10
Distilled water
990.0
ml
Na2MoO4 x 2 H2O
36.0
mg
NiCl2 x 6 H2O
24.0
mg
CuCl2 x 2 H2O
2.0
mg
CoCl2 x 6 H2O
190.0
mg
H3BO3
6.0
mg
MnCl2 x 4 H2O
100.0
mg
ZnCl2
70.0
mg
FeCl2 x 4 H2O
1.5
g
HCl (25%; 7.7 M)
10.0
ml
First dissolve FeCl2 in the HCl, then dilute in water, add and
dissolve the other salts. Finally make up to 1000.0 ml.
