Handbook of Microbiological Media, Fourth Edition part 110 pptx

3.0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L.. 20.0mg Preparation of Seven Vitamin Solution: Add components to d

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use inject, for each 5.0mL medium, 0.2mL sterile NaHCO3 solution,

0.05mL sterile Na2S·9H2O 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

NH4Cl 1.0g

NaCl 0.6g

Cysteine-HCl·H2O 0.5g

Sodium acetate 0.5g

K2HPO4 0.3g

KH2PO4 0.3g

MgCl2·6H2O 0.2g

CaCl2·2H2O 0.1g

KCl 0.1g

Resazurin 0.5mg

NaHCO3 solution 40.0mL

Na-formate solution 15.0mL

Trace elements solution 10.0mL

Vitamin solution 10.0mL

Na2S·9H2O solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Na-Formate Solution:

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% N2 Autoclave for 15 min at 15 psi pressure–121°C

Trace Elements Solution:

Compositionper liter:

MgSO4·7H2O 3.0g

Nitrilotriacetic acid 1.5g

NaCl 1.0g

MnSO4·2H2O 0.5g

CoSO4·7H2O 0.18g

ZnSO4·7H2O 0.18g

CaCl2·2H2O 0.1g

FeSO4·7H2O 0.1g

NiCl2·6H2O 0.025g

KAl(SO4)2·12H2O 0.02g

H3BO3 0.01g

Na2MoO4·4H2O 0.01g

CuSO4·5H2O 0.01g

Na2SeO3·5H2O 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:

Pyridoxine-HCl 10.0mg

Thiamine-HCl·2H2O 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 B12 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L Mix thoroughly Sparge with 80% H2 + 20% CO2 Filter sterilize

Na 2 S·9H 2 O Solution:

Composition per 10.0mL:

Na2S·9H2O 0.3g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to dis-tilled/deionized water and bring volume to 10.0mL Sparge with N2 Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C Store an-aerobically

NaHCO 3 Solution:

NaHCO3 10.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 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%

N2 + 20% CO2 gas mixture Add components, except Na-formate solu-tion, NaHCO3 solution, and Na2S·9H2O solution, to 1.0L distilled/de-ionized water Mix thoroughly Sparge with 80% N2 + 20% CO2 Dispense 5.0mL aliquots into Hungate tubes under 80% H2 + 20% CO2 gas mixture Autoclave for 15 min at 15 psi pressure–121°C Prior to use inject, for each 5.0mL medium, 0.2mL sterile NaHCO3 solution, 0.05mL sterile Na2S·9H2O 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

Yeast extract 1.0g

NH4Cl 1.0g NaCl 0.6g Cysteine-HCl·H2O 0.5g Sodium acetate 0.5g

K2HPO4 0.3g

KH2PO4 0.3g MgCl2·6H2O 0.2g CaCl2·2H2O 0.1g KCl 0.1g Resazurin 0.5mg NaHCO3 solution 40.0mL Na-formate solution 15.0mL Trace elements solution 10.0mL Vitamin solution 10.0mL

Na2S·9H2O solution 10.0mL Trypticase™ solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Trypticase™ Solution :

Trypticase™ 1.0g

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1086 Methanobrevibacter curvatus Medium

Preparation of Trypticase™ Solution : Add Trypticase™ to

Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C Cool

to room temperature

Na-Formate Solution:

Na-formate 5.0g

Preparation of Na-Formate Solution: Add Na-formate to

MgSO4·7H2O 3.0g

NaCl 1.0g

MnSO4·2H2O 0.5g

CoSO4·7H2O 0.18g

ZnSO4·7H2O 0.18g

CaCl2·2H2O 0.1g

FeSO4·7H2O 0.1g

NiCl2·6H2O 0.025g

KAl(SO4)2·12H2O 0.02g

H3BO3 0.01g

Na2MoO4·4H2O 0.01g

CuSO4·5H2O 0.01g

Na2SeO3·5H2O 0.3mg

Riboflavin 5.0mg

Lipoic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly Sparge

with 80% H2 + 20% CO2 Filter sterilize

Composition per 10.0mL:

Na2S·9H2O 0.3g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to

dis-tilled/deionized water and bring volume to 10.0mL Sparge with N2

Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C Store

an-aerobically

NaHCO3 10.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 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

N2 + 20% CO2 gas mixture Add components, except Trypticase™ so-lution, Na-formate soso-lution, NaHCO3 solution, and Na2S·9H2O solu-tion, to 1.0L distilled/deionized water Mix thoroughly Sparge with 80% N2 + 20% CO2 Dispense 5.0mL aliquots into Hungate tubes un-der 80% H2 + 20% CO2 gas mixture Autoclave for 15 min at 15 psi pressure–121°C Prior to use inject, for each 5.0mL medium, 0.2mL sterile NaHCO3 solution, 0.05mL sterile Trypticase™ solution, 0.05mL sterile Na2S·9H2O 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

NaCl 1.0g KCl 0.5g Casamino acids 0.5g Yeast extract 0.5g MgCl2·6H2O 0.4g

NH4Cl 0.3g

KH2PO4 0.2g

Na2SO4 0.15g CaCl2·2H2O 0.1g Resazurin 0.5mg NaHCO3 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

NaHCO3 5.8g

Preparation of NaHCO 3 Solution: Add NaHCO3 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 Compositionper liter:

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Preparation of Selenite-Tungstate Solution: Add components

to distilled/deionized water and bring volume to 1.0L Mix thoroughly Sparge with 100% N2 Filter sterilize

Dithionite Solution

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% N2 Filter sterilize

Trace Elements Solution SL-10:

FeCl2·4H2O 1.5g CoCl2·6H2O 190.0mg

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MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

Na2MoO4·2H2O 36.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

HCl (25% solution) 10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

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% N2 + 20% CO2 Autoclave for 15 min at 15

psi pressure–121°C

Seven Vitamin Solution:

Pyridoxine hydrochloride 300.0mg

Vitamin B12 100.0mg

Calcium pantothenate 100.0mg

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% N2

Mix thoroughly Filter sterilize

H2 + 20% CO2 gas atmosphere Add components, except NaHCO3

solu-tion, seven vitamin solusolu-tion, selenite-tungstate solusolu-tion, 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% H2 + 20%

CO2 Autoclave for 15 min at 15 psi pressure–121°C Aseptically and

an-aerobically add 40.0mL NaHCO3 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.

NaCl 1.0g

KCl 0.5g

Casamino acids 0.5g

Yeast extract 0.5g

MgCl2·6H2O 0.4g

NH4Cl 0.3g

KH2PO4 0.2g

Na2SO4 0.15g

CaCl2·2H2O 0.1g

Resazurin 0.5mg

Rumen fluid, bovine, clarified 400mL

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

NaHCO3 5.8g

Preparation of NaHCO 3 Solution: Add NaHCO3 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:

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Dithionite Solution:

Na-dithionite 2.0mg

FeCl2·4H2O 1.5g CoCl2·6H2O 190.0mg MnCl2·4H2O 100.0mg ZnCl2 70.0mg

Na2MoO4·2H2O 36.0mg NiCl2·6H2O 24.0mg

H3BO3 6.0mg CuCl2·2H2O 2.0mg HCl (25% solution) 10.0mL

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% N2 + 20% CO2 Autoclave for 15 min at 15 psi pressure–121°C

Pyridoxine hydrochloride 300.0mg Thiamine-HCl·2H2O 200.0mg Nicotinic acid 200.0mg Vitamin B12 100.0mg Calcium pantothenate 100.0mg

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% N2 Mix thoroughly Filter sterilize

Nutrient Supplement Solution:

Pancreatic digest of gelatin 5.0g Beef extract 3.0g

compo-nents to distilled/deionized water and bring volume to 1.0L Mix thor-oughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure– 121°C Cool to room tempterature

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1088 Methanobrevibacter curvatus Medium

MOPS Buffer:

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% N2 Adjust to pH 7.2 Filter sterilize

H2 + 20% CO2 gas atmosphere Add components, except clarified

bo-vine rumen fluid, MOPS buffer, nutrient supplement solution, NaHCO3

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% H2 +

20% CO2 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

NaHCO3 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)

NaCl 1.0g

KCl 0.5g

Casamino acids 0.5g

Yeast extract 0.5g

MgCl2·6H2O 0.4g

NH4Cl 0.3g

KH2PO4 0.2g

Na2SO4 0.15g

CaCl2·2H2O 0.1g

Resazurin 0.5mg

Rumen fluid, bovine, clarified 200mL

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

NaHCO3 5.8g

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:

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Dithionite Solution:

Na-dithionite 2.0mg

FeCl2·4H2O 1.5g CoCl2·6H2O 190.0mg MnCl2·4H2O 100.0mg ZnCl2 70.0mg

Na2MoO4·2H2O 36.0mg NiCl2·6H2O 24.0mg

H3BO3 6.0mg CuCl2·2H2O 2.0mg HCl (25% solution) 10.0mL

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% N2 + 20% CO2 Autoclave for 15 min at 15 psi pressure–121°C

Pyridoxine hydrochloride 300.0mg Thiamine-HCl·2H2O 200.0mg Nicotinic acid 200.0mg Vitamin B12 100.0mg Calcium pantothenate 100.0mg

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% N2 Mix thoroughly Filter sterilize

MOPS Buffer:

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% N2 Adjust to pH 7.7 Filter sterilize

H2 + 20% CO2 gas atmosphere Add components, except clarified bo-vine rumen fluid, MOPS buffer, NaHCO3 solution, seven vitamin solu-tion, selenite-tungstate solusolu-tion, 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% H2 + 20% CO2 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 NaHCO3 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

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Use: For the cultivation of Methanobrevibacter curvatus DSM 11111

DSM 11139 (strain RFM-1)

Methanocalculus halotolerans Medium

NaCl 50.0g

NH4Cl 1.0g

Na-acetate 0.5g

Yeast extract 0.5g

Trypticase™ 0.5g

K2HPO4 0.3g

KH2PO4 0.3g

KCl 0.17g

Resazurin 0.5mg

Magnesium chloride solution 30.0mL

Calcium chloride solution 10.0mL

L-Cysteine solution 10.0mL

pH 7.2-7.6 at 25°C

Magnesium Chloride Solution:

MgCl2·6H2O 3.2g

MgCl2·6H2O to distilled/deionized water and bring volume to 30.0mL

Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi

pressure–121°C

Calcium Chloride Solution:

CaCl2·2H2O 0.6g

Preparation of Calcium Chloride Solution: Add CaCl2·2H2O

to distilled/deionized water and bring volume to 10.0mL Mix

thor-oughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–

121°C

L -Cysteine Solution:

L-Cysteine·HCl·H2O 0.5g

Preparation of L -Cysteine Solution: Add L-cysteine·HCl·H2O to

distilled/deionized water and bring volume to 10.0mL Mix

thorough-ly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–

121°C

Na2S·9H2O 0.3g

NaHCO3 2.0g

with 80% N2 + 20% CO2 Autoclave for 15 min at 15 psi pressure–

121°C Cool to 25°C Must be prepared freshly

MgSO4·7H2O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO4·2H2O 0.5g CoSO4·7H2O 0.18g ZnSO4·7H2O 0.18g CaCl2·2H2O 0.1g FeSO4·7H2O 0.1g NiCl2·6H2O 0.025g KAl(SO4)2·12H2O 0.02g

H3BO3 0.01g

Na2MoO4·4H2O 0.01g CuSO4·5H2O 0.01g

Na2SeO3·5H2O 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% N2 + 20% CO2 Add components, except NaHCO3 so-lution, magnesium chloride soso-lution, calcium chloride soso-lution, L -cysteine-HCl·H2O solution, and Na2S·9H2O 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% N2 + 20% CO2 Add 10.0mL L-cysteine-HCl·H2O solution and 20.0mL NaHCO3 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 Na2S·9H2O Mix thoroughly After inoculation pres-surize vessels with 80% H2 + 20% CO2 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

NaCl 10.0g Yeast extract 2.0g Trypticase™ 2.0g

NH4Cl 0.9g

K2HPO4 0.4g MgCl2·6H2O 0.36g Resazurin 0.5mg

Na2CO3 solution 50.0mL

L-Cysteine-HCl·H2O solution 15.0mL Vitamin solution 10.0mL Trace elements solution 10.0mL

L -Cysteine Solution:

Preparation of L -Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 15.0mL Mix

thorough-ly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure– 121°C

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1090 Methanococcoides Medium

Na 2 CO 3 Solution:

Na2CO3 5.0g

Preparation of Na 2 CO 3 Solution: Add Na2CO3 to

with 80% N2 + 20% CO2 Filter sterilize

Na 2 S·9H 2 O Solution :

Composition per 15mL:

Na2S·9H2O 0.5g

Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C Cool

to room temperature

MgSO4·7H2O 3.0g

NaCl 1.0g

MnSO4·2H2O 0.5g

CoSO4·7H2O 0.18g

ZnSO4·7H2O 0.18g

CaCl2·2H2O 0.1g

FeSO4·7H2O 0.1g

NiCl2·6H2O 0.025g

KAl(SO4)2·12H2O 0.02g

H3BO3 0.01g

Na2MoO4·4H2O 0.01g

CuSO4·5H2O 0.01g

Na2SeO3·5H2O 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic

Riboflavin 5.0mg

Lipoic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

deionized water and bring volume to 1.0L Mix thoroughly Sparge

with 80% H2 + 20% CO2 Filter sterilize

Preparation of Medium: Add components, except Na2CO3

solu-tion, Na2S·9H2O solution, and L-cysteine solution, to

distilled/deion-ized water and bring volume to 1.0L Mix thoroughly Sparge with 100%

N2 for 30 min Autoclave for 15 min at 15 psi pressure–121°C

Asepti-cally distribute 5.0mL aliquots into Hungate tubes under 100% N2

Aseptically and anaerobically add per 5.0mL medium 0.25mL Na2CO3

solution, 0.075mL Na2S·9H2O solution, and 0.075mL L-cysteine

solu-tion Mix thoroughly Replace N2 atmosphere with atmosphere of 80%

H2 + 20% CO2 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% H2 + 20%

CO2 to 1.5 bar overpressure

Use: For the cultivation of Methanocalculus pumilus.

Methanococcal Complex Medium

See: Methanococcus McC Medium

Methanococcoides Medium

NaCl 18.0g NaHCO3 5.0g MgCl2·6H2O 4.0g MgSO4·7H2O 3.45g Trimethylamine·HCl 3.0g Trypticase™ 2.0g Yeast extract 2.0g Sodium acetate 1.0g

L-Cysteine·HCl 0.5g

Na2S·9H2O 0.5g KCl 0.335g

NH4Cl 0.25g CaCl2·2H2O 0.14g

K2HPO4 0.14g Fe(NH4)2(SO4)2·7H2O 2.0mg Resazurin 1.0mg Trace elements solution 10.0mL Vitamin solution 10.0mL

pH 7.0 ± 0.2 at 25°C

MgSO4·7H2O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO4·2H2O 0.5g CoSO4·7H2O 0.18g ZnSO4·7H2O 0.18g CaCl2·2H2O 0.1g FeSO4·7H2O 0.1g NiCl2·6H2O 0.025g KAl(SO4)2·12H2O 0.02g CuSO4·5H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Na2SeO3·5H2O 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

Pyridoxine·HCl 10.0mg Calcium DL-pantothenate 5.0mg Lipoic acid 5.0mg Nicotinic acid 5.0mg

Riboflavin 5.0mg Thiamine·HCl 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B12 0.1mg

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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% H2 + 20% CO2 Add components to distilled/deionized water and

bring volume to 1.0L Mix thoroughly Sparge with 80% H2 + 20%

CO2 Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation and maintenance of Methanococcoides

meth-ylutens.

Methanococcus deltae Medium

NaCl 35.0g

NaHCO3 5.0g

MgCl2·6H2O 4.0g

NH4Cl 2.7g

Sodium acetate 2.5g

K2HPO4 0.3g

KH2PO4 0.3g

Na2S·9H2O 0.3g

MgSO4·7H2O 0.13g

Resazurin 1.0mg

(NH4)2SO4 0.3mg

Vitamin solution 10.0mL

L-Cysteine·HCl solution 10.0mL

pH 6.9 ± 0.2 at 25°C

MgSO4·7H2O 3.0g

NaCl 1.0g

MnSO4·2H2O 0.5g

CoSO4·7H2O 0.18g

ZnSO4·7H2O 0.18g

CaCl2·2H2O 0.1g

FeSO4·7H2O 0.1g

NiCl2·6H2O 0.025g

KAl(SO4)2·12H2O 0.02g

CuSO4·5H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Na2SeO3·5H2O 0.3mg

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

Pyridoxine·HCl 10.0mg

Calcium DL-pantothenate 5.0mg

Lipoic acid 5.0mg

Riboflavin 5.0mg

Thiamine·HCl 5.0mg

Biotin 2.0mg

Folic acid 2.0mg Vitamin B12 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:

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% N2 for 15 min at 15 psi pressure–121°C

Na2S·9H2O 0.3g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

H2 + 20% CO2 Add components, except L-cysteine·HCl solution and

Na2S·9H2O 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 Na2S·9H2O solution to each liter of medium

or, using a syringe, inject the appropriate amount of sterile L -cysteine·HCl solution and sterile Na2S·9H2O solution into individual tubes containing medium

Use: For the cultivation and maintenance of Methanococcus deltae.

Methanococcus jannaschii Medium

NaCl 30.0g MgSO4·7H2O 3.40g MgCl2·2H2O 2.7g NaHCO3 1.0g

Na2S·9H2O 0.5g KCl 0.33g

NH4Cl 0.25g CaCl2·2H2O 0.14g

K2HPO4 0.14g Fe(NH4)2(SO4)2·6H2O 0.01g Resazurin 1.0mg

Na2SeO3·5H2O 0.5mg NiCl2·6H2O 0.5mg Trace elements solution 10.0mL Vitamin solution 10.0mL

L-Cysteine·HCl solution 10.0mL

pH 6.0 ± 0.2 at 25°C

MgSO4·7H2O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO4·2H2O 0.5g CoSO4·7H2O 0.18g ZnSO4·7H2O 0.18g CaCl2·2H2O 0.1g

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1092 Methanococcus jannaschii Medium

FeSO4·7H2O 0.1g

NiCl2·6H2O 0.025g

KAl(SO4)2·12H2O 0.02g

CuSO4·5H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Na2SeO3·5H2O 0.3mg

KOH Add remaining components Adjust pH to 7.0 Add

distilled/de-ionized water to 1.0L

Pyridoxine·HCl 10.0mg

Calcium DL-pantothenate 5.0mg

Lipoic acid 5.0mg

Riboflavin 5.0mg

Thiamine·HCl 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

deionized water and bring volume to 1.0L Adjust pH to 7.0 Mix

thor-oughly

L -Cysteine·HCl Solution:

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% N2 for 15 min at 15 psi pressure–121°C

Na2S·9H2O 0.3g

H2 + 20% CO2 Add components, except L-cysteine·HCl solution and

Na2S·9H2O 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

Na2S·9H2O solution to each liter of medium or, using a syringe, inject

the appropriate amount of sterile L-cysteine·HCl solution and sterile

Na2S·9H2O solution into individual tubes containing medium

Use: For the cultivation and maintenance of Methanococcus species.

Methanococcus jannaschii Medium

PIPES

(piperazine-N,N´-bis[2-ethanesulfonic acid]) buffer 15.12g

MgCl2·6H2O 4.3g

MgSO4·7H2O 3.4g

NaCl 3.0g

NH4Cl 0.25g

K2HPO4 0.14g

CaCl2·2H2O 0.14g KCl 0.33g Minerals solution 10.0mL

Na2S2O3 solution 10.0mL β-Mercaptoethanol solution 10.0mL SeO2 solution 1.0mL

pH 7.0 ± 0.2 at 25°C

Minerals Solution:

Nitrilotriacetic acid 4.5g FeCl2·4H2O 0.4g CoCl2·2H2O 0.17g MnCl2·4H2O 0.1g ZnCl2 0.1g NaMoO4·6H2O 36.0mg CaCl2·H2O 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:

Na2S2O3·5H2O 0.63g

Preparation of Na 2 S 2 O 3 Solution: Add Na2S2O3·5H2O to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

β-Mercaptoethanol Solution:

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% N2 Autoclave for 15 min at 15 psi pres-sure–121°C

SeO 2 Solution:

SeO2 0.011g

Preparation of SeO 2 Solution: Add SeO2 to distilled/deionized water and bring volume to 100.0mL Mix thoroughly

Preparation of Medium: Prepare and dispense medium under 100% N2 Add components, except Na2S2O3 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% N2 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 anaerobicalAseptical-ly add 1.0mL of sterile Na2S2O3 solution and 1.0mL of sterile β-mercaptoethanol solution to each bottle Mix thoroughly Prior to inoculation, flush each bottle with 80% H2 + 20% CO2

Use: For the cultivation of Methanococcus jannaschii

Methanococcus McC Medium

NaHCO3 5.0g Yeast extract 2.0g

L-Cysteine·HCl·H2O 0.5g General salts solution 500.0mL NaCl solution 75.0mL

K2HPO4 solution 10.0mL

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Trace minerals solution 10.0mL

Sodium acetate solution 10.0mL

Iron stock solution 5.0mL

Resazurin solution 1.0mL

General Salts Solution:

MgSO4·7H2O 6.9g

MgCl2·6H2O 5.5g

NH4Cl 1.0g

KCl 0.67g

CaCl2·2H2O 0.28g

Preparation of General Salts Solution: Add components to

dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly

NaCl Solution:

NaCl 29.3g

Preparation of NaCl Solution: Add NaCl to distilled/deionized

water and bring volume to 100.0mL Mix thoroughly

NaOH 1 pellet

Na2S·9H2O 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%N2 Dissolve 1 pellet of NaOH in the anaerobic

wa-ter Weigh out a little more than 2.5g of Na2S·9H2O Briefly rinse the

crystals in distilled/deionized water Dry the crystals by blotting on

pa-per towels or filter papa-per Add 2.5g of washed Na2S·9H2O 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% N2 Autoclave for 15 min at 15 psi

pressure–121°C Store at room temperature in an anaerobic chamber

K 2 HPO 4 Solution:

K2HPO4 1.4g

Preparation of K 2 HPO 4 Solution: Add K2HPO4 to

Trace Minerals Solution:

Na2WO4·2H2O 1.0g

Fe(NH4)2(SO4)2·6H2O 0.2g

Na2SeO3 0.2g

Na2MoO4·2H2O 0.1g

Mn4·2H2O 0.1g

Zn4·7H2O 0.1g

NiCl2·7H2O 0.025g

CuSO4·5H2O 0.01g

Preparation of Trace Minerals Solution: Add nitrilotriacetic

KOH Add remaining components Add distilled/deionized water to

1.0L Adjust pH to 7.0

Sodium Acetate Solution:

Sodium acetate·3H2O 13.6g

ace-tate·3H2O to distilled/deionized water and bring volume to 100.0mL Mix thoroughly

Iron Stock Solution:

Fe(NH4)2(SO4)2·6H2O 0.2g

Preparation of Iron Stock Solution: Add Fe(NH4)2(SO4)2·6H2O

to 5.0mL of distilled H2O containing 2 drops of concentrated HCl Mix thoroughly When the Fe(NH4)2(SO4)2·6H2O has dissolved, bring the volume to 100.0mL with distilled/deionized water

Resazurin Solution:

Resazurin 10.0mg

Preparation of Resazurin Solution: Add resazurin to distilled/ deionized water and bring volume to 10.0mL Mix thoroughly

H2 + 20% CO2 Add components, except NaHCO3 and Na2S·9H2O 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% H2 + 20%

CO2 Add NaHCO3 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 Na2S·9H2O solution to each tube Mix thoroughly

Use: For the cultivation of Methanococcus species.

Methanococcus McN Medium

NaHCO3 5.0g

L-Cysteine·HCl·H2O 0.5g General salts solution 500.0mL NaCl solution 75.0mL

K2HPO4 solution 10.0mL Trace minerals solution 10.0mL Iron stock solution 5.0mL Resazurin solution 1.0mL

MgSO4·7H2O 6.9g MgCl2·6H2O 5.5g

NH4Cl 1.0g KCl 0.67g CaCl2·2H2O 0.28g

Preparation of General Salts Solution: Add components to dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly

NaCl Solution:

NaCl 29.3g

Preparation of NaCl Solution: Add NaCl to distilled/deionized water and bring volume to 100.0mL Mix thoroughly

Na2S·9H2O 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

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1094 Methanococcus McNail Medium