Handbook of Microbiological Media, Fourth Edition part 174 pot

2.0mg Cyanocobalamin ...100.0μg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L.. 2.0mg Cyanocobalamin ...100.0μg Preparatio

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Thermodesulfotobacterium Agar 1725

Na2S·9H2O solution 10.0mL

Na2S2O4 solution 10.0mL

Trace elements solution SL-10 1.0mL

NaHCO3 solution variable

pH 6.8 ± 0.2 at 25°C

NaHCO 3 Solution:

Composition per 20.0mL:

NaHCO3 1.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to

distilled/de-ionized water and bring volume to 20.0mL Mix thoroughly Sparge

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

121°C

Na 2 S·9H 2 O Solution:

Na2S·9H2O 0.15g

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

Na 2 S 2 O 3 Solution:

Na2S2O3·5H2O 2.0g

Preparation of Na 2 S 2 O 3 Solution: Add Na2S2O3·5H2O to

Trace Elements Solution SL-10:

Composition per liter:

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

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

Preparation of Medium: Prepare and dispense medium under

100% N2 Add components, except Na2S·9H2O solution, Na2S2O3

solu-tion, and NaHCO3 solution, to distilled/deionized water and bring

vol-ume to 980.0mL Mix thoroughly Gently heat and bring to boiling Cool

to room temperature while sparging with 100% N2 Anaerobically

dis-tribute 9.8mL volumes into tubes Autoclave for 15 min at 15 psi

pres-sure–121°C Aseptically and anaerobically add 0.1mL of sterile

Na2S·9H2O solution and 0.1mL of sterile Na2S2O4 solution to each tube

Aseptically and anaerobically add a sufficient volume of sterile NaHCO3

solution to each tube to bring the pH to 6.8

Use: For the cultivation of Thermodesulforhabdus norvegicus

Thermodesulfotobacterium Agar

Compositionper liter:

Na2SO4 30.0g

Agar 20.0g

Sodium lactate 4.0g

Yeast extract 1.0g

Mineral solution 2 50.0mL

Na2CO3 solution 50.0mL Mineral solution 1 25.0mL Cysteine-sulfide reducing agent 20.0mL Wolfe’s mineral solution 10.0mL Wolfe’s vitamin solution 10.0mL Resazurin (0.025% solution) 4.0mL

pH 7.2 ± 0.2 at 25°C

Mineral Solution 1:

Composition per liter:

K2HPO4 6.0g

Preparation of Mineral Solution 1: Add K2HPO4 to distilled/de-ionized water and bring volume to 1.0L Mix thoroughly

NaCl 12.0g

KH2PO4 6.0g (NH4)2SO4 6.0g MgSO4·7H2O 2.4g CaCl2·2H2O 1.6g

Preparation of Mineral Solution 2: Add components to distilled/ deionized water and bring volume to 1.0L Mix thoroughly

Na 2 CO 3 Solution:

Compositionper 100.0mL:

Na2CO3 8.0g

Preparation of Na 2 CO 3 Solution: Add Na2CO3 to distilled/deion-ized water and bring volume to 100.0mL Mix thoroughly

Cysteine-Sulfide Reducing Agent:

L-Cysteine·HCl·H2O 300.0mg

Na2S·9H2O 300.0mg

Preparation of Cysteine-Sulfide Reducing Agent: Add

L-cysteine·HCl·H2O to 10.0mL of distilled/deionized water Mix thor-oughly In a separate tube, add Na2S·9H2O to 10.0mL of distilled/de-ionized water Mix thoroughly Gas both solutions with 100% N2 and cap tubes Autoclave both solutions for 15 min at 15 psi pressure– 121°C using fast exhaust Cool to 50°C Aseptically combine the two solutions under 100% N2

Wolfe’s Mineral Solution:

MgSO4·7H2O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO4·H2O 0.5g FeSO4·7H2O 0.1g CoCl2·6H2O 0.1g CaCl2 0.1g ZnSO4·7H2O 0.1g CuSO4·5H2O 0.01g AlK(SO4)2·12H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Preparation of Wolfe’s Mineral 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

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1726 Thermodesulfotobacterium Broth

Wolfe’s Vitamin Solution:

Pyridoxine·HCl 10.0mg

Thiamine·HCl 5.0mg

Riboflavin 5.0mg

Nicotinic acid 5.0mg

Calcium pantothenate 5.0mg

p-Aminobenzoic acid 5.0mg

Thioctic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Cyanocobalamin 100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to

distilled/deionized water and bring volume to 1.0L Mix thoroughly

Filter sterilize

Preparation of Medium: Add components, except vitamin

solu-tion and cysteine-sulfide reducing agent, to distilled/deionized water

and bring volume to 970.0mL Mix thoroughly Gently heat and bring

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

50°–55°C under 80% N2 + 20% CO2 Aseptically add the sterile

vita-min solution and then the sterile cysteine-sulfide reducing agent

Ad-just the pH to 7.2 Distribute aseptically and anaerobically into sterile

tubes

Use: For the cultivation and maintenance of Thermodesulfobacterium

commune and other Thermodesulfobacterium species.

Thermodesulfotobacterium Broth

Compositionper liter:

Na2SO4 30.0g

Sodium lactate 4.0g

Yeast extract 1.0g

Na2CO3 solution 50.0mL

Cysteine-sulfide reducing agent 20.0mL

Wolfe’s mineral solution 10.0mL

Wolfe’s vitamin solution 10.0mL

Resazurin (0.025% solution) 4.0mL

pH 7.2 ± 0.2 at 25°C

K2HPO4 6.0g

Preparation of Mineral Solution 1: Add K2HPO4 to

distilled/de-ionized water and bring volume to 1.0L Mix thoroughly

NaCl 12.0g

KH2PO4 6.0g

(NH4)2SO4 6.0g

MgSO4·7H2O 2.4g

CaCl2·2H2O 1.6g

Preparation of Mineral Solution 2: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly

Na 2 CO 3 Solution:

Compositionper 100.0mL:

Na2CO3 8.0g

Preparation of Na 2 CO 3 Solution: Add Na2CO3 to

distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly

Cysteine-Sulfide Reducing Agent:

L-Cysteine·HCl·H2O 300.0mg

Na2S·9H2O 300.0mg

Preparation of Cysteine-Sulfide Reducing Agent: Add

L-cysteine·HCl·H2O to 10.0mL of distilled/deionized water Mix thor-oughly In a separate tube, add Na2S·9H2O to 10.0mL of distilled/de-ionized water Mix thoroughly Gas both solutions with 100% N2 and cap tubes Autoclave both solutions for 15 min at 15 psi pressure– 121°C using fast exhaust Cool to 50°C Aseptically combine the two solutions under 100% N2

MgSO4·7H2O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO4·H2O 0.5g FeSO4·7H2O 0.1g CoCl2·6H2O 0.1g CaCl2 0.1g ZnSO4·7H2O 0.1g CuSO4·5H2O 0.01g AlK(SO4)2·12H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Pyridoxine·HCl 10.0mg Thiamine·HCl 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg Calcium pantothenate 5.0mg

Thioctic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg Cyanocobalamin 100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except vitamin solu-tion and cysteine-sulfide reducing agent, to distilled/deionized water and bring volume to 970.0mL Mix thoroughly Autoclave for 15 min

at 15 psi pressure–121°C Cool under 80% N2 + 20% CO2 Aseptically add the sterile vitamin solution and then the sterile cysteine-sulfide re-ducing agent Adjust the pH to 7.2 Distribute aseptically and anaero-bically into sterile tubes

Use: For the cultivation and maintenance of Thermodesulfobacterium

commune and other Thermodesulfobacterium species.

Thermodesulfovibrio yellowstonii Medium

(DSMZ Medium 749) Composition per liter:

Na2SO4 4.0g Na-lactate 2.5g

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Thermofilum pendens Medium 1727

NaHCO3 1.3g

KCl 0.5g

Yeast extract 0.5g

MgCl2·6H2O 0.4g

NH4Cl 0.25g

Na2HPO4 0.2g

Na-thioglycolate 0.2g

L-Ascorbic acid 0.2g

CaCl2·2H2O 0.15g

Resazurin 0.5mg

Trace elements solution 10.0mL

Vitamin solution 10.0mL

pH 7.5 ± 0.2 at 25°C

Trace Elements Solution:

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

D-Ca-pantothenate 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

Preparation of Medium: Prepare and dispense medium under an

oxygen-free 100% N2 Add components, except vitamin solution,

Na-thioglycolate, NaHCO3, and L-Ascorbic acid, to distilled/deionized

water and bring volume to 990.0L Mix thoroughly Gently heat and

bring to boiling Cool while sparging with 100% N2 Add 0.2g

Na-thio-glycolate, 1.3g NaHCO3, and 0.2g L-ascorbic acid Mix thoroughly

Adjust pH to 7.5 Autoclave for 15 min at 15 psi pressure–121°C Cool

to 25°C Aseptically and anaerobically add 10.0mL sterile vitamin

so-lution Mix thoroughly Aseptically and anaerobically distribute into

sterile tubes or flasks

Use: For the cultivation of Thermodesulfovibrio yellowstonii.

Thermofilum pendens Medium

Sulfur, powdered 10.0g (NH4)2SO4 1.3g

KH2PO4 0.28g MgSO4·7H2O 0.25g CaCl2·2H2O 0.07g

Na2S·9H2O 0.3g FeCl3·6H2O 0.02g

Na2B4O7·10H2O 4.5mg MnCl2·4H2O 1.8mg ZnSO4·7H2O 0.22mg CuCl2·2H2O 0.05mg

Na2MoO4·2H2O 0.03mg VOSO4·2H2O 0.03mg CoSO4·7H2O 0.01mg Yeast extract solution 20.0mL Sucrose solution 20.0mL Polar lipid fraction 6.0–12.0mL

pH 5.2 ± 0.2 at 25°C

Yeast Extract Solution:

Yeast extract 2.0g

Preparation of Yeast Extract Solution: Add yeast extract to dis-tilled/deionized water and bring volume to 20.0mL Mix thoroughly Gently heat and bring to boiling Boil for a few minutes Sparge with 100% N2 Do not autoclave

Sucrose Solution:

Sucrose 2.0g

Preparation of Sucrose Solution: Add sucrose to distilled/deion-ized water and bring volume to 20.0mL Mix thoroughly Filter steril-ize Sparge with 100% N2

Preparation of Sulfur: Add 10.0g of powdered sulfur to a flask and sterilize by steaming for 3 hr on 3 consecutive days

Polar Lipid Fraction:

Thermoproteus tenax cells (wet weight) 10.0g

Chloroform 500.0mL Acetone 500.0mL Methanol 500.0mL

TA buffer solution 80.0mL Chloroform/methanol 1:1 (v/v) 20.0mL

TA Buffer Solution:

Tris·HCl 0.79g β-mercaptoethanol 0.78g

NH4Cl 0.118g EDTA 0.029g

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

Preparation of Polar Lipid Fraction: Add 10.0g (wet weight) of

Thermoproteus tenax cells to 20.0mL of TA buffer solution Mix

thor-oughly Sonicate for 2 min Centrifuge at 20,000 rpm for 20 min Re-suspend pellet in 20.0mL of fresh TA buffer solution Recentrifuge at 20,000 rpm for 20 min Again resuspend pellet in 20.0mL of fresh TA buffer solution Recentrifuge at 20,000 rpm for 20 min Resuspend pel-let in 20.0mL of fresh TA buffer solution Centrifuge at 5,000 rpm for

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1728 Thermogymnomonas Medium

5 min Decant the supernatant solution and discard the pellet Extract

the supernatant solution twice with 20.0mL of chloroform/methanol

(1:1) each time Chromatograph the extract on a SIL-LC (325 mesh)

si-licic acid column (20cm × 2cm) using 500.0mL of chloroform,

fol-lowed by 500.0mL of acetone, folfol-lowed by 500.0mL of methanol The

methanol fraction is further purified by DEAE chromatography using

chloroform/methanol 1:1 and methanol

Preparation of Medium: Prepare and dispense medium under

100% N2 Add components, except yeast extract solution, sucrose

so-lution, sulfur, and polar lipid fraction, to distilled/deionized water and

bring volume to 950.0mL Mix thoroughly Sparge with 100% N2

An-aerobically distribute into tubes or bottles Autoclave for 15 min at 15

psi pressure–121°C Aseptically and anaerobically add to 1.0L of

me-dium 20.0mL of sterile yeast extract solution, 20.0mL of sterile sucrose

solution, 10.0g of sterile sulfur, and 6.0–12.0mL of sterile polar lipid

fraction Mix thoroughly

Use: For the cultivation and maintenance ofThermofilum pendens.

Thermogymnomonas Medium

(DSMZ Medium 1141) Composition per liter:

KH2PO4 3.0g

(NH4)2SO4 0.2g

MgSO4·7H2O 0.5g

CaCl2·2H2O 025g

Glucose solution 50.0mL

Yeast extract solution 50.0mL

pH 3.0 ± 0.2 at 25°C

Glucose Solution :

Glucose 10.0g

Preparation of Glucose Solution: Add glucose to

distilled/deion-ized water and bring volume to 50.0mL Mix thoroughly Filter

steril-ize

Yeast Extract Solution :

Yeast extract 1.0g

Preparation of Yeast Extract Solution: Add yeast extract to

Filter sterilize

Preparation of Medium: Add components, except glucose and

yeast extract solutions, to distilled/deionized water and bring volume

to 900.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–

121°C Cool to room temperature Aspetically add glucose and yeast

extract solutions Mix thoroughly Adjust pH to 3.0 with sterile 10N

H2SO4

Use: For the cultivation of Thermogymnomonas spp.

Thermoleophilum Medium

NaNO2 2.0g

Na2HPO4 0.21g

MgSO4·7H2O 0.2g

KCl 0.04g

NaH2PO4 90.0mg

CaCl2 15.0mg

FeSO4·7H2O 1.0mg

ZnSO4·7H2O 70.0μg

H3BO3 10.0μg MnSO4·5H2O 10.0μg MoO3 10.0μg CuSO4·5H2O 5.0μg

n-Heptadecane 1.0mL

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except n-heptadecane,

to distilled/deionized water and bring volume to 1.0L Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Aseptically add 1.0mL

of n-heptadecane Mix thoroughly Aseptically distribute into sterile

tubes or flasks

Use: For the cultivation of Thermoleophilum album and

Thermoleo-philum minutum.

Thermomicrobium fosteri Agar

Agar 20.0g

NH4Cl 2.0g

Na2HPO4 0.21g MgSO4·7H2O 0.2g NaH2PO4 0.09g KCl 0.04g CaCl2 0.015g ZnSO4·7H2O 70.0μg

H3BO3 10.0μg MnSO4·5H2O 10.0μg MoO3 10.0μg CuSO4·5H2O 5.0μg FeSO4·7H2O 1.0mg Heptadecane, filter sterilized 20.0mL

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except heptadecane, to distilled/deionized water and bring volume to 980.0mL Mix

thorough-ly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°–55°C Aseptically add 20.0mL of sterile heptadecane Mix thoroughly Aseptically distribute into sterile tubes Cool tubes rapidly in a slanted position

Use: For the cultivation and maintenance of Thermomicrobium

fos-teri.

Thermomicrobium fosteri Broth

NH4Cl 2.0g

Na2HPO4 0.21g MgSO4·7H2O 0.2g NaH2PO4 0.09g KCl 0.04g CaCl2 0.015g ZnSO4·7H2O 70.0μg

H3BO3 10.0μg MnSO4·5H2O 10.0μg MoO3 10.0μg CuSO4·5H2O 5.0μg FeSO4·7H2O 1.0mg Heptadecane, filter sterilized 20.0mL

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except heptadecane, to distilled/deionized water and bring volume to 980.0mL Mix

thorough-ly Autoclave for 15 min at 15 psi pressure–121°C Aseptically add

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Thermophilic Hydrogen-Bacteria Medium 1729

20.0mL of sterile heptadecane Mix thoroughly Aseptically distribute

into sterile tubes or flasks

Use: For the cultivation of Thermomicrobium fosteri.

Thermomicrobium roseum Agar

Agar 20.0g

(NH4)2SO4 (sublimed) 1.3g

Yeast extract 1.0g

Tryptone 1.0g

MgSO4·7H2O 0.247g

KH2PO4 0.280g

CaCl2·2H2O 0.074g

FeCl3·6H2O 0.019g

Salt solution 1.0mL

pH 8.5 ± 0.2 at 25°C

Salt Solution:

Na2B4O7·10H2O 4.4g

MnCl2·4H2O 1.8g

ZnSO4·7H2O 0.22g

CuCl2·H2O 0.05g

Na2MoO4·4H2O 0.03g

VOSO4·2H2O 0.03g

Preparation of Salt Solution: Add components to

distilled/deion-ized water and bring volume to 100.0mL Adjust pH to 2.0 Mix

thor-oughly

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L Mix thoroughly Adjust pH to 8.5

Gently heat and bring to boiling Autoclave for 15 min at 15 psi

pres-sure–121°C Pour into Petri dishes or pour short slants with a long butt

in screw-capped tubes

Use: For the cultivation and maintenance of Thermomicrobium roseum.

Thermomicrobium roseum Medium

(NH4)2SO4 (sublimed) 1.3g

Yeast extract 1.0g

Tryptone 1.0g

MgSO4·7H2O 0.247g

KH2PO4 0.280g

CaCl2·2H2O 0.074g

FeCl3·6H2O 0.019g

Salt solution 1.0mL

pH 8.5 ± 0.2 at 25°C

Salt Solution:

Na2B4O7·10H2O 4.4g

MnCl2·4H2O 1.8g

ZnSO4·7H2O 0.22g

CuCl2·H2O 0.05g

Na2MoO4·4H2O 0.03g

VOSO4·2H2O 0.03g

Preparation of Salt Solution: Add components to

distilled/deion-ized water and bring volume to 100.0mL Adjust pH to 2.0 Mix

thor-oughly

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 8.5 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C

Use: For the cultivation of Thermomicrobium roseum.

Thermomonospora Medium

Sucrose 30.0g Agar 15.0g Casamino acids 6.0g NaNO3 3.0g Yeast extract 2.0g

K2HPO4 1.0g MgSO4·7H2O 0.5g KCl 0.5g FeSO4·7H2O 0.01g

pH 8.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Distribute into tubes

or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation of Thermomonospora alba and

Thermomono-spora mesophila.

Thermophilic Bacillus Medium

Peptone 8.0g Yeast extract 4.0g NaCl 3.0g

pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Distribute into tubes

or flasks Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation and maintenance of a variety of thermophilic

Bacillus species.

Thermophilic Hydrogen-Bacteria Medium Composition per 1000.5mL:

Na2HPO4·12H2O 4.5g

KH2PO4 1.5g NaCl 1.0g

NH4NO3 1.0g MgSO4·7H2O 0.2g CaCl2·2H2O 10.0mg FeSO4·7H2O 10.0mg Trace elements solution 0.5mL

pH 7.0 ± 0.2 at 25°C

ZnSO4·7H2O 28.0mg CoCl2·6H2O 4.0mg

H3BO3 4.0mg MnSO4·5H2O 4.0mg MoO3 4.0mg CuSO4·5H2O 2.0mg

Preparation of Trace Elements Solution : Add components to

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1730 Thermophilic Maintenance Medium

water and bring volume to 1.0L Mix thoroughly Distribute into tubes

or flasks Autoclave for 15 min at 15 psi pressure–121°C Incubate

cul-tures in 5% O2 + 80% H2 + 10% CO2

Use: For the cultivation and maintenance of Hydrogenobacter

ther-mophilus and Pseudomonas species.

Thermophilic Maintenance Medium

NaHCO3 3.0g

Yeast extract 1.0g

NH4Cl 1.0g

KH2PO4 0.4g

K2HPO4 0.4g

MgSO4·7H2O 0.1g

Cysteine-sulfide reducing solution 40.0mL

Fructose solution 25.0mL

Wolfe’s vitamin solution 10.0mL

Wolfe’s mineral solution 10.0mL

Resazurin (0.01% solution) 1.0mL

pH 5.6 ± 0.2 at 25°C

Cysteine-Sulfide Reducing Solution:

Composition per 100.0mL:

L-Cysteine·HCl·H2O 1.25g

Na2S·9H2O 1.25g

Preparation of Cysteine-Sulfide Reducing Solution: Add L

-cysteine·HCl·H2O and Na2S·9H2O to distilled/deionized water and bring

volume to 100.0mL Mix thoroughly

Fructose Solution:

Fructose 20.0g

Preparation of Fructose Solution: Add fructose to

distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly Filter

sterilize

Pyridoxine·HCl 0.01g

Thiamine·HCl 5.0mg

Riboflavin 5.0mg

Calcium pantothenate 5.0mg

Thioctic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Cyanocobalamin 100.0μg

Preparation of Wolfe’s Vitamin Solution: Add components to

MgSO4·7H2O 3.0g

Nitrilotriacetic acid 1.5g

NaCl 1.0g

MnSO4·H2O 0.5g

FeSO4·7H2O 0.1g

CoCl2·6H2O 0.1g

CaCl2 0.1g

ZnSO4·7H2O 0.1g

CuSO4·5H2O 0.01g AlK(SO4)2·12H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Preparation of Medium: Add components, except cysteine-sulfide reducing solution and fructose solution, to distilled/deionized water and bring volume to 935.0mL Mix thoroughly Gently heat and bring

to boiling Continue boiling until resazurin turns colorless, indicating reduction Add 40.0mL of the cysteine-sulfide reducing solution Au-toclave for 15 min at 15 psi pressure–121°C Cool to 50°C under O2 -free 90% N2 + 10% CO2 Add 25.0mL of the sterile fructose solution Adjust the pH to 5.6 if necessary Aseptically and anaerobically distrib-ute into sterile tubes Cap with rubber stoppers

Use: For the cultivation and maintenance of a variety of thermophilic

anaerobes, including Clostridium thermoautotrophicum.

Thermophilic Methanosarcina Medium

NaCl 2.25g Pancreatic digest of casein 2.0g Yeast extract 2.0g NaHCO3 0.85g MgSO4·7H2O 0.5g

NH4Cl 0.5g

K2HPO4 0.348g CaCl2·2H2O 0.25g

KH2PO4 0.227g FeSO4·7H2O 2.0mg Resazurin 1.0mg Rumen fluid, clarified 50.0mL Methanol solution 10.0mL Vitamin solution 10.0mL

L-Cysteine·HCl·H2O solution 10.0mL

Na2S·9H2O solution 10.0mL Trace elements solution SL-10 1.0mL

pH 6.5–6.8 at 25°C

Methanol Solution:

Methanol 5.0mL

Preparation of Methanol Solution: Add methanol to distilled/de-ionized water and bring volume to 10.0mL Sparge with 100% N2 Au-toclave for 15 min at 15 psi pressure–121°C

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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Thermophilic Methanothrix Medium 1731

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly Filter

ster-ilize Sparge with 80% N2 + 20% CO2

L -Cysteine·HCl·H 2 O Solution:

L-Cysteine·HCl·H2O 0.3g

Preparation of L -Cysteine·HCl·H 2 O Solution: Add L

-cyste-ine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL

Mix thoroughly Autoclave under 100% N2 for 15 min at 15 psi

pres-sure–121°C

Na2S·9H2O 0.3g

Trace Elements Solution SL-10:

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

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 100% N2

Preparation of Medium: Add components, except methanol

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

Sparge under 80% N2 + 20% CO2 for 3–4 min Autoclave for 15 min

at 15 psi pressure–121°C Aseptically and anaerobically add 20.0mL of

sterile vitamin solution, 10.0mL of sterile L-cysteine·HCl·H2O

solu-tion, and 10.0mL of sterile Na2S·9H2O solution Mix thoroughly

Aseptically and anaerobically distribute into sterile screw-capped

bot-tles under 80% N2 + 20% CO2

Use: For the cultivation and maintenance of Methanosarcina

thermo-phila.

Thermophilic Methanothrix Medium

NH4Cl 0.5g

K2HPO4 0.4g

MgCl2·6H2O 0.1g

Resazurin 1.0mg

NaHCO3 solution 20.0mL

Trace elements solution .10.0mL

CaCl2·2H2O solution 10.0mL

Sodium acetate solution 10.0mL

Vitamin solution 10.0mL

Coenzyme M solution 10.0mL

Na2S·9H2O solution 5.0mL

pH 6.5 ± 0.2 at 25°C

NaHCO 3 Solution:

NaHCO3 1.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to distilled/de-ionized water and bring volume to 20.0mL Mix thoroughly Sparge with 80% N2 + 20% CO2 for 15 min Autoclave for 15 min at 15 psi pressure–121°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 approximately 500.0mL of distilled/deionized water Dissolve

by adding KOH and adjust pH to 6.5 Add remaining components Bring volume to 1.0L with additional distilled/deionized water Adjust

pH to 7.0 with KOH

CaCl 2 ·2H2O Solution:

CaCl2·2H2O 0.1g

Preparation of CaCl 2 ·2H2O Solution: Add CaCl2·2H2O 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

Sodium Acetate Solution:

Sodium acetate 3.3g

Preparation of Sodium Acetate Solution: Add sodium acetate 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

Preparation of Vitamin Solution: Add components to distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Sparge with 80%

N2 + 20% CO2

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1732 Thermophilic Spirochete Medium

Coenzyme M Solution:

Coenzyme M 0.142g

Preparation of Coenzyme M Solution: Add coenzyme M to

Na2S·9H2O 0.5g

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

pres-sure–121°C

Preparation of Medium: Add components, except NaHCO3

solu-tion, CaCl2·2H2O solution, sodium acetate solution, vitamin solution,

coenzyme M solution, and Na2S·9H2O solution, to distilled/deionized

water and bring volume to 935.0mL Gently heat and bring to boiling

Continue boiling for 10 min Cool to room temperature while sparging

with 80% N2 + 20% CO2 Gas the medium until the pH reaches 5.8

An-aerobically distribute the medium into serum bottles Autoclave for 15

min at 15 psi pressure–121°C Aseptically and anaerobically add

20.0mL of sterile NaHCO3 solution, 10.0mL of sterile CaCl2·2H2O

so-lution, 10.0mL of sterile sodium acetate soso-lution, 10.0mL of sterile

vi-tamin solution, 10.0mL of sterile coenzyme M solution, and 5.0mL of

sterile Na2S·9H2O solution Bring gas atmosphere in each bottle to

30% CO2

Use: For the cultivation and maintenance of Methanothrix

thermo-phila.

Thermophilic Spirochete Medium

Solution A 920.0mL

Solution D 50.0mL

Solution E (Vitamin solution) 20.0mL

Solution F 10.0mL

Solution G 10.0mL

Solution B (Trace elements solution SL-10) 1.0mL

Solution C (Selenite-tungstate solution) 1.0mL

pH 6.9 ± 0.2 at 25°C

Solution A:

NaCl 4.0g

MgCl2·6H2O 0.8g

KCl 0.5g

NH4Cl 0.3g

KH2PO4 0.2g

CaCl2·2H2O 0.03g

Resazurin 1.0mg

Preparation of Solution A : Prepare and dispense under 80% N2 +

20% CO2 Add components to distilled/deionized water and bring

vol-ume to 920.0mL Mix thoroughly Gently heat and bring to boiling

Continue boiling for a few minutes Cool to room temperature while

sparging with 80% N2 + 20% CO2 Anaerobically distribute into tubes

or bottles Autoclave for 15 min at 15 psi pressure–121°C

Solution B (Trace Elements Solution SL-10):

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

Preparation of Solution B (Trace Elements Solution SL-10):

Add FeCl2·4H2O to 10.0mL of HCl solution Mix thoroughly Add dis-tilled/deionized water and bring volume to 1.0L Add remaining com-ponents Mix thoroughly Sparge with 100% N2 Autoclave for 15 min

at 15 psi pressure–121°C

Solution C (Selenite-Tungstate Solution):

NaOH 0.5g

Na2SeO3·5H2O 3.0mg

Na2WO4·2H2O 4.0mg

Preparation of Solution C (Selenite-Tungstate Solution): Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at

15 psi pressure–121°C

Solution D:

NaHCO3 3.0mg

Preparation of Solution D : Add NaHCO3 to distilled/deionized water and bring volume to 50.0mL Mix thoroughly Sparge with 80%

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

Solution E (Vitamin Solution):

Preparation of Solution E (Vitamin Solution) : Add components

to distilled/deionized water and bring volume to 1.0L Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Solution F:

Starch, soluble 1.0g

Preparation of Solution F : Add starch to distilled/deionized water

and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Solution G:

Na2S·9H2O 0.3g

Preparation of Solution G : Add Na2S·9H2O to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100%

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

Preparation of Medium: Prepare and dispense medium under 80% N2 + 20% CO2 To 920.0mL of sterile solution A, aseptically and anaerobically add 1.0mL of sterile solution B, 1.0mL of sterile

Trang 9

Thermoplasma acidophilum Medium 1733

solution C, 50.0mL of sterile solution D, 20.0mL of sterile solution

E, 10.0mL of sterile solution F, and 10.0mL of sterile solution G in

that order Mix thoroughly

Use: For the cultivation and maintenance of Spirochaeta thermophila.

Thermophilic Streptomycete Medium

Agar 20.0g

Maltose 20.0g

Soybean meal 5.0g

Yeast extract 2.0g

pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to tap water and bring

volume to 1.0L Mix thoroughly Gently heat and bring to boiling

Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–

121°C Pour into sterile Petri dishes or leave in tubes

Use: For the isolation and cultivation of thermophilic streptomycetes

Thermophilic Streptomycete Medium

Soybean oil meal 20.0g

Glucose 10.0g

NaCl 10.0g

Pancreatic digest of casein 10.0g

Silica solution (Ludox) 500.0mL

Preparation of Medium: Add components, except silica solution,

to distilled/deionized water and bring volume to 500.0mL Mix

thor-oughly Gently heat until dissolved Autoclave this solution and the

500.0mL of silica solution separately for 15 min at 15 psi pressure–

121°C Cool to 25°C Adjust the pH of both solutions to 7.0

Aseptical-ly combine the two sterile solutions Mix thoroughAseptical-ly Pour into sterile

Petri dishes in 40.0mL volumes

Thermophilic Streptomycete Medium IA

Agar 20.0g

Sucrose 5.0g

Pancreatic digest of casein 5.0g

Yeast extract 3.0g

MgSO4·7H2O 0.5g

FeSO4·7H2O 0.01g

Dung extract 5.0mL

Molasses 5.0mL

Trace elements solution 1.0mL

pH 7.2 ± 0.2 at 25°C

Dung Extract:

Sheep manure, dried 25.0g

Preparation of Dung Extract: Add dried sheep manure to

100.0mL of tap water Mix thoroughly Autoclave for 30 min at 15 psi

pressure–121°C Filter through Whatman #1 filter paper Store at 4°C

under toluene

Fe(NH4)2SO4 0.1g

ZnSO4 0.1g

MnSO4 0.05g CoSO4 0.01g

H3BO3 0.01g CuSO4 8.0mg

Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100.0mL Mix

thorough-ly

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring

to boiling Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes

Thermoplasma acidophilum Growth Medium 7B

Sucrose 17.0g (NH4)2SO4 6.8g KOH 1.22g Yeast extract 1.0g MgSO4 0.5g CaCl2·2H2O 0.25g

H3PO4 solution 1.5mL Antifoam A 10.0μL

pH 1.60 ± 0.2 at 25°C

H 3 PO 4 Solution:

H3PO4 85.0g

Preparation of H 3 PO 4 Solution: Add H3PO4 to distilled/deion-ized water and bring volume to 100.0mL Mix thoroughly

Preparation of Medium: Add components, except Antifoam A, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 1.60 with 50% H2SO4 Distribute into tubes or flasks Sterilize by heating to 100°C for 30 min Allow to stand at room tem-perature for 24 hr Add 10.0μL of Antifoam A per liter

Use: For the cultivation of Thermococcus acidophilum.

Thermoplasma acidophilum Medium

(NH4)2SO4 1.32g Yeast extract solution 1.0g

KH2PO4 0.372g MgSO4·7H2O 0.247g CaCl2·2H2O 0.074g Glucose solution 20.0mL Yeast extract solution 10.0mL Trace elements solution 10.0mL

pH 1.0–2.0 at 25°C

Glucose Solution:

Glucose 10.0g

Preparation of Glucose Solution : Add glucose to

distilled/deion-ized water and bring volume to 20.0mL Mix thoroughly Autoclave for

15 min at 15 psi pressure–121°C

Yeast Extract Solution:

Yeast extract 1.0g

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1734 Thermoplasma acidophilum Medium 7A

Preparation of Yeast Extract Solution : Add yeast extract to

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

FeCl3·6H2O 1.93g

Na2B4O7·10H2O 0.45g

MnCl2·4H2O 0.18g

ZnSO4·7H2O 22.0mg

CuCl2·2H2O 5.0mg

VOSO4·5H2O 3.8mg

Na2MoO4·2H2O 3.0mg

CoSO4·7H2O 2.0mg

Preparation of Trace Elements Solution: Add components to

Preparation of Medium: Add components, except glucose

solu-tion and yeast extract solusolu-tion, to distilled/deionized water and bring

volume to 970.0mL Mix thoroughly Adjust pH to 1.0–2.0 with 10N

H2SO4 Autoclave for 15 min at 15 psi pressure–121°C Aseptically

add 20.0mL of sterile glucose solution and 10.0mL of sterile yeast

ex-tract solution Mix thoroughly Aseptically distribute into sterile tubes

or flasks

Use: For the cultivation and maintenance of Thermoplasma

acidophi-lum.

Thermoplasma acidophilum Medium 7A

Glucose 10.0g

(NH4)2SO4 6.8g

KH2PO4 3.0g

Yeast extract 1.0g

MgSO4 0.5g

CaCl2·2H2O 0.25g

pH 1.65 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Adjust pH to 1.65

with 50% H2SO4 Distribute into tubes or flasks Sterilize by heating to

100°C for 30 min

Use: For the cultivation of Thermococcus acidophilum.

Thermoplasma Agar

Basal solution 450.0mL

Solution B 450.0mL

Solution C 100.0mL

pH 2.0 ± 0.2 at 25°C

Basal Solution:

KH2PO4 3.0g

Yeast extract 1.0g

MgSO4·7H2O 0.5g

CaCl2·2H2O 0.25g

(NH4)2SO4 0.2g

Preparation of Basal Solution: Add components to

distilled/de-ionized water and bring volume to 500.0mL Mix thoroughly Adjust

pH to 2.0 with 10N H2SO4 Autoclave for 15 min at 15 psi pressure–

121°C Cool to 55°C

Solution B:

Noble agar 12.0g

Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 450.0mL Mix thoroughly Gently heat and bring

to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 55°C

Solution C:

Glucose 10.0g

Preparation of Solution C: Add glucose to distilled/deionized wa-ter and bring volume to 100.0mL Mix thoroughly Filwa-ter swa-terilize

Preparation of Medium: Aseptically combine the cooled, sterile basal medium with sterile solution B and sterile solution C Mix thor-oughly Pour into sterile Petri dishes or distribute into sterile tubes

acidophi-lum and other Thermoplasma species.

Thermoplasma Broth

Basal solution 500.0mL Solution C 100.0mL

pH 2.0 ± 0.2 at 25°C

Basal Solution:

KH2PO4 3.0g Yeast extract 1.0g MgSO4·7H2O 0.5g CaCl2·2H2O 0.25g (NH4)2SO4 0.2g

Preparation of Basal Solution: Add components to distilled/de-ionized water and bring volume to 500.0mL Mix thoroughly Adjust

pH to 2.0 with 10N H2SO4

Solution C:

Glucose 10.0g

Preparation of Solution C: Add glucose to distilled/deionized wa-ter and bring volume to 100.0mL Mix thoroughly Filwa-ter swa-terilize

Preparation of Medium: Add 500.0mL of basal solution to 400.0mL of distilled/deionized water Autoclave for 15 min at 15 psi pressure–121°C Cool to 55°C Aseptically add 100.0mL of sterile glu-cose solution Mix thoroughly Aseptically distribute into sterile tubes

acidophi-lum and other Thermoplasma species.

Thermoplasma volcanium Medium

KH2PO4 3.0g MgSO4·7H2O 1.0g CaCl2·2H2O 0.25g (NH4)2SO4 0.2g Glucose solution 10.0mL Yeast extract solution 10.0mL

pH 6.5 ± 0.2 at 25°C

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