Handbook of Microbiological Media, Fourth Edition part 54 pptx

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

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Desulfohalobium Medium 525

KCl 0.5g

NH4Cl 0.25g

KH2PO4 0.2g

CaCl2·2H2O 0.15g

Yeast extract 0.05g

Resazurin 0.5mg

Wolfe’s vitamin solution 10.0mL

NaHCO3 solution 10.0mL

Na2S·9H2O solution 10.0mL

L-Cysteine·HCl solution 10.0mL

Wolfe's Mineral Elixir 1.0mL

n-Decane 1.0mL

pH 7.1 ± 0.2 at 25°C

L -Cysteine·HCl Solution:

Compositionper 10.0mL:

L-Cysteine·HCl 0.1g

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

dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly

Sparge with 100% N2 Autoclave under 100% N2 for 15 min at 15 psi

pressure–121°C

Na 2 S·9H 2 O Solution :

Na2S·9H2O 0.1g

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

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

to room temperature

NaHCO 3 Solution :

NaHCO3 3.0g

Preparation of NaHCO 3 Solution: Add components to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Sparge

with 20% CO2 + 80% H2 Autoclave for 15 min at 15 psi pressure–

121°C Cool to room temperature

Wolfe’s Mineral Elixir:

Compositionper liter:

MgSO4·7H2O 30.0g

NaCl 10.0g

MnSO4·2H2O 5.0g

(NH4)2NiSO4·6H2O 2.8g

CoCl2·6H2O 1.8g

ZnSO4·7H2O 1.8g

FeSO4·7H2O 1.0g

CaCl2·2H2O 1.0g

KAl(SO4)2·12H2O 0.18g

CuSO4·5H2O 0.1g

H3BO3 0.1g

Na2MoO4·2H2O 0.1g

Na2SeO4 0.1g

Na2WO4·2H2O 0.1g

Preparation of Wolfe’s Mineral Elixir: Adjust pH of 1.0L of

dis-tilled/deionized water to 1.0 with dilute H2SO4 Add remaining

com-ponents one at a time Mix thoroughly to dissolve

Wolfe’s Vitamin Solution:

Composition per liter:

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 bicarbonate, vi-tamins, and reducing agents, to distilled/deionized water and bring vol-ume to 960.0mL Adjust pH to 7.2 Gently heat and bring to boiling Boil for 1 min Mix thoroughly Cool to room temperature while sparg-ing with 80% N2 + 20% CO2 Dispense into culture vessels under an atmosphere of 80% N2 + 20% CO2 (e.g., Balch tubes or serum vials) Add n-decane to each vessel Close the vessels using butyl rubber stop-pers fixed with aluminum crimps Autoclave for 15 min at 15 psi pres-sure–121°C Add vitamins and reducing agents from sterile, anoxic stock solutions Adjust pH to 7.1 Use 20% as inoculum and incubate vials in an inverted position

Use: For the cultivation of Desulfoglaeba spp.

Desulfohalobium Medium

NaCl 100.0g MgCl2·6H2O 20.0g KCl 4.0g

Na2SO4 3.0g CaCl2·2H2O 2.7g

NH4Cl 1.0g Sodium acetate 1.0g Trypticase™ 1.0g Yeast extract 1.0g

K2HPO4 0.3g

KH2PO4 0.3g Sodium (L)-lactate 2.5g Resazurin 1.0mg

Na2SeO3·5H2O 3.0μg

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

pH 7.0 ± 0.2 at 25°C

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

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526 Desulfohalobium utahense Medium

Na 2 S·9H 2 O Solution:

Na2S·9H2O 1.0mg

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

Preparation of Medium: Add components, except Na2S·9H2O

so-lution, to distilled/deionized water and bring volume to 1.0L Mix

thor-oughly Gas under 100% N2 Autoclave for 15 min at 15 psi pressure–

121°C Aseptically and anaerobically add 10.0mL of sterile

Na2S·9H2O solution Mix thoroughly Aseptically and anaerobically

distribute into sterile tubes or flasks

Use: For the cultivation and maintenance ofMethanohalophilus

ore-gonense

Desulfohalobium utahense Medium

(DSMZ Medium 1055)

NaCl 10.0g

MgSO4·7H2O 10.0g

KCl 6.0g

NH4Cl 1.0g

KH2PO4 0.1g

CaCl2·2H2O 0.4g

Yeast extract 0.5g

Resazurin 0.5mg

NaHCO3 solution 10.0mL

Lactate solution 10.0mL

Trace elements solution SL-10 1.0mL

Selenite-tungstate solution 1.0mL

pH 7.1 ± 0.2 at 25°C

Lactate Solution :

Na-(L)-lactate 2.5g

Preparation of Lactate Solution: Add Na-(L)-lactate to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly

Auto-clave under 100% N2 for 15 min at 15 psi pressure–121°C Cool to

room temperature

Na2S·9H2O 0.1g

to room temperature

NaHCO3 4.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to

distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Sparge

Selenite/Tungstate Solution:

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

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

Preparation of Medium: Add components, except bicarbonate, vi-tamins, and reducing agents, to distilled/deionized water and bring vol-ume to 960.0mL Adjust pH to 7.2 Gently heat and bring to boiling Boil for 1 min Mix thoroughly Cool to room temperature while sparg-ing with 80% N2 + 20% CO2 Dispense into culture vessels under an atmosphere of 80% N2 + 20% CO2 Autoclave for 15 min at 15 psi pres-sure–121°C Aseptically add bicarbonate, sodium lactate, and sulfide Adjust the final pH of the medium to 7.0–7.2

Use: For the cultivation of Desulfohalobium utahense.

Desulfohalobium utahense Medium with Malate

NaCl 10.0g MgSO4·7H2O 10.0g KCl 6.0g

NH4Cl 1.0g

KH2PO4 0.1g CaCl2·2H2O 0.4g Yeast extract 0.5g Resazurin 0.5mg NaHCO3 solution 10.0mL Malate solution 10.0mL

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

pH 7.1 ± 0.2 at 25°C

Malate Solution :

Na2(DL)-malate 1.0g

Preparation of Malate Solution: Add Na2(DL)-malate to distilled/ deionized water and bring volume to 10.0mL Mix thoroughly Auto-clave under 100% N2 for 15 min at 15 psi pressure–121°C Cool to room temperature

Na2S·9H2O 0.1g

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

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Desulfomicrobium WHB Medium 527

to room temperature

NaHCO3 4.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to

distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Sparge

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

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

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 100% N2 Autoclave for 15 min at 15 psi pressure–

121°C

Preparation of Medium: Add components, except bicarbonate,

vi-tamins, and reducing agents, to distilled/deionized water and bring

vol-ume to 960.0mL Adjust pH to 7.2 Gently heat and bring to boiling

Boil for 1 min Mix thoroughly Cool to room temperature while

sparg-ing with 80% N2 + 20% CO2 Dispense into culture vessels under an

atmosphere of 80% N2 + 20% CO2 Autoclave for 15 min at 15 psi

pres-sure–121°C Aseptically add bicarbonate, sodium lactate, and sulfide

Adjust the final pH of the medium to 7.0–7.2

Use: For the cultivation of Desulfovermiculus halophilus.

Desulfoluna Medium

NaCl 20.0g

MgSO4·7H2O 2.0g

NH4Cl 1.0g

Na2SO4 1.0g

KH2PO4 0.5g

Yeast extract 0.5g

CaCl2·2H2O 0.1g

Resazurin 1.0mg

L-Cysteine·HCl solution 10.0mL

Na-lactate, 60% solution 2.9mL

Trace elements solution SL-10 1.0mL

pH 6.5 ± 0.2 at 25°C

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

L -Cysteine·HCl Solution:

L-Cysteine·HCl 0.5g

Preparation of L -Cysteine Solution : Add L-cysteine·HCl to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C

Preparation of Medium: Add components, except cyseine solu-tion, to distilled/deionized water and bring volume to 990.0mL Gently heat and bring to boiling Boil for 1 min Mix thoroughly Cool to room temperature while sparging with 100% N2 Dispense into culture ves-sels under an atmosphere of 100% N2 Autoclave for 15 min at 15 psi pressure–121°C Aseptically add 10.0mL cysteine solution Adjust the final pH of the medium to 6.5

Use: For the cultivation of Desulfoluna spp.

Desulfomicrobium WHB Medium

Solution A 870.0mL Solution C 100.0mL Solution D 10.0mL Solution E (Vitamin solution) 10.0mL Solution F 10.0mL Solution B (Trace elements solution SL-10) 1.0mL Solution G 1.0mL Solution H 1.0mL

pH 7.1–7.4 at 25°C

Solution A:

NaCl 21.0g MgCl2·6H2O 3.1g

Na2SO4 3.0g KCl 0.5g

NH4Cl 0.3g

KH2PO4 0.2g CaCl2·2H2O 0.15g Resazurin 1.0mg

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 870.0mL Mix thoroughly Gently heat and bring to boiling Continue boiling for 3–4 min Allow to cool to room temperature while gassing under 80% N2 + 20% CO2 Continue gas-sing until pH reaches below 6.0 Seal the flask under 80% N2 + 20%

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

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528 Desulfomonile Medium

Solution B (Trace Elements Solution SL-10):

FeCl2·4H2O 1.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

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:

NaHCO3 5.0g

Preparation of Solution C: Add NaHCO3 to distilled/deionized

water and bring volume to 100.0mL Mix thoroughly Filter sterilize

Sparge with 80% N2 + 20% CO2

Solution D:

Sodium lactate 4.0g

Preparation of Solution D: Add sodium lactate to

distilled/deion-ized water and bring volume to 10.0mL Mix thoroughly Sparge with

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

Solution E (Vitamin Solution):

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

Preparation of Solution E (Vitamin Solution): Add

compo-nents to distilled/deionized water and bring volume to 1.0L Mix

thor-oughly Filter sterilize Sparge with 100% N2

Solution F:

Na2S·9H2O 0.4g

Preparation of Solution F: 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

Solution G (Selenite-Tungstate Solution):

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Preparation of Solution G (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 H (Seven Vitamins):

Pyridoxine·HCl 0.3g Thiamine·HCl 0.2g Nicotinic acid 0.2g Calcium DL-pantothenate 0.1g Vitamin B12 0.1g

Biotin 20.0mg

Preparation of Solution H (Seven Vitamins): Add components

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

Preparation of Medium: Aseptically and anaerobically combine solution A with solution B, solution C, solution D, solution E, solution

F, solution G, and solution H, in that order Mix thoroughly Anaerobi-cally distribute into sterile tubes or flasks under 80% N2 + 20% CO2

Use: For the cultivation of Desulfomicrobium species.

Desulfomonile Medium

Solution A 870.0mL Solution C 100.0mL Solution D 10.0mL Solution E (Vitamin solution) 10.0mL Solution F 10.0mL Solution B (Trace elements solution SL-10) 1.0mL Solution G 1.0mL

pH 6.8–7.0 at 25°C

Solution A:

Na2SO4 3.0g NaCl 1.0g KCl 0.5g MgCl2·6H2O 0.4g

NH4Cl 0.3g

KH2PO4 0.2g CaCl2·2H2O 0.15g Resazurin 1.0mg

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 870.0mL Mix thoroughly Gently heat and bring to boiling Continue boiling for 3–4 min Allow to cool to room temperature while gassing under 80% N2 + 20% CO2 Continue gas-sing until pH reaches below 6.0 Seal the flask under 80% N2 + 20%

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

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Desulfomonile tiedjei Medium 529

com-ponents Mix thoroughly Gas under 100% N2 Autoclave for 15 min at

Solution C:

NaHCO3 2.5g

Preparation of Solution C: Add NaHCO3 to distilled/deionized

water and bring volume to 100.0mL Mix thoroughly Filter sterilize

Gas under 80% N2 + 20% CO2

Solution D:

Sodium pyruvate 4.0g

Preparation of Solution D: Add sodium pyruvate to

distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Gas under

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

Nicotinamide 50.0mg

1,4-Naphthoquinone 20.0mg

Calcium DL-pantothenate 5.0mg

Thioctic acid 5.0mg

Riboflavin 5.0mg

Thiamine·HCl 5.0mg

Biotin 5.0mg

Folic acid 5.0mg

Vitamin B12 5.0mg

Hemin 5.0mg

Preparation of Solution E (Vitamin Solution): Add

1,4-naphtho-quinone and hemin to 10.0mL of 0.1N NaOH Mix thoroughly Add

re-maining components and bring volume to 1.0L with distilled/deionized

water Mix thoroughly Gas under 100% N2 Autoclave for 15 min at

Solution F:

Na2S·9H2O 0.4g

Preparation of Solution F: Add Na2S·9H2O to distilled/deionized

water and bring volume to 10.0mL Mix thoroughly Gas under 100%

Solution G:

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Preparation of Solution G: Add components to distilled/deionized

water and bring volume to 10.0mL Mix thoroughly Gas under 100%

Preparation of Medium: Aseptically and anaerobically combine

870.0mL of sterile solution A with 1.0mL of sterile solution B,

100.0mL of sterile solution C, 10.0mL of sterile solution D, 10.0mL of

sterile solution E, and 10.0mL of sterile solution F, in that order Mix

thoroughly Anaerobically distribute into sterile tubes or flasks under

100% N2 Add 50.0mg/L of sodium dithionite prior to inoculation

Use: For the cultivation and maintenance ofDesulfomonile tiedjei.

Desulfomonile tiedjei Medium

NaHCO3 3.0g

PIPES (piperazine-N,N´-bis-2-ethanesulfonic acid) buffer 1.5g

Na2SO4 1.42g Yeast extract 1.0g Mineral solution 20.0mL Trace metal solution 10.0mL

Na2S2O4 solution 10.0mL Vitamin solution 10.0mL Sodium pyruvate solution 10.0mL Resazurin (0.1% solution) 1.0mL

pH 7.3 ± 0.2 at 25°C

Mineral Solution:

NH4Cl 50.0g NaCl 40.0g MgCl2·6H2O 8.3g KCl 5.0g

KH2PO4 5.0g CaCl2·2H2O 1.0g

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

Trace Metal Solution:

Nitrilotriacetic acid 2.0g MnSO4·H2O 1.0g Fe(NH4)2(SO4)2·6H2O 0.8g CoCl2·6H2O 0.2g ZnSO4·7H2O 0.2g CuCl2·2H2O 0.02g

Na2MoO4·H2O 0.02g

Na2SeO4 0.02g

Na2WO4 0.02g NiCl2·6H2O 0.02g

Preparation of Trace Metal 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

Vitamin Solution:

Nicotinamide 0.05g 1,4-Naphthoquinone 0.02g

Biotin 5.0mg Calcium pantothenate 5.0mg Cyanocobalamin 5.0mg Folic acid 5.0mg Hemin 5.0mg Pyridoxine·HCl 5.0mg Riboflavin 5.0mg Thioctic acid 5.0mg

NaOH (0.1N solution) 5.0mL

Preparation of Vitamin Solution: Add thioctic acid,

1,4-naphtho-quinone, and hemin to 5.0mL of 0.1N NaOH solution Mix thoroughly.

Bring volume to 1.0L with distilled/deionized water Add remaining components Mix thoroughly

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530 Desulfomusa hansenii Medium

Na 2 S 2 O 4 Solution:

Na2S2O4 0.087g

Preparation of Na 2 S 2 O 4 Solution: Add Na2S2O4 to

distilled/de-ionized water and bring volume to 10.0mL Filter sterilize Prepare

freshly

Sodium Pyruvate Solution:

Sodium pyruvate 4.4g

Preparation of Sodium Pyruvate Solution: Add sodium

pyru-vate to distilled/deionized water and bring volume to 10.0mL Filter

sterilize

Preparation of Medium: Add PIPES buffer, Na2SO4, yeast extract,

mineral solution, and trace metal solution to distilled/deionized water

and bring volume to 970.0mL Mix thoroughly Adjust pH to 7.3 with

HCl Add NaHCO3 and resazurin Gently heat and bring to boiling

un-der 80% N2 + 20% CO2 Replace headspace with 2 atm pressure of the

same gas phase Autoclave for 15 min at 15 psi pressure–121°C Cool

to 25°C Anaerobically and aseptically add sterile vitamin solution,

so-dium pyruvate solution, and Na2S2O4 solution Mix thoroughly

Use: For the cultivation and maintenance of Desulfomonile tiedjei.

Desulfomusa hansenii Medium

Solution A 870.0mL

Solution D 50.0mL

Solution F 50.0mL

Solution E (Vitamin solution) 10.0mL

Solution G 10.0mL

Solution H 10.0mL

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

Solution C (Selenite-tungstate solution) 1.0mL

pH 7.2 ± 0.2 at 25°C

Solution A:

Composition per 870.0mL:

NaCl 20.0g

Na2SO4 1.42g

KCl 0.67g

NH4Cl 0.1g

KH2PO4 0.01g

MgSO4·7H2O 0.02g

Resazurin 0.5mg

Preparation of Solution A: Add components to 870.0mL of

dis-tilled/deionized water Mix thoroughly Gently heat and bring to boiling

Boil for 3 min Cool to room temperature while sparging with 80% N2 +

20% CO2 Autoclave for 15 min at 15 psi pressure–121°C Cool to room

temperature

FeCl2·4H2O 1.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

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

Solution C (Selenite-Tungstate Solution):

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Preparation of Solution C (Selenite-Tungstate Solution):

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

Solution D:

NaHCO3 5.0g

Preparation of Solution D: Add NaHCO3 to distilled/deionized water and bring volume to 50.0mL Mix thoroughly Sparge with 100%

Pyridoxine-HCl 10.0mg Thiamine-HCl·2H2O 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg

D-Ca-pantothenate 5.0mg

Lipoic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B12 0.1mg

Preparation of Solution E (Vitamin Solution): Add compo-nents to distilled/deionized water and bring volume to 1.0L Mix thor-oughly Sparge with 100% N2 Filter sterilize

Solution F:

MgCl2·6H2O 10.6g CaCl2·2H2O 1.52g

Preparation of Solution F: Add components to distilled/deionized water and bring volume to 50.0mL Mix thoroughly Sparge with 100%

Solution G:

FeCl2·4H2O 0.52g

Preparation of Solution G: Add FeCl2·4H2O to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Adjust pH to 2.0 Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Solution H:

Na-propionate 0.96g

Preparation of Solution H: Add Na-propionate to distilled/deion-ized 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: Aseptically and anaerobically under 80%

N2 + 20% CO2 sequentially add to 860.0mL sterile solution A, 1.0mL solution B, 1.0mL solution C, 50.0mL solution D, 10.0mL solution E,

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Desulfonatronospira Medium 531

50.0mL solution F, 10.0mL solution G, and 10.0mL solution H

Asep-tically and anaerobically distribute under 80% N2 + 20% CO2 into

ap-propriate vessels The final pH should be 7.2 Addition of 10–20mg

sodium dithionite per liter from a 5% (w/v) solution, freshly prepared

under N2 and filter sterilized, may stimulate growth

Use: For the cultivation of Desulfomusa hansenii.

Desulfonatronospira Medium

Na2CO3 95.0g

NaHCO3 15.0g

NaCl 15.0g

K2HPO4 0.5g

Ammonium chloride solution 10.0mL

Magnesium chloride solution 10.0mL

Yeast extract solution 10.0mL

Pyruvate solution 10.0mL

Thiosulfate solution 10.0mL

Vitamin solution 10.0mL

Trace elements solution SL-10 with -EDTA 1.0mL

Selenite/tungstate solution .1.0mL

pH 10.0 ± 0.2 at 25°C

Trace Elements Solution SL-10 with EDTA:

FeCl2·4H2O 1.5g

Na2-EDTA 0.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

Preparation of Trace Elements Solution SL-10 with EDTA:

at 15 psi pressure–121°C Adjsut pH to 7.0

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Pyridoxine-HCl 10.0mg

Thiamine-HCl·2H2O 5.0mg

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

Na2S·9H2O 0.24g

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

to room temperature

Ammonium Chloride Solution:

Composition per10.0mL:

NH4Cl 0.2g

Preparation of Ammonium Chloride Solution: Add NH4Cl 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

Magnesium Chloride Solution:

MgCl2·6H2O 0.2g

Preparation of Magnesium Chloride Solution: Add MgCl2·6H2O

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

Yeast Extract Solution:

Yeast extract 0.5g

Preparation of Yeast Extract Solution: Add yeast extract 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

Lactate Solution:

Na-lactate 2.2g

Preparation of Lactate Solution: Add Na-lactate to distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Sulfite Solution:

Na2SO3 1.3g

Preparation of Sulfite: Add Na2SO3 to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Filter sterilize

Preparation of Medium: Add components, except ammonium chloride, magnesium chloride, yeast extract, trace elements, vitamins, lactate, sulfite, and sulfide solutions, to distilled/deionized water and bring volume to 930.0mL Gently heat and bring to boiling Boil for 1 min Mix thoroughly Cool to room temperature while sparging with 100% N2 Dispense into culture vessels under an atmosphere of 100%

N2 Autoclave for 15 min at 15 psi pressure–121°C Aseptically add the ammonium chloride, magnesium chloride, yeast extract, trace ele-ments, vitamins, lactate, sulfite, and sulfide solutions Adjust the pH to

10

Use: For the cultivation of Desulfonatronospira spp.

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532 Desulfonatronospira Medium

Desulfonatronospira Medium

Na2CO3 95.0g

NaHCO3 15.0g

NaCl 15.0g

K2HPO4 0.5g

Ammonium chloride solution 10.0mL

Magnesium chloride solution 10.0mL

Lactate solution 10.0mL

Sulfite solution 10.0mL

Trace elements solution SL-10 with EDTA 1.0mL

Selenite/tungstate solution .1.0mL

pH 10.0 ± 0.2 at 25°C

Trace Elements Solution SL-10 with EDTA:

FeCl2·4H2O 1.5g

Na2-EDTA 0.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

Preparation of Trace Elements Solution SL-10 with EDTA:

at 15 psi pressure–121°C Adjsut pH to 7.0

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

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

Lactate Solution:

Na-lactate 2.2g

Preparation of Lactate Solution: Add Na-lactate to distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Sulfite Solution:

Na2SO3 1.3g

Preparation of Sulfite: Add Na2SO3 to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Filter sterilize

Na2S·9H2O 0.24g

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

to room temperature

Ammonium Chloride Solution:

NH4Cl 0.2g

Preparation of Ammonium Chloride Solution: Add NH4Cl 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

Magnesium Chloride Solution:

MgCl2·6H2O 0.2g

Preparation of Magnesium Chloride Solution: Add MgCl2·6H2O

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

Yeast extract 0.5g

Preparation of Yeast Extract Solution: Add yeast extract 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

Pyruvate Solution:

Na-pyruvate 2.2g

Preparation of Pyruvate Solution: Add Na-pyruvate 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

Thiosulfate Solution:

Na2S2O3 5.0g

Preparation of Thiosulfate Solution: Add Na2S2O3 to distilled/ deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Filter sterilize

Preparation of Medium: Add components, except ammonium chloride, magnesium chloride, yeast extract, trace elements, vitamin, pyruvate, thiosulfate, and sulfide solutions, to distilled/deionized water and bring volume to 930.0mL Gently heat and bring to boiling Boil

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Desulfonatronum Medium 533

for 1 min Mix thoroughly Cool to room temperature while sparging

with 100% N2 Dispense into culture vessels under an atmosphere of

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

add the ammonium chloride, magnesium chloride, yeast extract, trace

elements, vitamin, pyruvate, thiosulfate, and sulfide solutions Adjust

the pH to 10

Use: For the cultivation of Desulfonatronospira delicata.

Desulfonatronovibrio Medium

NaHCO3 15.0g

Na2CO3 10.0g

NaCl 10.0g

Na2SO4 3.0g

NH4Cl 1.0g

Na2HPO4 0.2g

KCl 0.2g

Resazurin 0.5mg

Na-formate solution 10.0mL

Trace elements solution 10.0mL

pH 9.6 ± 0.2 at 25°C

Na-Formate Solution:

Na-formate 5.0g

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

Yeast extract 1.5g

Preparation of Yeast Extract Solution: Add yeast extract to

Sparge with 100% N2 Autoclave under 100% N2 for 15 min at 15 psi

pressure–121°C Cool to room temperature

Na 2 S·9H 2 O Solution:

Composition per 10.0mL:

Na2S·9H2O 1.0g

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

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

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 Filter sterilize

Pyridoxine-HCl 10.0mg Thiamine-HCl·2H2O 5.0mg Riboflavin 5.0mg Nicotinic 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 100% N2 Filter sterilize

Preparation of Medium: Prepare and dispense medium under an oxygen-free atmosphere of 100% N2 Add components, except vitamin solution, yeast extract solution, Na-formate solution, trace elements so-lution, and Na2S·9H2O solution, to distilled/deionized water and bring volume to 950.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C Aseptically and an-aerobically add 10.0mL sterile vitamin solution, 10.0mL of sterile yeast extract solution, 10.0mL sterile Na-formate solution, 10.0mL sterile trace elements solution, and 10.0mL of sterile Na2S·9H2O solu-tion Mix thoroughly Adjust pH to 9.6 Aseptically and anaerobically distribute into sterile tubes or flasks

Use: For the cultivation of Desulfonatronovibrio hydrogenovorans.

Desulfonatronum Medium

Composition per 1010.0mL:

NaCl 10.0g

Na2SO4 5.0g

Na2CO3 3.5g

NH4Cl 1.0g Yeast extract 1.0g

Na2S·9H2O 0.5g

KH2PO4 0.2g KCl 0.2g MgCl2·6H2O 0.1g Resazurin 0.5mg Na-formate solution 10.0mL Vitamin solution 10.0mL Trace elements solution SL-10 1.0mL

pH 8.9 ± 0.2 at 25°C

Na-Formate Solution:

Na-formate 4.0g

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

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534 Desulfonema ishimotoi Medium

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

FeCl2·4H2O 1.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

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 Autoclave for 15 min at 15 psi pressure–

121°C

Preparation of Medium: Prepare and dispense medium under an

oxygen-free atmosphere of 100% N2 Add components, except

Na-for-mate solution and Na2S·9H2O, to distilled/deionized water and bring

volume to 1.0L Mix thoroughly Sparge with 100% N2 for 30 min Add

the Na2S·9H2O Mix thoroughly Adjust pH to 8.8–9.0 Dispense into

Hungate tubes under 100% N2 Autoclave for 15 min at 15 psi

pres-sure–121°C Cool to 25°C Aseptically and anaerobically add

Na-for-mate solution, 0.1mL per 10.0mL medium

Use: For the cultivation of Desulfonatronum lacustre.

Desulfonema ishimotoi Medium

Solution A 850.0mL

Agar solution 50.0mL

Solution C 50.0mL

Solution H 20.0mL

Solution D 10.0mL

Solution F (Vitamin solution) 10.0mL

Solution I 10.0mL

Solution G (Artificial sediment) 6.6mL

Solution E 1.0mL

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

pH 6.9 ± 0.2 at 25°C

Solution A:

NaCl 21.0g

MgCl2·6H2O 5.5g

Na2SO4 3.0g CaCl2·2H2O 1.35g KCl 0.5g

NH4Cl 0.3g

KH2PO4 0.2g Resazurin 0.5mg

Na2SeO3·5H2O 3µg

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 800.0mL Mix thoroughly

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

Solution C:

NaHCO3 5.0g

Preparation of Solution C: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Filter sterilize Flush with 80% N2 + 20% CO2 to remove dissolved oxygen

Solution D:

Na-acetate·3H2O 2.5g Isobutyric acid 0.18g

Na2-succinate 0.1g

Preparation of Solution D: Add components to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100%

N2 Filter sterilize

Solution E:

Na2-succinate 1.0g

Preparation of Solution E: Add Na2-succinate to distilled/deion-ized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Solution F (Vitamin Solution):

Pyridoxine-HCl 10.0mg Vitamin B12 5.1mg Thiamine-HCl·2H2O 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg

Lipoic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg

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