Handbook of Microbiological Media, Fourth Edition part 110 pptx

Methanobacterium II Medium 1085
use inject, for each 5.0mL medium, 0.2mL sterile NaHCO
3
solution,
0.05mL sterile Na
2
S·9H
2
O solution, and 0.05mL sterile methanol so-
lution. The pH should be 7.0.
Use: For the cultivation of Methanosarcina barkeri DSM 10131 and
Methanosarcina mazei=Methanococcus mazei (Methanosarcina fri-
sia) DSM 10132.
Methanobacterium II Medium
(DSMZ Medium 825)
Composition per 1065.0mL:
Yeast extract 1.0g
NH
4
Cl 1.0g
NaCl 0.6g
Cysteine-HCl·H
2
O 0.5g
Sodium acetate 0.5g
K
2
HPO
4
0.3g
KH

2
PO
4
0.3g
MgCl
2
·6H
2
O 0.2g
CaCl
2
·2H
2
O 0.1g
KCl 0.1g
Resazurin 0.5mg
NaHCO
3
solution 40.0mL
Na-formate solution 15.0mL
Trace elements solution 10.0mL
Vitamin solution 10.0mL
Na
2
S·9H
2
O solution 10.0mL
pH 7.0 ± 0.2 at 25°C
Na-Formate Solution:
Composition

per 10.0mL:
Na-formate 5.0g
Preparation of Na-Formate Solution: Add Na-formate to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4
·7H

2
O 0.18g
CaCl
2
·2H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g
NiCl
2
·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
H
3
BO
3
0.01g
Na

2
MoO
4
·4H
2
O 0.01g
CuSO
4
·5H
2
O 0.01g
Na
2
SeO
3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH
to 6.5 with KOH. Add remaining components. Add distilled/deionized
water to 1.0L. Mix thoroughly.
Vitamin Solution:
Composition
per liter:
Pyridoxine-HCl 10.0mg
Thiamine-HCl·2H
2
O 5.0mg
Riboflavin 5.0mg

Nicotinic acid 5.0mg
D-Ca-pantothenate 5.0mg
p-Aminobenzoic acid 5.0mg
Lipoic acid 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge
with 80% H
2
+ 20% CO
2
. Filter sterilize.
Na
2
S·9H
2
O Solution:
Composition per 10.0mL:
Na
2
S·9H
2
O 0.3g
Preparation of Na
2
S·9H

2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Sparge with N
2
.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an-
aerobically.
NaHCO
3
Solution:
Composition
per 100.0mL:
NaHCO
3
10.0g
Preparation of NaHCO
3
Solution: Add NaHCO
3
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Auto-
clave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be
prepared freshly.
Preparation of Medium: Prepare and dispense medium under 80%
N
2

+ 20% CO
2
gas mixture. Add components, except Na-formate solu-
tion, NaHCO
3
solution, and Na
2
S·9H
2
O solution, to 1.0L distilled/de-
ionized water. Mix thoroughly. Sparge with 80% N
2
+ 20% CO
2
.
Dispense 5.0mL aliquots into Hungate tubes under 80% H
2
+ 20% CO
2
gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Prior to
use inject, for each 5.0mL medium, 0.2mL sterile NaHCO
3
solution,
0.05mL sterile Na
2
S·9H
2
O solution, and 0.075mL sterile Na-formate
solution. The pH should be 7.0.
Use: For the cultivation of Methanobacterium formicicum DSM 10111.

Methanobacterium II Medium
(DSMZ Medium 825)
Composition per 1075.0mL:
Yeast extract 1.0g
NH
4
Cl 1.0g
NaCl 0.6g
Cysteine-HCl·H
2
O 0.5g
Sodium acetate 0.5g
K
2
HPO
4
0.3g
KH
2
PO
4
0.3g
MgCl
2
·6H
2
O 0.2g
CaCl
2
·2H

2
O 0.1g
KCl 0.1g
Resazurin 0.5mg
NaHCO
3
solution 40.0mL
Na-formate solution 15.0mL
Trace elements solution 10.0mL
Vitamin solution 10.0mL
Na
2
S·9H
2
O solution 10.0mL
Trypticase™ solution 10.0mL
pH 7.0 ± 0.2 at 25°C
Trypticase™ Solution:
Composition per 10.0mL:
Trypticase™ 1.0g
© 2010 by Taylor and Francis Group, LLC
1086 Methanobrevibacter curvatus Medium
Preparation of Trypticase™ Solution: Add Trypticase™ to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Autoclave under 100% N
2
for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
Na-Formate Solution:
Composition

per 10.0mL:
Na-formate 5.0g
Preparation of Na-Formate Solution: Add Na-formate to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4
·7H

2
O 0.18g
CaCl
2
·2H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g
NiCl
2
·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
H
3
BO
3
0.01g
Na

2
MoO
4
·4H
2
O 0.01g
CuSO
4
·5H
2
O 0.01g
Na
2
SeO
3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH
to 6.5 with KOH. Add remaining components. Add distilled/deionized
water to 1.0L. Mix thoroughly.
Vitamin Solution:
Composition
per liter:
Pyridoxine-HCl 10.0mg
Thiamine-HCl·2H
2
O 5.0mg
Riboflavin 5.0mg

Nicotinic acid 5.0mg
D-Ca-pantothenate 5.0mg
p-Aminobenzoic acid 5.0mg
Lipoic acid 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge
with 80% H
2
+ 20% CO
2
. Filter sterilize.
Na
2
S·9H
2
O Solution:
Composition per 10.0mL:
Na
2
S·9H
2
O 0.3g
Preparation of Na
2
S·9H

2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Sparge with N
2
.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an-
aerobically.
NaHCO
3
Solution:
Composition
per 100.0mL:
NaHCO
3
10.0g
Preparation of NaHCO
3
Solution: Add NaHCO
3
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Auto-
clave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be
prepared freshly.
Preparation of Medium: Prepare and dispense medium under 80%
N
2

+ 20% CO
2
gas mixture. Add components, except Trypticase™ so-
lution, Na-formate solution, NaHCO
3
solution, and Na
2
S·9H
2
O solu-
tion, to 1.0L distilled/deionized water. Mix thoroughly. Sparge with
80% N
2
+ 20% CO
2
. Dispense 5.0mL aliquots into Hungate tubes un-
der 80% H
2
+ 20% CO
2
gas mixture. Autoclave for 15 min at 15 psi
pressure–121°C. Prior to use inject, for each 5.0mL medium, 0.2mL
sterile NaHCO
3
solution, 0.05mL sterile Trypticase™ solution,
0.05mL sterile Na
2
S·9H
2
O solution, and 0.075mL sterile Na-formate

solution. The pH should be 7.0.
Use: For the cultivation of Methanobacterium oryzae DSM 11106.
Methanobrevibacter curvatus Medium
(DSMZ Medium 734)
Composition per 1010.0mL:
NaCl 1.0g
KCl 0.5g
Casamino acids 0.5g
Yeast extract 0.5g
MgCl
2
·6H
2
O 0.4g
NH
4
Cl 0.3g
KH
2
PO
4
0.2g
Na
2
SO
4
0.15g
CaCl
2
·2H

2
O 0.1g
Resazurin 0.5mg
NaHCO
3
solution 40.0mL
Dithionite solution 10.0mL
Trace elements solution SL-10 1.0mL
Selenite-tungstate solution 1.0mL
Seven vitamin solution 1.0mL
pH 7.4 ± 0.2 at 25°C
NaHCO
3
Solution:
Composition
per 40.0mL:
NaHCO
3
5.8g
Preparation of NaHCO
3
Solution: Add NaHCO
3
to distilled/de-
ionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave
for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared
freshly.
Selenite-Tungstate Solution
Composition
per liter:

NaOH 0.5g
Na
2
WO
4
·2H
2
O 4.0mg
Na
2
SeO
3
·5H
2
O 3.0mg
Preparation of Selenite-Tungstate Solution: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Dithionite Solution
Composition
per 10.0mL:
Na-dithionite 2.0mg
Preparation of Dithionite Solution: Add Na-dithionite to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Trace Elements Solution SL-10:

Composition
per liter:
FeCl
2
·4H
2
O 1.5g
CoCl
2
·6H
2
O 190.0mg
© 2010 by Taylor and Francis Group, LLC
Methanobrevibacter curvatus Medium 1087
MnCl
2
·4H
2
O 100.0mg
ZnCl
2
70.0mg
Na
2
MoO
4
·2H
2
O 36.0mg
NiCl

2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10: Add FeCl
2
·4H
2
O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. Sparge with 80% N
2
+ 20% CO
2
. Autoclave for 15 min at 15
psi pressure–121°C.
Seven Vitamin Solution:
Composition

per liter:
Pyridoxine hydrochloride 300.0mg
Thiamine-HCl·2H
2
O 200.0mg
Nicotinic acid 200.0mg
Vitamin B
12
100.0mg
Calcium pantothenate 100.0mg
p-Aminobenzoic acid 80.0mg
D(+)-Biotin 20.0mg
Preparation of Seven Vitamin Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Sparge with 100% N
2
.
Mix thoroughly. Filter sterilize.
Preparation of Medium: Prepare and dispense medium under 80%
H
2
+ 20% CO
2
gas atmosphere. Add components, except NaHCO
3
solu-
tion, seven vitamin solution, selenite-tungstate solution, and trace ele-
ments solution SL-10, to distilled/deionized water and bring volume to
947.0mL. Mix thoroughly. Adjust pH to 7.6. Sparge with 80% H
2
+ 20%

CO
2
. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and an-
aerobically add 40.0mL NaHCO
3
solution, 1.0mL selenite-tungstate
solution, 1.0mL seven vitamin solution, and 1.0mL trace elements so-
lution SL-10. Mix thoroughly. Aseptically and anaerobically distribute
into sterile tubes or bottles. Adjust pH to 7.6. Prior to inoculation add di-
thionite solution (0.1mL per 10mL medium) as reductant.
Use: For the cultivation of Methanobrevibacter curvatus.
Methanobrevibacter curvatus Medium
(DSMZ Medium 734)
Composition per 1022.0mL:
NaCl 1.0g
KCl 0.5g
Casamino acids 0.5g
Yeast extract 0.5g
MgCl
2
·6H
2
O 0.4g
NH
4
Cl 0.3g
KH
2
PO
4

0.2g
Na
2
SO
4
0.15g
CaCl
2
·2H
2
O 0.1g
Resazurin 0.5mg
Rumen fluid, bovine, clarified 400mL
NaHCO
3
solution 40.0mL
Dithionite solution 10.0mL
MOPS buffer 10.0mL
Nutrient supplement solution 2.0mL
Trace elements solution SL-10 1.0mL
Selenite-tungstate solution 1.0mL
Seven vitamin solution 1.0mL
pH 7.2 ± 0.2 at 25°C
NaHCO
3
Solution:
Composition
per 40.0mL:
NaHCO
3

5.8g
Preparation of NaHCO
3
Solution: Add NaHCO
3
to distilled/de-
ionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave
for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared
freshly.
Selenite-Tungstate Solution:
Composition
per liter:
NaOH 0.5g
Na
2
WO
4
·2H
2
O 4.0mg
Na
2
SeO
3
·5H
2
O 3.0mg
Preparation of Selenite-Tungstate Solution: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N

2
. Filter sterilize.
Dithionite Solution:
Composition
per 10.0mL:
Na-dithionite 2.0mg
Preparation of Dithionite Solution: Add Na-dithionite to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Trace Elements Solution SL-10:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
CoCl
2
·6H
2
O 190.0mg
MnCl
2
·4H
2
O 100.0mg
ZnCl

2
70.0mg
Na
2
MoO
4
·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10: Add FeCl
2
·4H
2
O

to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. Sparge with 80% N
2
+ 20% CO
2
. Autoclave for 15 min at 15
psi pressure–121°C.
Seven Vitamin Solution:
Composition
per liter:
Pyridoxine hydrochloride 300.0mg
Thiamine-HCl·2H
2
O 200.0mg
Nicotinic acid 200.0mg
Vitamin B
12
100.0mg
Calcium pantothenate 100.0mg
p-Aminobenzoic acid 80.0mg
D(+)-Biotin 20.0mg
Preparation of Seven Vitamin Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Sparge with 100% N
2
.
Mix thoroughly. Filter sterilize.
Nutrient Supplement Solution:
Composition
per liter:

Pancreatic digest of gelatin 5.0g
Beef extract 3.0g
Preparation of Nutrient Supplement Solution: Add compo-
nents to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–
121°C. Cool to room tempterature.
© 2010 by Taylor and Francis Group, LLC
1088 Methanobrevibacter curvatus Medium
MOPS Buffer:
Composition
per 10.0mL:
MOPS [3-(N-morpholino) propane
sulfonic acid] 2.1g
Na-acetate 0.3g
EDTA 0.1g
Preparation of MOPS Buffer: Add components to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 100% N
2
. Adjust to pH 7.2. Filter sterilize.
Preparation of Medium: Prepare and dispense medium under 80%
H
2
+ 20% CO
2
gas atmosphere. Add components, except clarified bo-
vine rumen fluid, MOPS buffer, nutrient supplement solution, NaHCO
3

solution, seven vitamin solution, selenite-tungstate solution, and trace
elements solution SL-10, to distilled/deionized water and bring volume
to 547.0mL. Mix thoroughly. Adjust pH to 7.2. Sparge with 80% H
2
+
20% CO
2
. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically
and anaerobically add 400.0mL sterile clarified bovine rumen fluid,
10.0mL MOPS buffer, 2.0mL nutrient supplement solution, 40.0mL
NaHCO
3
solution, 1.0mL selenite-tungstate solution, 1.0mL seven vi-
tamin solution, and 1.0mL trace elements solution SL-10. Mix thor-
oughly. Aseptically and anaerobically distribute into sterile tubes or
bottles. Adjust pH to 7.2. Prior to inoculation add dithionite solution
(0.1mL per 10mL medium) as reductant.
Use: For the cultivation of Methanobrevibacter curvatus DSM 11111
(strain RFM-2).
Methanobrevibacter curvatus Medium
(DSMZ Medium 734)
Composition per 1020.0mL:
NaCl 1.0g
KCl 0.5g
Casamino acids 0.5g
Yeast extract 0.5g
MgCl
2
·6H
2

O 0.4g
NH
4
Cl 0.3g
KH
2
PO
4
0.2g
Na
2
SO
4
0.15g
CaCl
2
·2H
2
O 0.1g
Resazurin 0.5mg
Rumen fluid, bovine, clarified 200mL
NaHCO
3
solution 40.0mL
Dithionite solution 10.0mL
MOPS buffer 10.0mL
Trace elements solution SL-10 1.0mL
Selenite-tungstate solution 1.0mL
Seven vitamin solution 1.0mL
pH 7.7 ± 0.2 at 25°C

NaHCO
3
Solution:
Composition
per 40.0mL:
NaHCO
3
5.8g
Preparation of NaHCO
3
Solution: Add NaHCO
3
to distilled/de-
ionized water and bring volume to 40.0mL. Mix thoroughly. Autoclave
for 15 min at 15 psi pressure–121°C. Cool to 25°C. Must be prepared
freshly.
Selenite-Tungstate Solution:
Composition
per liter:
NaOH 0.5g
Na
2
WO
4
·2H
2
O 4.0mg
Na
2
SeO

3
·5H
2
O 3.0mg
Preparation of Selenite-Tungstate Solution: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Dithionite Solution:
Composition
per 10.0mL:
Na-dithionite 2.0mg
Preparation of Dithionite Solution: Add Na-dithionite to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Filter sterilize.
Trace Elements Solution SL-10:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
CoCl
2
·6H
2

O 190.0mg
MnCl
2
·4H
2
O 100.0mg
ZnCl
2
70.0mg
Na
2
MoO
4
·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2

O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10: Add FeCl
2
·4H
2
O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. Sparge with 80% N
2
+ 20% CO
2
. Autoclave for 15 min at 15
psi pressure–121°C.
Seven Vitamin Solution:
Composition
per liter:
Pyridoxine hydrochloride 300.0mg
Thiamine-HCl·2H
2
O 200.0mg
Nicotinic acid 200.0mg
Vitamin B
12
100.0mg
Calcium pantothenate 100.0mg
p-Aminobenzoic acid 80.0mg
D(+)-Biotin 20.0mg
Preparation of Seven Vitamin Solution: Add components to dis-

tilled/deionized water and bring volume to 1.0L. Sparge with 100% N
2
.
Mix thoroughly. Filter sterilize.
MOPS Buffer:
Composition
per 10.0mL:
MOPS [3-(N-morpholino) propane sulfonic acid] 2.1g
Na-acetate 0.3g
EDTA 0.1g
Preparation of MOPS Buffer: Add components to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 100% N
2
. Adjust to pH 7.7. Filter sterilize.
Preparation of Medium: Prepare and dispense medium under 80%
H
2
+ 20% CO
2
gas atmosphere. Add components, except clarified bo-
vine rumen fluid, MOPS buffer, NaHCO
3
solution, seven vitamin solu-
tion, selenite-tungstate solution, and trace elements solution SL-10, to
distilled/deionized water and bring volume to 747.0mL. Mix thoroughly.
Adjust pH to 7.7. Sparge with 80% H
2
+ 20% CO
2

. Autoclave for 15 min
at 15 psi pressure–121°C. Aseptically and anaerobically add 200.0mL
sterile clarified bovine rumen fluid, 10.0mL MOPS buffer, 240.0mL
NaHCO
3
solution, 1.0mL selenite-tungstate solution, 1.0mL seven vi-
tamin solution, and 1.0mL trace elements solution SL-10. Mix thor-
oughly. Aseptically and anaerobically distribute into sterile tubes or
bottles. Adjust pH to 7.7. Prior to inoculation add dithionite solution
(0.1mL per 10mL medium) as reductant.
© 2010 by Taylor and Francis Group, LLC
Methanocalculus pumilus Medium 1089
Use: For the cultivation of Methanobrevibacter curvatus DSM 11111
DSM 11139 (strain RFM-1)
Methanocalculus halotolerans Medium
(DSMZ Medium 905)
Composition per 1010.0mL:
NaCl 50.0g
NH
4
Cl 1.0g
Na-acetate 0.5g
Yeast extract 0.5g
Trypticase™ 0.5g
K
2
HPO
4
0.3g
KH

2
PO
4
0.3g
KCl 0.17g
Resazurin 0.5mg
Magnesium chloride solution 30.0mL
NaHCO
3
solution 20.0mL
Trace elements solution 10.0mL
Calcium chloride solution 10.0mL
Na
2
S·9H
2
O solution 10.0mL
L-Cysteine solution 10.0mL
pH 7.2-7.6 at 25°C
Magnesium Chloride Solution:
Composition
per 30.0mL:
MgCl
2
·6H
2
O 3.2g
Preparation of Magnesium Chloride Solution: Add
MgCl
2

·6H
2
O to distilled/deionized water and bring volume to 30.0mL.
Mix thoroughly. Sparge with 100% N
2
. Autoclave for 15 min at 15 psi
pressure–121°C.
Calcium Chloride Solution:
Composition
per 10.0mL:
CaCl
2
·2H
2
O 0.6g
Preparation of Calcium Chloride Solution: Add CaCl
2
·2H
2
O
to distilled/deionized water and bring volume to 10.0mL. Mix thor-
oughly. Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–
121°C.
L-Cysteine Solution:
Composition
per 10.0mL:
L-Cysteine·HCl·H
2

O 0.5g
Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H
2
O to
distilled/deionized water and bring volume to 10.0mL. Mix thorough-
ly. Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–
121°C.
Na
2
S·9H
2
O Solution:
Composition
per 10.0mL:
Na
2
S·9H
2
O 0.3g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H
2
O to dis-

tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
NaHCO
3
Solution:
Composition
per 20.0mL:
NaHCO
3
2.0g
Preparation of NaHCO
3
Solution: Add NaHCO
3
to distilled/de-
ionized water and bring volume to 20.0mL. Mix thoroughly. Sparge
with 80% N
2
+ 20% CO
2
. Autoclave for 15 min at 15 psi pressure–
121°C. Cool to 25°C. Must be prepared freshly.
Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H

2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4
·7H
2
O 0.18g
CaCl
2
·2H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g
NiCl

2
·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
H
3
BO
3
0.01g
Na
2
MoO
4
·4H
2
O 0.01g
CuSO
4
·5H
2
O 0.01g
Na
2

SeO
3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH
to 6.5 with KOH. Add remaining components. Add distilled/deionized
water to 1.0L. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under
sparge with 80% N
2
+ 20% CO
2
. Add components, except NaHCO
3
so-
lution, magnesium chloride solution, calcium chloride solution,
L-
cysteine-HCl·H
2
O solution, and Na
2
S·9H
2
O solution, to distilled/de-
ionized water and bring volume to 950.0mL. Mix thoroughly. Gently
heat and bring to boiling. Boil for 3 min. Cool to room temperature while
sparging with 80% N
2

+ 20% CO
2
. Add 10.0mL L-cysteine-HCl·H
2
O
solution and 20.0mL NaHCO
3
solution. Mix thoroughly. Adjust pH to
7.5. Distribute into anaerobic tubes or bottles. Autoclave for 15 min at
15 psi pressure–121°C. Aseptically and anaerobically add per liter,
30.0mL magnesium chloride solution, 10.0mL calcium chloride solu-
tion, and 10.0mL Na
2
S·9H
2
O. Mix thoroughly. After inoculation pres-
surize vessels with 80% H
2
+ 20% CO
2
gas mixture to 1 bar
overpressure and add sulfide from a sterile, anaerobic stock solution.
The final pH of the medium should be 7.2–7.6.
Use: For the cultivation of Methanocalculus halotolerans.
Methanocalculus pumilus Medium
(DSMZ Medium 892)
Composition per 1080.0mL:
NaCl 10.0g
Yeast extract 2.0g
Trypticase™ 2.0g

NH
4
Cl 0.9g
K
2
HPO
4
0.4g
MgCl
2
·6H
2
O 0.36g
Resazurin 0.5mg
Na
2
CO
3
solution 50.0mL
Na
2
S·9H
2
O solution 15.0mL
L-Cysteine-HCl·H
2
O solution 15.0mL
Vitamin solution 10.0mL
Trace elements solution 10.0mL
L-Cysteine Solution:

Composition
per 15.0mL:
L-Cysteine·HCl·H
2
O 0.5g
Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H
2
O to
distilled/deionized water and bring volume to 15.0mL. Mix thorough-
ly. Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–
121°C.
© 2010 by Taylor and Francis Group, LLC
1090 Methanococcoides Medium
Na
2
CO
3
Solution:
Composition
per 50.0mL:
Na
2
CO
3
5.0g
Preparation of Na
2
CO

3
Solution: Add Na
2
CO
3
to distilled/de-
ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 80% N
2
+ 20% CO
2
. Filter sterilize.
Na
2
S·9H
2
O Solution:
Composition per 15mL:
Na
2
S·9H
2
O 0.5g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H

2
O to dis-
tilled/deionized water and bring volume to 15.0mL. Mix thoroughly.
Autoclave under 100% N
2
for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4
·7H

2
O 0.18g
CaCl
2
·2H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g
NiCl
2
·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
H
3
BO
3
0.01g
Na

2
MoO
4
·4H
2
O 0.01g
CuSO
4
·5H
2
O 0.01g
Na
2
SeO
3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH
to 6.5 with KOH. Add remaining components. Add distilled/deionized
water to 1.0L. Mix thoroughly.
Vitamin Solution:
Composition
per liter:
Pyridoxine-HCl 10.0mg
Thiamine-HCl·2H
2
O 5.0mg
Riboflavin 5.0mg

Nicotinic acid 5.0mg
D-Ca-pantothenate 5.0mg
p-Aminobenzoic acid 5.0mg
Lipoic acid 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge
with 80% H
2
+ 20% CO
2
. Filter sterilize.
Preparation of Medium: Add components, except Na
2
CO
3
solu-
tion, Na
2
S·9H
2
O solution, and L-cysteine solution, to distilled/deion-
ized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100%
N
2
for 30 min. Autoclave for 15 min at 15 psi pressure–121°C. Asepti-

cally distribute 5.0mL aliquots into Hungate tubes under 100% N
2
.
Aseptically and anaerobically add per 5.0mL medium 0.25mL Na
2
CO
3
solution, 0.075mL Na
2
S·9H
2
O solution, and 0.075mL L-cysteine solu-
tion. Mix thoroughly. Replace N
2
atmosphere with atmosphere of 80%
H
2
+ 20% CO
2
. Repeat atmosphere replacement several times with
overpressurization. The initial pH of 9.0 will decrease over a 30 min
period to 7.3–7.5. After inoculation use atmosphere of 80% H
2
+ 20%
CO
2
to 1.5 bar overpressure.
Use: For the cultivation of Methanocalculus pumilus.
Methanococcal Complex Medium
See: Methanococcus McC Medium

Methanococcoides Medium
Composition per liter:
NaCl 18.0g
NaHCO
3
5.0g
MgCl
2
·6H
2
O 4.0g
MgSO
4
·7H
2
O 3.45g
Trimethylamine·HCl 3.0g
Trypticase™ 2.0g
Yeast extract 2.0g
Sodium acetate 1.0g
L-Cysteine·HCl 0.5g
Na
2
S·9H
2
O 0.5g
KCl 0.335g
NH
4
Cl 0.25g

CaCl
2
·2H
2
O 0.14g
K
2
HPO
4
0.14g
Fe(NH
4
)
2
(SO
4
)
2
·7H
2
O 2.0mg
Resazurin 1.0mg
Trace elements solution 10.0mL
Vitamin solution 10.0mL
pH 7.0 ± 0.2 at 25°C
Trace Elements Solution:
Composition
per liter:
MgSO
4

·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4
·7H
2
O 0.18g
CaCl
2
·2H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g

NiCl
2
·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
CuSO
4
·5H
2
O 0.01g
H
3
BO
3
0.01g
Na
2
MoO
4
·2H
2
O 0.01g
Na

2
SeO
3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Adjust pH to 7.0. Add distilled/de-
ionized water to 1.0L.
Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 10.0mg
Calcium
DL-pantothenate 5.0mg
Lipoic acid 5.0mg
Nicotinic acid 5.0mg
p-Aminobenzoic acid 5.0mg
Riboflavin 5.0mg
Thiamine·HCl 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
© 2010 by Taylor and Francis Group, LLC
Methanococcus jannaschii Medium 1091
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thor-

oughly.
Preparation of Medium: Prepare the medium anaerobically under
80% H
2
+ 20% CO
2
. Add components to distilled/deionized water and
bring volume to 1.0L. Mix thoroughly. Sparge with 80% H
2
+ 20%
CO
2
. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation and maintenance of Methanococcoides meth-
ylutens.
Methanococcus deltae Medium
Composition per liter:
NaCl 35.0g
NaHCO
3
5.0g
MgCl
2
·6H
2
O 4.0g
NH
4
Cl 2.7g
Sodium acetate 2.5g

L-Cysteine·HCl 0.3g
K
2
HPO
4
0.3g
KH
2
PO
4
0.3g
Na
2
S·9H
2
O 0.3g
MgSO
4
·7H
2
O 0.13g
Resazurin 1.0mg
(NH
4
)
2
SO
4
0.3mg
Trace elements solution 10.0mL

Vitamin solution 10.0mL
L-Cysteine·HCl solution 10.0mL
Na
2
S·9H
2
O solution 10.0mL
pH 6.9 ± 0.2 at 25°C
Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4

·7H
2
O 0.18g
CaCl
2
·2H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g
NiCl
2
·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
CuSO
4
·5H
2
O 0.01g

H
3
BO
3
0.01g
Na
2
MoO
4
·2H
2
O 0.01g
Na
2
SeO
3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Adjust pH to 7.0. Add distilled/de-
ionized water to 1.0L.
Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 10.0mg
Calcium DL-pantothenate 5.0mg
Lipoic acid 5.0mg
Nicotinic acid 5.0mg

p-Aminobenzoic acid 5.0mg
Riboflavin 5.0mg
Thiamine·HCl 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thor-
oughly.
L-Cysteine·HCl Solution:
Composition
per 10.0mL:
L-Cysteine·HCl 0.3g
Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to
distilled/deionized water and bring volume to 10.0mL. Mix thorough-
ly. Autoclave under 100% N
2
for 15 min at 15 psi pressure–121°C.
Na
2
S·9H
2
O Solution:
Composition
per 10.0mL:
Na
2
S·9H

2
O 0.3g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
Preparation of Medium: Prepare and dispense medium under 80%
H
2
+ 20% CO
2
. Add components, except L-cysteine·HCl solution and
Na
2
S·9H
2
O solution, to distilled/deionized water and bring volume to
980.0mL. Mix thoroughly. Anaerobically distribute into tubes or flasks
fitted with butyl rubber stoppers. Autoclave for 15 min at 15 psi pres-
sure–121°C. Anaerobically add 10.0mL of sterile
L-cysteine·HCl solu-

tion and 10.0mL of sterile Na
2
S·9H
2
O solution to each liter of medium
or, using a syringe, inject the appropriate amount of sterile L-
cysteine·HCl solution and sterile Na
2
S·9H
2
O solution into individual
tubes containing medium.
Use: For the cultivation and maintenance of Methanococcus deltae.
Methanococcus jannaschii Medium
Composition per liter:
NaCl 30.0g
MgSO
4
·7H
2
O 3.40g
MgCl
2
·2H
2
O 2.7g
NaHCO
3
1.0g
Na

2
S·9H
2
O 0.5g
KCl 0.33g
NH
4
Cl 0.25g
CaCl
2
·2H
2
O 0.14g
K
2
HPO
4
0.14g
Fe(NH
4
)
2
(SO
4
)
2
·6H
2
O 0.01g
Resazurin 1.0mg

Na
2
SeO
3
·5H
2
O 0.5mg
NiCl
2
·6H
2
O 0.5mg
Trace elements solution 10.0mL
Vitamin solution 10.0mL
L-Cysteine·HCl solution 10.0mL
Na
2
S·9H
2
O solution 10.0mL
pH 6.0 ± 0.2 at 25°C
Trace Elements Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g

NaCl 1.0g
MnSO
4
·2H
2
O 0.5g
CoSO
4
·7H
2
O 0.18g
ZnSO
4
·7H
2
O 0.18g
CaCl
2
·2H
2
O 0.1g
© 2010 by Taylor and Francis Group, LLC
1092 Methanococcus jannaschii Medium
FeSO
4
·7H
2
O 0.1g
NiCl
2

·6H
2
O 0.025g
KAl(SO
4
)
2
·12H
2
O 0.02g
CuSO
4
·5H
2
O 0.01g
H
3
BO
3
0.01g
Na
2
MoO
4
·2H
2
O 0.01g
Na
2
SeO

3
·5H
2
O 0.3mg
Preparation of Trace Elements Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Adjust pH to 7.0. Add distilled/de-
ionized water to 1.0L.
Vitamin Solution:
Composition
per liter:
Pyridoxine·HCl 10.0mg
Calcium
DL-pantothenate 5.0mg
Lipoic acid 5.0mg
Nicotinic acid 5.0mg
p-Aminobenzoic acid 5.0mg
Riboflavin 5.0mg
Thiamine·HCl 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Adjust pH to 7.0. Mix thor-
oughly.
L-Cysteine·HCl Solution:
Composition
per 10.0mL:

L-Cysteine·HCl 0.5g
Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to
distilled/deionized water and bring volume to 10.0mL. Mix thorough-
ly. Autoclave under 100% N
2
for 15 min at 15 psi pressure–121°C.
Na
2
S·9H
2
O Solution:
Composition
per 10.0mL:
Na
2
S·9H
2
O 0.3g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2

. Autoclave for 15 min at 15 psi pressure–121°C.
Preparation of Medium: Prepare and dispense medium under 80%
H
2
+ 20% CO
2
. Add components, except L-cysteine·HCl solution and
Na
2
S·9H
2
O solution, to distilled/deionized water and bring volume to
980.0mL. Mix thoroughly. Anaerobically distribute into tubes or
flasks. Autoclave for 15 min at 15 psi pressure–121°C. Anaerobically
add 10.0mL of sterile
L-cysteine·HCl solution and 10.0mL of sterile
Na
2
S·9H
2
O solution to each liter of medium or, using a syringe, inject
the appropriate amount of sterile
L-cysteine·HCl solution and sterile
Na
2
S·9H
2
O solution into individual tubes containing medium.
Use: For the cultivation and maintenance of Methanococcus species.
Methanococcus jannaschii Medium

Composition per 1020.0mL:
PIPES (piperazine-N,N´-
bis[2-ethanesulfonic acid]) buffer 15.12g
MgCl
2
·6H
2
O 4.3g
MgSO
4
·7H
2
O 3.4g
NaCl 3.0g
NH
4
Cl 0.25g
K
2
HPO
4
0.14g
CaCl
2
·2H
2
O 0.14g
KCl 0.33g
Minerals solution 10.0mL
Na

2
S
2
O
3
solution 10.0mL
β-Mercaptoethanol solution 10.0mL
SeO
2
solution 1.0mL
pH 7.0 ± 0.2 at 25°C
Minerals Solution:
Composition
per liter:
Nitrilotriacetic acid 4.5g
FeCl
2
·4H
2
O 0.4g
CoCl
2
·2H
2
O 0.17g
MnCl
2
·4H
2
O 0.1g

ZnCl
2
0.1g
NaMoO
4
·6H
2
O 36.0mg
CaCl
2
·H
2
O 27.0mg
Preparation of Minerals Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
Na
2
S
2
O
3
Solution:
Composition
per 10.0mL:
Na
2
S
2
O
3

·5H
2
O 0.63g
Preparation of Na
2
S
2
O
3
Solution: Add Na
2
S
2
O
3
·5H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
β-Mercaptoethanol Solution:
Composition
per 10.0mL:
β-Mercaptoethanol 0.39g
Preparation of β-Mercaptoethanol Solution: Add β-mercapto-
ethanol to distilled/deionized water and bring volume to 10.0mL. Mix
thoroughly. Sparge with 100% N
2

. Autoclave for 15 min at 15 psi pres-
sure–121°C.
SeO
2
Solution:
Composition
per 100.0mL:
SeO
2
0.011g
Preparation of SeO
2
Solution: Add SeO
2
to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Add components, except Na
2
S
2
O
3
solution and β-mercapto-
ethanol solution, to distilled/deionized water and bring volume to 1.0L.
Mix thoroughly. Gently heat and bring to boiling. Continue boiling for
3 min. Cool to room temperature while sparging with 100% N
2

. Adjust
pH to 7.0 with KOH. Anaerobically distribute 100.0mL volumes into an-
aerobic bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptical-
ly and anaerobically add 1.0mL of sterile Na
2
S
2
O
3
solution and 1.0mL of
sterile β-mercaptoethanol solution to each bottle. Mix thoroughly. Prior to
inoculation, flush each bottle with 80% H
2
+ 20% CO
2
.
Use: For the cultivation of Methanococcus jannaschii.
Methanococcus McC Medium
Composition per 1100.0mL:
NaHCO
3
5.0g
Yeast extract 2.0g
L-Cysteine·HCl·H
2
O 0.5g
General salts solution 500.0mL
NaCl solution 75.0mL
Na
2

S·9H
2
O solution 20.0mL
K
2
HPO
4
solution 10.0mL
© 2010 by Taylor and Francis Group, LLC
Methanococcus McN Medium 1093
Trace minerals solution 10.0mL
Sodium acetate solution 10.0mL
Iron stock solution 5.0mL
Resazurin solution 1.0mL
General Salts Solution:
Composition
per liter:
MgSO
4
·7H
2
O 6.9g
MgCl
2
·6H
2
O 5.5g
NH
4
Cl 1.0g

KCl 0.67g
CaCl
2
·2H
2
O 0.28g
Preparation of General Salts Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly.
NaCl Solution:
Composition
per 100.0mL:
NaCl 29.3g
Preparation of NaCl Solution: Add NaCl to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly.
Na
2
S·9H
2
O Solution:
Composition
per 100.0mL:
NaOH 1 pellet
Na
2
S·9H
2
O 2.5g
Preparation of Na
2
S·9H

2
O Solution: Bring 100.0mL of dis-
tilled/deionized water to boiling. Cool to room temperature while
sparging with 100%N
2
. Dissolve 1 pellet of NaOH in the anaerobic wa-
ter. Weigh out a little more than 2.5g of Na
2
S·9H
2
O. Briefly rinse the
crystals in distilled/deionized water. Dry the crystals by blotting on pa-
per towels or filter paper. Add 2.5g of washed Na
2
S·9H
2
O crystals to
100.0mL of anaerobic NaOH solution. Distribute into serum bottles fit-
ted with butyl rubber stoppers and aluminum seals. Do not grease stop-
pers. Pressurize to 60kPa with 100% N
2
. Autoclave for 15 min at 15 psi
pressure–121°C. Store at room temperature in an anaerobic chamber.
K
2
HPO
4
Solution:
Composition
per 100.0mL:

K
2
HPO
4
1.4g
Preparation of K
2
HPO
4
Solution: Add K
2
HPO
4
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly.
Trace Minerals Solution:
Composition
per liter:
Nitrilotriacetic acid 1.5g
Na
2
WO
4
·2H
2
O 1.0g
Fe(NH
4
)
2

(SO
4
)
2
·6H
2
O 0.2g
Na
2
SeO
3
0.2g
Na
2
MoO
4
·2H
2
O 0.1g
Mn
4
·2H
2
O 0.1g
Zn
4
·7H
2
O 0.1g
NiCl

2
·7H
2
O 0.025g
CuSO
4
·5H
2
O 0.01g
Preparation of Trace Minerals Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Add distilled/deionized water to
1.0L. Adjust pH to 7.0.
Sodium Acetate Solution:
Composition
per 100.0mL:
Sodium acetate·3H
2
O 13.6g
Preparation of Sodium Acetate Solution: Add sodium ace-
tate·3H
2
O to distilled/deionized water and bring volume to 100.0mL.
Mix thoroughly.
Iron Stock Solution:
Composition
per 100.0mL:
Fe(NH
4
)

2
(SO
4
)
2
·6H
2
O 0.2g
Preparation of Iron Stock Solution: Add Fe(NH
4
)
2
(SO
4
)
2
·6H
2
O
to 5.0mL of distilled H
2
O containing 2 drops of concentrated HCl. Mix
thoroughly. When the Fe(NH
4
)
2
(SO
4
)
2

·6H
2
O has dissolved, bring the
volume to 100.0mL with distilled/deionized water.
Resazurin Solution:
Composition
per 10.0mL:
Resazurin 10.0mg
Preparation of Resazurin Solution: Add resazurin to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under 80%
H
2
+ 20% CO
2
. Add components, except NaHCO
3
and Na
2
S·9H
2
O so-
lution, to distilled/deionized water and bring volume to 1080.0mL.
Mix thoroughly. Gently heat and bring to boiling. Continue boiling for
3 min. Cool to room temperature while sparging with 80% H
2
+ 20%
CO
2
. Add NaHCO

3
. Mix thoroughly. Anaerobically distribute 9.8mL
volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure–
121°C. Aseptically and anaerobically add 0.2mL of sterile Na
2
S·9H
2
O
solution to each tube. Mix thoroughly.
Use: For the cultivation of Methanococcus species.
Methanococcus McN Medium
Composition per 1100.0mL:
NaHCO
3
5.0g
L-Cysteine·HCl·H
2
O 0.5g
General salts solution 500.0mL
NaCl solution 75.0mL
Na
2
S·9H
2
O solution 20.0mL
K
2
HPO
4
solution 10.0mL

Trace minerals solution 10.0mL
Iron stock solution 5.0mL
Resazurin solution 1.0mL
General Salts Solution:
Composition
per liter:
MgSO
4
·7H
2
O 6.9g
MgCl
2
·6H
2
O 5.5g
NH
4
Cl 1.0g
KCl 0.67g
CaCl
2
·2H
2
O 0.28g
Preparation of General Salts Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly.
NaCl Solution:
Composition
per 100.0mL:

NaCl 29.3g
Preparation of NaCl Solution: Add NaCl to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly.
Na
2
S·9H
2
O Solution:
Composition
per 100.0mL:
Na
2
S·9H
2
O 2.5g
NaOH 1 pellet
Preparation of Na
2
S·9H
2
O Solution: Bring 100.0mL of dis-
tilled/deionized water to boiling. Cool to room temperature while
© 2010 by Taylor and Francis Group, LLC
1094 Methanococcus McNail Medium
sparging with 100%N
2
. Dissolve 1 pellet of NaOH in the anaerobic wa-
ter. Weigh out a little more than 2.5g of Na
2
S·9H

2
O. Briefly rinse the
crystals in distilled/deionized water. Dry the crystals by blotting on pa-
per towels or filter paper. Add 2.5g of washed Na
2
S·9H
2
O crystals to
100.0mL of anaerobic NaOH solution. Distribute into serum bottles fit-
ted with butyl rubber stoppers and aluminum seals. Do not grease stop-
pers. Pressurize to 60kPa with 100% N
2
. Autoclave for 15 min at 15 psi
pressure–121°C. Store at room temperature in an anaerobic chamber.
K
2
HPO
4
Solution:
Composition
per 100.0mL:
K
2
HPO
4
1.4g
Preparation of K
2
HPO
4

Solution: Add K
2
HPO
4
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly.
Trace Minerals Solution:
Composition
per liter:
Nitrilotriacetic acid 1.5g
Na
2
WO
4
·2H
2
O 1.0g
Fe(NH
4
)
2
(SO
4
)
2
·6H
2
O 0.2g
Na
2

SeO
3
0.2g
Na
2
MoO
4
·2H
2
O 0.1g
Mn
4
·2H
2
O 0.1g
Zn
4
·7H
2
O 0.1g
NiCl
2
·7H
2
O 0.025g
CuSO
4
·5H
2
O 0.01g

Preparation of Trace Minerals Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Add distilled/deionized water to
1.0L. Adjust pH to 7.0.
Iron Stock Solution:
Composition
per 100.0mL:
Fe(NH
4
)
2
(SO
4
)
2
·6H
2
O 0.2g
Preparation of Iron Stock Solution: Add Fe(NH
4
)
2
(SO
4
)
2
·6H
2
O
to 5.0mL of distilled H

2
O containing 2 drops of concentrated HCl. Mix
thoroughly. When the Fe(NH
4
)
2
(SO
4
)
2
·6H
2
O has dissolved, bring the
volume to 100.0mL with distilled/deionized water.
Resazurin Solution:
Composition
per 10.0mL:
Resazurin 10.0mg
Preparation of Resazurin Solution: Add resazurin to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under 80%
H
2
+ 20% CO
2
. Add components, except NaHCO
3
and Na
2
S·9H

2
O so-
lution, to distilled/deionized water and bring volume to 1080.0mL.
Mix thoroughly. Gently heat and bring to boiling. Continue boiling for
3 min. Cool to room temperature while sparging with 80% H
2
+ 20%
CO
2
. Add NaHCO
3
. Mix thoroughly. Anaerobically distribute 9.8mL
volumes into anaerobic tubes. Autoclave for 15 min at 15 psi pressure–
121°C. Aseptically and anaerobically add 0.2mL of sterile Na
2
S·9H
2
O
solution to each tube. Mix thoroughly.
Use: For the cultivation of Methanococcus species.
Methanococcus McNail Medium
Composition per 1100.0mL:
NaHCO
3
5.0g
L-Leucine 1.0g
L-Isoleucine 0.5g
L-Cysteine·HCl·H
2
O 0.5g

General salts solution 500.0mL
NaCl solution 75.0mL
Na
2
S·9H
2
O solution 20.0mL
K
2
HPO
4
solution 10.0mL
Trace minerals solution 10.0mL
Sodium acetate solution 10.0mL
Pantoyllactone solution 10.0mL
Iron stock solution 5.0mL
Resazurin solution 1.0mL
General Salts Solution:
Composition
per liter:
MgSO
4
·7H
2
O 6.9g
MgCl
2
·6H
2
O 5.5g

NH
4
Cl 1.0g
KCl 0.67g
CaCl
2
·2H
2
O 0.28g
Preparation of General Salts Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly.
NaCl Solution:
Composition
per 100.0mL:
NaCl 29.3g
Preparation of NaCl Solution: Add NaCl to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly.
Na
2
S·9H
2
O Solution:
Composition
per 100.0mL:
NaOH 1 pellet
Na
2
S·9H
2
O 2.5g

Preparation of Na
2
S·9H
2
O Solution: Bring 100.0mL of dis-
tilled/deionized water to boiling. Cool to room temperature while
sparging with 100%N
2
. Dissolve 1 pellet of NaOH in the anaerobic wa-
ter. Weigh out a little more than 2.5g of Na
2
S·9H
2
O. Briefly rinse the
crystals in distilled/deionized water. Dry the crystals by blotting on pa-
per towels or filter paper. Add 2.5g of washed Na
2
S·9H
2
O crystals to
100.0mL of anaerobic NaOH solution. Distribute into serum bottles fit-
ted with butyl rubber stoppers and aluminum seals. Do not grease stop-
pers. Pressurize to 60kPa with 100% N
2
. Autoclave for 15 min at 15 psi
pressure–121°C. Store at room temperature in an anaerobic chamber.
K
2
HPO
4

Solution:
Composition
per 100.0mL:
K
2
HPO
4
1.4g
Preparation of K
2
HPO
4
Solution: Add K
2
HPO
4
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly.
Trace Minerals Solution:
Composition
per liter:
Nitrilotriacetic acid 1.5g
Na
2
WO
4
·2H
2
O 1.0g
Fe(NH

4
)
2
(SO
4
)
2
·6H
2
O 0.2g
Na
2
SeO
3
0.2g
Na
2
MoO
4
·2H
2
O 0.1g
Mn
4
·2H
2
O 0.1g
Zn
4
·7H

2
O 0.1g
NiCl
2
·7H
2
O 0.025g
CuSO
4
·5H
2
O 0.01g
Preparation of Trace Minerals Solution: Add nitrilotriacetic
acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with
KOH. Add remaining components. Add distilled/deionized water to
1.0L. Adjust pH to 7.0.
© 2010 by Taylor and Francis Group, LLC