Handbook of Microbiological Media, Fourth Edition part 103 pps

0.1g Preparation of Solution C: Add components to distilled/deionized water and bring volume to 10.0mL.. 3.0mg HCl 25%,w/v...8.0mL Preparation of Trace Elements Solution: Add components

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Marine Flagellate Medium with B-Vitamins 1015

sterile CaCl2·2H2O solution Mix thoroughly Pour into sterile Petri

dishes or distribute into sterile tubes

Use: For the cultivation of Cytophaga species, Flexibacter species,

Microscilla species, and Saprospira grandis.

Marine Cytophaga Medium A

Compositionper liter:

Agar 15.0g

Pancreatic digest of casein 2.0g

Beef extract 0.5g

Yeast extract 0.5g

Sodium acetate 0.2g

Seawater 700.0mL

pH 7.2 ± 0.2 at 25°C

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

Use: For the cultivation of Flexibacter maritimus.

Marine Cytophaga Medium B

Agar 15.0g

Beef extract 0.5g

Yeast extract 0.5g

Sodium acetate 0.2g

Seawater 500.0mL

pH 7.2 ± 0.2 at 25°C

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

Use: For the cultivation of Vibrio ordalii.

Marine Cytophaga Medium C

Agar 15.0g

Beef extract 0.5g

Yeast extract 0.5g

Sodium acetate 0.2g

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to seawater 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 cultivation of Cytophaga agarovorans, Cytophaga

fer-mentans, and Cytophaga salmonicolor.

Marine Desulfovibrio Medium

Solution A 980.0mL

Solution B 10.0mL

Solution C 10.0mL

pH 7.8 ± 0.2 at 25°C

Solution A:

Compositionper 980.0mL:

NaCl 25.0g

DL-Sodium lactate 2.0g MgSO4·7H2O 2.0g

Na2SO4 1.0g

NH4Cl 1.0g Yeast extract 1.0g

K2HPO4 0.5g CaCl2·2H2O 0.1g Resazurin 1.0mg

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 980.0mL Mix thoroughly Gently heat and bring to boiling Continue boiling for 3–4 min Allow to cool to room temperature while gassing under 100% N2

Solution B:

FeSO4·7H2O 0.5g

Preparation of Solution B: Add FeSO4·7H2O to distilled/deion-ized water and bring volume to 10.0mL Mix thoroughly

Solution C:

Ascorbic acid 0.1g Sodium thioglycolate 0.1g

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

Preparation of Medium: To 980.0mL of cooled solution A, anaer-obically add 10.0mL of solution B and 10.0mL of solution C Mix thor-oughly Adjust pH to 7.8 with NaOH Distribute into tubes or flasks During distribution, swirl the medium to keep the precipitate in suspen-sion Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation and maintenance of Desulfovibrio desulfuri-cans, Desulfovibrio salexigens, and Desulfovibrio vulgaris.

Marine Flagellate Medium Compositionper 15.0mL:

Rice grains 2.0g Seawater 15.0mL

Preparation of Medium: Autoclave rice grains for 15 min at 15 psi pressure–121°C Add 2.0g of sterile rice grains to 15.0mL of filter-ster-ilized seawater Aseptically distribute into T-25 tissue culture flasks

Use: For the cultivation of Acanthoecopsis unguiculata, Amastigomonas species, Bicosoeca vacillans, Bodo designis, Bodo variabilis, Caecitellus parvulus, Choanoeca perplexa, Codosiga gracilis, Diaphanoeca grandis, Entosiphon species, Goniomonas species, Procryptobia species, Pseudo-bodo tremulans, Rhynchomonas nasuta, Salpingoeca urceolata, Stepha-noeca diplocostata, and Stephanopogon apogon.

Marine Flagellate Medium with B-Vitamins Compositionper liter:

Seawater 990.0mL Vitamin solution 10.0mL

Vitamin Solution:

Thiamine·HCl 0.15g Calcium D-(+)-pantothenate 0.05g Nicotinamide 0.05g

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1016 Marine Glucose Trypticase™ Yeast Extract Agar

Pyridoxal·HCl 0.05g

Riboflavin 0.05g

Folic acid 0.025g

Pyridoxamine·HCl 0.025g

Biotin 12.5mg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 100.0mL Mix thoroughly Filter

sterilize

Preparation of Medium: Allow natural seawater to age for 2

months Filter sterilize Aseptically add 100.0mL of sterile vitamin

so-lution Mix thoroughly Aseptically distribute into sterile tubes or

flasks

Use: For the cultivation of Oikomonas species.

Marine Glucose Trypticase ™ Yeast Extract Agar

(MGTY Agar) Compositionper liter:

Agar 8.0g

Glucose 2.0g

Yeast extract 1.0g

L-Cysteine·HCl·H2O 0.5g

Seawater 750.0mL

Tris-HCl buffer (5.0 mM, pH 7.5) 50.0mL

Resazurin (0.1% solution) 1.0mL

pH 7.5 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Gently heat while

stirring and bring to boiling Distribute into tubes or flasks under 97%

N2 + 3% H2 Cap with rubber stoppers and place tubes in a press

Au-toclave for 15 min at 15 psi pressure–121°C with fast exhaust

Use: For the cultivation and maintenance of Spirochaeta isovalerica

Marine Glucose Trypticase ™ Yeast Extract Broth

(MGTY Broth) Compositionper liter:

Glucose 2.0g

Yeast extract 1.0g

Seawater 750.0mL

Tris-HCl buffer (5.0 mM, pH 7.5) 50.0mL

Resazurin (0.1% solution) 1.0mL

pH 7.5 ± 0.2 at 25°C

stirring and bring to boiling Distribute into tubes or flasks under 97%

N2 + 3% H2 Cap with rubber stoppers and place tubes in a press

Au-toclave for 15 min at 15 psi pressure–121°C with fast exhaust

Use: For the cultivation and maintenance of Spirochaeta isovalerica

Marine Methanogenium Alcohol Medium

NaCl 21.0g

MgCl2·6H2O 3.0g

NaCl 1.0g

KCl 0.5g

MgCl2·6H2O 0.5g

NH4Cl 0.4g Sodium acetate·3H2O 0.4g

KH2PO4 0.2g CaCl2·2H2O 0.1g NaHCO3 solution 60.0mL 2-Propanol 5.0mL

Na2S·9H2O solution 3.0mL Cyanocobalamin solution 1.0mL Selenite-molybdate-tungstate solution 1.0mL Thiamine solution 1.0mL Trace elements solution 1.0mL Vitamin solution 1.0mL

Trace Elements Solution:

FeSO4·7H2O 1400.0mg ZnSO4·7H2O 145.0mg CoCl2·6H2O 120.0mg MnCl2·4H2O 100.0mg NiCl2·6H2O 50.0mg

H3BO3 6.0mg CuSO4·5H2O 3.0mg HCl (25%,w/v) 8.0mL

Preparation of Trace Elements 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

Selenite-Molybdate-Tungstate Solution:

NaOH 0.2g

Na2MoO4·2H2O 40.0mg

Na2WO4·2H2O 33.0mg

Na2SeO3·2H2O 5.0mg

Preparation of Selenite-Molybdate-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 pres-sure–121°C

NaHCO 3 Solution:

NaHCO3 84.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to distilled/de-ionized water and bring volume to 10.0mL 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 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 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

Preparation of 2-Propanol: Filter sterilize 10.0mL of 2-propanol Sparge with 100% N2

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Marine Methylotroph Broth 1017

Vitamin Solution:

Sodium 2-mercaptoethanesulfonate 0.25g

Pyridoxine·HCl 0.15g

Calcium pantothenate 0.1g

Nicotinic acid 0.1g

p-Aminobenzoic acid 40.0mg

Biotin 10.0mg

Potassium phosphate buffer

(25mM solution, pH 7.0) 1.0L

Preparation of Vitamin Solution: Combine components Mix

thoroughly Filter sterilize.Sparge with 100% N2

Thiamine Solution:

Thiamine·HCl 0.1g

Sodium phosphate buffer

(0.1M solution, pH 3.6) 1.0L

Preparation of Thiamine Solution: Combine components Mix

thoroughly Filter sterilize.Sparge with 100% N2

Cyanocobalamin Solution:

Cyanocobalamin 50.0mg

Preparation of Cyanocobalamin Solution: Add

cyanocobala-min to distilled/deionized water and bring volume to 1.0L Mix

thor-oughly Filter sterilize Sparge with 100% N2

Preparation of Medium: Prepare and dispense medium under 80%

N2 + 20% CO2 Add components, except NaHCO3 solution,

2-propa-nol, Na2S·9H2O solution, cyanocobalamin solution,

selenite-molyb-date-tungstate solution, thiamine solution, trace elements solution, and

vitamin solution, to distilled/deionized water and bring volume to

930.0mL Mix thoroughly Sparge with 80% N2 + 20% CO2 Autoclave

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

60.0mL of sterile NaHCO3 solution, 5.0mL of sterile 2-propanol,

3.0mL of sterile Na2S·9H2O solution, 1.0mL of sterile

cyanocobala-min solution, 1.0mL of sterile selenite-molybdate-tungstate solution,

1.0mL of sterile thiamine solution, 1.0mL of sterile trace elements

so-lution, and 1.0mL of sterile vitamin solution Mix thoroughly

Asepti-cally and anaerobiAsepti-cally distribute into sterile tubes or bottles

Use: For the cultivation of marine Methanogenium species

Marine Methanol Medium

NaCl 20.0g

(NH4)2SO4 2.0g

K2HPO4 2.0g

KH2PO4 1.0g

MgSO4·7H2O 0.3g

Methanol 10.0mL

Vitamin B12 solution 10.0mL

Trace metals solution 1.0mL

pH 7.0 ± 0.2 at 25°C

Vitamin B 12 Solution:

Vitamin B12 10.0μg

Preparation of Vitamin B 12 Solution: Add the vitamin B12 to

dis-tilled/deionized water and bring volume to 100.0mL Adjust pH to 5

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

Trace Metals Solution:

ZnSO4·7H2O 1.4g MnSO4·H2O 0.84g FeSO4·7H2O 0.28g CuSO4·5H2O 0.25g

Na2MoO4·2H2O 0.24g CoCl2·6H2O 0.24g CaCl2·2H2O 0.15g

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

Preparation of Medium: Add components, except vitamin B12 so-lution and methanol, to distilled/deionized water and bring volume to 980.0mL Adjust pH to 7.0 with NaOH Autoclave for 15 min at 15 psi pressure–121°C Filter sterilize methanol Aseptically add sterile vita-min B12 solution and filter-sterilized methanol Distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Methylophaga thalassica.

Marine Methylotroph Agar Compositionper 1003.0mL:

Agarose 12.0g Bis (2-hydroxyethyl) aminotris

(hydroxy-methyl) methane 2.0g

KH2PO4 0.14g Ferric ammonium citrate 0.06g Methanol 2.0mL Vitamin B12 solution 1.0mL

pH 7.4 ± 0.2 at 25°C

Vitamin B12 0.1mg

Preparation of Vitamin Solution: Add vitamin B12 to distilled/ deionized water and bring volume to 100.0mL Mix thoroughly Filter sterilize Store at 5°C

Preparation of Medium: Add components, except methanol and vitamin B12 solution, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.4 Gently heat and bring to boil-ing Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°C Aseptically add 2.0mL of filter-sterilized methanol and 1.0mL of ster-ile vitamin B12 solution Mix thoroughly Pour into sterile Petri dishes

or distribute into sterile tubes

Use: For the cultivation of Alteromonas species, Methylophaga marina, Methylophaga thalassica, and Methylophilus species.

Marine Methylotroph Broth Compositionper 1003.0mL:

Bis (2-hydroxyethyl) aminotris (hydroxy-methyl) methane 2.0g

KH2PO4 0.14g Ferric ammonium citrate 0.06g Methanol 2.0mL Vitamin B12 solution 1.0mL

pH 7.4 ± 0.2 at 25°C

Vitamin B12 0.1mg

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1018 Marine Oxidation Fermentation HiVeg Medium

Preparation of Vitamin Solution: Add vitamin B12 to distilled/

deionized water and bring volume to 100.0mL Mix thoroughly Filter

sterilize Store at 5°C

Preparation of Medium: Add components, except methanol and

vitamin B12 solution, to distilled/deionized water and bring volume to

1.0L Mix thoroughly Adjust pH to 7.4 Autoclave for 15 min at 15 psi

pressure–121°C Cool to 50°C Aseptically add 2.0mL of

filter-steril-ized methanol and 1.0mL of sterile vitamin B12 solution Mix

thor-oughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of Alteromonas species, Methylophaga

marina, Methylophaga thalassica, and Methylophilus species.

Marine Oxidation Fermentation HiVeg Medium

(MOF HiVeg Medium) Compositionper liter:

NaCl 9.7g

MnCl2 4.4g

Agar 3.0g

Na2SO4 1.6g

Plant hydrolysate 1.0g

CaCl2 0.9g

(NH4)2SO4 0.5g

Tris hydroxymethyl aminomethane 0.5g

KCl 0.275g

Yeast extract 0.1g

NaHCO3 0.08g

KBr 0.04g

SrCl2 0.017g

H3BO3 0.011g

Phenol Red 0.01g

Na2HPO4 4.0mg

Sodium silicate 2.0mg

NaFl 1.2mg

NH4NO3 0.8mg

pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from

Hi-Media

stirring and bring to boiling Distribute into tubes or flasks Autoclave

for 15 min at 15 psi pressure–121°C

Use: For the differentiation of marine bacteria on the basis of

fermen-tative and oxidative metabolism of carbohydrates

Marine Peptone Succinate Salts Medium

(PSS Medium) Compositionper liter:

Peptone 10.0g

Succinic acid 1.0g

(NH4)2SO4 1.0g

MgSO4·7H2O 1.0g

FeCl3·6H2O 2.0mg

MnSO4·H2O 2.0mg

Synthetic seawater 1.0L

pH 6.8 ± 0.2 at 25°C

Synthetic Seawater:

Composition per liter:

NaCl 27.5g

MgCl2 5.0g

MgSO4·7H2O 2.0g KCl 1.0g CaCl2 0.5g FeSO4 1.0mg

Preparation of Synthetic Seawater: Add components to dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components to 1.0L of synthetic sea-water Mix thoroughly Gently heat while stirring and bring to boiling Adjust pH to 6.8 with KOH Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation and maintenance of Oceanospirillum beijer-inckii and Oceanospirillum multiglobuliferum.

Marine Peptone Yeast Medium with Magnesium Sulfate Compositionper liter:

NaCl 20.0g Peptone 10.0g MgSO4·7H2O 2.0g (NH4)2SO4 2.0g Yeast extract 1.0g

pH 7.0 ± 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

Use: For the cultivation and maintenance of Oceanospirillum pusil-lum.

Marine Pseudomonas Medium

Agar 15.0g Nutrient broth 8.0g Yeast extract 5.0g Salt solution 1.0L

Salt Solution:

NaCl 12.86g MgCl2 2.48g KCl 0.75g CaCl2 0.56g Fe(SO4)2(NH4)2 0.048g

Preparation of Salt Solution: Add components to distilled/deion-ized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components to 1.0L of salt solution 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

Use: For the cultivation and maintenance of Alteromonas haloplank-tis.

Marine Rhodococcus Medium

Yeast extract 10.0g Malt extract 4.0g Glucose 4.0g Seawater 750.0mL

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Marine Salts Medium for Sporosarcina halophila 1019

Use: For the cultivation and maintenance of Rhodococcus

marinona-scens.

Marine Rhodopseudomonas Medium

NaCl 30.4g

Yeast extract 1.0g

Disodium succinate 1.0g

KH2PO4 0.5g

MgSO4·7H2O 0.4g

NH4Cl 0.4g

CaCl2·2H2O 0.05g

Ferric citrate (0.1% solution) 5.0mL

Trace elements solution SL-6 1.0mL

Ethanol 0.5mL

pH 6.8 ± 0.2 at 25°C

Trace Elements Solution SL-6:

H3BO3 0.3g

CoCl2·6H2O 0.2g

ZnSO4·7H2O 0.1g

MnCl2·4H2O 0.03g

Na2MoO4·H2O 0.03g

NiCl2·6H2O 0.02g

CuCl2·2H2O 0.01g

Preparation of Trace Elements Solution SL-6: Add components

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

Adjust pH to 3.4

Use: For the cultivation and maintenance of Rhodopseudomonas

marina.

Marine Rhodopseudomonas Medium

NaCl 30.0g

Peptone 2.5g

Yeast extract 2.5g

pH 7.0 ± 0.4 at 25°C

water and bring volume to 1.0L Mix thoroughly Distribute into

screw-capped tubes Autoclave for 15 min at 15 psi pressure–121°C Before

inoculating, loosen the screw caps, heat the medium to drive out O2,

and screw down the cap tightly

Use: For the cultivation of Rhodopseudomonas marina.

Marine Salts Medium Compositionper liter:

NaCl 81.0g

Yeast extract 10.0g

MgSO4 9.6g

MgCl2 7.0g Proteose peptone No.3 5.0g KCl 2.0g Glucose 1.0g CaCl2 0.36g NaHCO3 0.06g NaBr 0.026g

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C

Use: For the cultivation of marine bacteria

Marine Spirochete Medium (DSMZ Medium 1008) Composition per liter:

Trypticase peptone 2.0g Yeast extract 1.0g Na-thioglycolate 1.0g Resazurin 0.5mg Charcoal-filtered, natural seawater 800.0mL Cellobiose solution 20.0mL

pH 7.5 ± 0.2 at 25°C

Cellobiose Solution:

Cellobiose 10.0g

Preparation of Cellobiose Solution: Add components to dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly Sparge with 100% N2 Filter sterilize

Preparation of Medium: Add components, except thioglycolate and cellobiose solution, to seawater and bring volume to 800.0mL Mix thoroughly Bring volume to 980.0mL with distilled/deionized water (Note: Bottled water from Biomaris GmbH can be used instead of fil-tered seawater.) Gently heat and bring to boiling Boil for 3 min Cool

to room temperature while sparging with 100% N2 Add the

thioglyco-late Adjust pH to 7.5 with 10N NaOH Dispense into tubes or bottles

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

to room temperature Aseptically and anoxically add cellobiose solu-tion

Use: For the cultivation of Spirochaeta bajacaliforniensis.

Marine Salts Medium for Sporosarcina halophila

Marine salts mix 100.0g Agar 20.0g Yeast extract 10.0g Proteose peptone No 3 5.0g Glucose 1.0g

pH 7.0 ± 0.2 at 25°C

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

Use: For the cultivation and maintenance of Sporosarcina halophila.

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1020 Marine Spirochete Medium

Marine Spirochete Medium

Cellobiose 2.0g

Peptone 2.0g

Yeast extract 1.0g

Sodium thioglycolate 1.0g

Seawater, charcoal filtered 800.0mL

pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components, except sodium

thiogly-colate, to glass-distilled water and bring volume to 1.0L Mix

thor-oughly Bubble 100% N2 into medium for 1.5 min Add sodium

thioglycolate Adjust pH to 7.5 with 10N KOH Distribute into tubes or

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

Use: For the cultivation and maintenance of Spirochaeta

bajacaliforn-iensis.

Marine Thermococcus Medium

(DSMZ Medium 760) Compositionper liter:

NaCl 19.45g

MgCl2 8.8g

Sulfur 5.0g

Peptone 5.0g

Na2SO3 3.24g

CaCl2 1.8g

Yeast extract 1.0g

KCl 0.55g

NaHCO3 0.16g

Ferric citrate 0.1g

KBr 0.08g

SrCl2 0.03g

H3BO3 0.02g

Na2HPO4 8.0mg

Na2SiO3 4.0mg

NaF 2.4mg

NH4NO3 1.6mg

Na2S·9H2O solution 0.5mL

pH 6.0 ± 0.2 at 25°C

Na2S·9H2O 1.0g

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

Neutralize to pH 7.0 with sterile HCl

Preparation of Medium: Add components, except sulfur and

Na2S·9H2O solution, to distilled/deionized water and bring volume to

1.0L Mix thoroughly Filter through normal filter paper An iron

sedi-ment will collect in the filter Gently heat while stirring and bring to

boiling Boil for 5 min Cool under an anaerobic gas mixture of N2

Ad-just pH to 6.0 Distribute the medium into Hungate tubes or serum

bot-tles containing finely divided sulfur (0.5% w/v) Seal the tubes or

bottles under the same anaerobic gas used when cooling the medium

Sterilize the medium at 100°C for 3 hr on 3 consecutive days Reduce

the medium by adding 10% neutralized Na2S·9H2O solution to a final

concentration of 0.05% The medium should not give a heavy black

precipitate; if it does the iron sediment was not adequately removed by

filtering in the initial stages and the medium should be made again,

making sure that the iron is removed by filtering

Use: For the cultivation and maintenance of Thermococcus aegaeus

DSM 12767

NaCl 19.45g MgCl2 8.8g Sulfur 5.0g Peptone 5.0g

Na2SO3 3.24g CaCl2 1.8g Yeast extract 1.0g KCl 0.55g NaHCO3 0.16g Ferric citrate 0.1g KBr 0.08g SrCl2 0.03g

H3BO3 0.02g

Na2HPO4 8.0mg

Na2SiO3 4.0mg NaF 2.4mg

NH4NO3 1.6mg

pH 6.5 ± 0.2 at 25°C

Na2S·9H2O 1.0g

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 Neutralize to pH 7.0 with sterile HCl

Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Filter through normal filter paper An iron sedi-ment will collect in the filter Gently heat while stirring and bring to boiling Boil for 5 min Cool under an anaerobic gas mixture of N2 Ad-just pH to 6.5 Distribute the medium into Hungate tubes or serum bot-tles containing finely divided sulfur (0.5% w/v) Seal the tubes or bottles under the same anaerobic gas used when cooling the medium Sterilize the medium at 100°C for 3 hr on 3 consecutive days Reduce the medium by adding 10% neutralized Na2S·9H2O solution to a final concentration of 0.05% The medium should not give a heavy black precipitate; if it does the iron sediment was not adequately removed by filtering in the initial stages and the medium should be made again, making sure that the iron is removed by filtering

Use: For the cultivation and maintenance of Thermococcus pacificus DSM 10394 and Thermococcus gorgonarius DSM 10395.

Na2SO3 3.24g CaCl2 1.8g Yeast extract 1.0g

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Marine Thermococcus Medium 1021

KCl 0.55g

NaHCO3 0.16g

Ferric citrate 0.1g

KBr 0.08g

SrCl2 0.03g

H3BO3 0.02g

Na2HPO4 8.0mg

Na2SiO3 4.0mg

NaF 2.4mg

NH4NO3 1.6mg

pH 6.5 ± 0.2 at 25°C

Na2S·9H2O 1.0g

boiling Boil for 5 min Cool under an anaerobic gas mixture of 80%

N2 + 20% CO2 Adjust pH to 6.5 Distribute the medium into Hungate

tubes or serum bottles containing finely divided sulfur (0.5% w/v) Seal

the tubes or bottles under the same anaerobic gas used when cooling

the medium Sterilize the medium at 100°C for 3 hr on 3 consecutive

days Reduce the medium by adding 10% neutralized Na2S·9H2O

so-lution to a final concentration of 0.05% The medium should not give

a heavy black precipitate; if it does the iron sediment was not

adequate-ly removed by filtering in the initial stages and the medium should be

made again, making sure that the iron is removed by filtering

Use: For the cultivation and maintenance of Thermococcus stetteri

DSM 5262

NaCl 19.45g

MgCl2 8.8g

Sulfur 5.0g

Peptone 5.0g

Na2SO3 3.24g

CaCl2 1.8g

Yeast extract 1.0g

KCl 0.55g

NaHCO3 0.16g

Ferric citrate 0.1g

KBr 0.08g

SrCl2 0.03g

H3BO3 0.02g

Na2HPO4 8.0mg

Na2SiO3 4.0mg

NaF 2.4mg

NH4NO3 1.6mg

pH 5.8 ± 0.2 at 25°C

Na2S·9H2O 1.0g

Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Filter through normal filter paper An iron sedi-ment will collect in the filter Gently heat while stirring and bring to boiling Boil for 5 min Cool under an anaerobic gas mixture of N2 Ad-just pH to 5.8 Distribute the medium into Hungate tubes or serum bot-tles containing finely divided sulfur (0.5% w/v) Seal the tubes or bottles under the same anaerobic gas used when cooling the medium Sterilize the medium at 100°C for 3 hr on 3 consecutive days Reduce the medium by adding 10% neutralized Na2S·9H2O solution to a final concentration of 0.05% The medium should not give a heavy black precipitate; if it does the iron sediment was not adequately removed by filtering in the initial stages and the medium should be made again, making sure that the iron is removed by filtering

Use: For the cultivation and maintenance of Thermococcus celer DSM

2476

Na2SO3 3.24g CaCl2 1.8g Yeast extract 1.0g KCl 0.55g NaHCO3 0.16g Ferric citrate 0.1g KBr 0.08g SrCl2 0.03g

H3BO3 0.02g

Na2HPO4 8.0mg

Na2SiO3 4.0mg NaF 2.4mg

NH4NO3 1.6mg

pH 7.2 ± 0.2 at 25°C

Na2S·9H2O 1.0g

Na2S·9H2O solution, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Filter through normal filter paper An iron sedi-ment will collect in the filter Gently heat while stirring and bring to boiling Boil for 5 min Cool under an anaerobic gas mixture of N2 Ad-just pH to 7.2 Distribute the medium into Hungate tubes or serum

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bot-1022 Marine Thermococcus Medium

tles containing finely divided sulfur (0.5% w/v) Seal the tubes or

Use: For the cultivation and maintenance of Thermococcus profundus

DSM 9503, Thermococcus peptonophilus DSM 10343, Thermococcus

guaymasensis 11113, and Thermococcus aggregans DSM 12819

NaCl 19.45g

MgCl2 8.8g

Sulfur 5.0g

Peptone 5.0g

Na2SO3 3.24g

CaCl2 1.8g

Yeast extract 1.0g

KCl 0.55g

NaHCO3 0.16g

Ferric citrate 0.1g

KBr 0.08g

SrCl2 0.03g

H3BO3 0.02g

Na2HPO4 8.0mg

Na2SiO3 4.0mg

NaF 2.4mg

NH4NO3 1.6mg

pH 7.5 ± 0.2 at 25°C

Na2S·9H2O 1.0g

boiling Boil for 5 min Cool under an anaerobic gas mixture of N2

Ad-just pH to 7.5 Distribute the medium into Hungate tubes or serum

bot-tles containing finely divided sulfur (0.5% w/v) Seal the tubes or

Use: For the cultivation and maintenance of Thermococcus litoralis

DSM 5473, Thermococcus litoralis 5474, Thermococcus fumicolans

DSM 12820, and Thermococcus sibiricus DSM 12597

Marinithermus hydrothermalis Medium

NaCl 30.0g MgCl2·6H2O 4.18g MgSO4·7H2O 3.4g Yeast extract 1.0g Tryptone 1.0g KCl 0.33g

NH4Cl 0.25g

K2HPO4 0.14g CaCl2·2H2O 0.14g Fe(NH4)2(SO4)2·6H2O 10.0mg NiCl2·6H2O 0.5mg Na2Se3·5H2O 0.5mg Trace elements solution 10.0mL

pH 7.0 ± 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

Use: For the cultivation of Marinithermus hydrothermalis.

Marinitoga Medium

(DSMZ Medium 904) Composition per 1045.0mL:

Sea salts 30.0g PIPES 6.0g Yeast extract 1.0g Tryptone 1.0g Resazurin 0.5mg Glucose solution 25.0mL

L-Cysteine solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Glucose Solution:

Glucose 2.5g

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Marinitoga piezophila Medium 1023

Preparation of Glucose Solution: Add sucrose to

distilled/deion-ized water and bring volume to 25.0mL Mix thoroughly Sparge with

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

L -Cysteine Solution:

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 0.5g

Preparation of Medium: Prepare and dispense medium under 100%

N2 Add components, except glucose solution, L-cysteine-HCl·H2O

so-lution, and Na2S·9H2O solution, to distilled/deionized water and bring

volume to 1.0L Mix thoroughly Adjust pH to 7.0 Distribute into

anaer-obe tubes or bottles Autoclave for 15 min at 15 psi pressure–121°C

Aseptically and anaerobically add per liter, 50.0mL glucose solution,

10.0mL L-cysteine-HCl·H2O solution, and 10.0mL Na2S·9H2O Mix

thoroughly The final pH should be 7.0

Use: For the cultivation of Marinitoga camini and Caloranaerobacter

azorensis.

Marinitoga piezophila Medium

NaCl 30.0g

Yeast extract 5.0g

Trypticase™ 5.0g

MES 1.95g

NH4Cl 1.0g

Na-acetate 0.83g

K2HPO4 0.3g

KH2PO4 0.3g

MgCl2·6H2O 0.2g

CaCl2·2H2O 0.1g

KCl 0.1g

Resazurin 0.5mg

Maltose solution 100.0mL

Cysteine solution 10.0mL

pH 6.0 ± 0.2 at 25°C

Maltose Solution:

Maltose 4.96g

Preparation of Maltose Solution: Add maltose to

distilled/deion-ized water and bring volume to 100.0mL Mix thoroughly Sparge with

100% N2 Filter sterilize

Cysteine Solution:

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 Cool to 25°C

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

Preparation of Medium: Add components, except maltose solution, NaHCO3 solution, and Na2S·9H2O solution, to 880.0mL distilled/deion-ized water Mix thoroughly Sparge for 30 min with 100% N2 Adjust pH

to 6.0 with concentrated NaOH Distribute under 100% N2 into anaero-bic tubes or bottles Autoclave for 15 min at 15 psi pressure–121°C Cool

to 25°C Aseptically and anaerobically add 100.0mL sterile maltose so-lution, 10.0mL sterile Na2S·9H2O solution, and 10.0mL sterile cysteine solution per liter medium Mix thoroughly

Use: For the cultivation of Marinitoga piezophila.

Marinitoga piezophila Medium

NaCl 30.0g Sulfur 10.0g Yeast extract 5.0g Trypticase™ 5.0g MES 1.95g

NH4Cl 1.0g Na-acetate 0.83g

K2HPO4 0.3g

KH2PO4 0.3g MgCl2·6H2O 0.2g CaCl2·2H2O 0.1g KCl 0.1g Resazurin 0.5mg

Na2S·9H2O solution 10.0mL Cysteine solution 10.0mL

pH 6.0 ± 0.2 at 25°C

Cysteine Solution:

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 Cool to 25°C

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

Preparation of Sulfur: Sterilize sulfur by steaming for 3 hr on each

of 3 successive days

Preparation of Medium: Add components, except sulfur, cysteine solution, and Na2S·9H2O solution, to 980.0mL distilled/deionized wa-ter Mix thoroughly Sparge for 30 min with 100% N2 Adjust pH to 6.0

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1024 Marinobacter lutaoensis Medium

with concentrated NaOH Distribute under 100% N2 into anaerobic

tubes or bottles containing appropriate amounts of sterile sulfur (1g

steam-sterilized sulfur per 100mL medium) Autoclave for 20 min at

110°C Cool to room temperature Aseptically and anaerobically add

10.0mL sterile Na2S·9H2O solution and 10.0mL sterile cysteine

solu-tion per liter medium Mix thoroughly

Use: For the cultivation of Marinitoga piezophila.

Marinobacter lutaoensis Medium

(DSMZ Medium 1066) Composition per liter:

Peptone 4.0g

Yeast extract 2.0g

NaCl 25.0g

MgCl2·6H2O 2.0g

pH 7.0 ± 0.2 at 25°C

wa-ter and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0 Distribute

into tubes or flasks Gently heat while stirring and bring to boiling Mix

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

Use: For the cultivation of Marinobacter lutaoensis.

Marinobacter Medium

NaCl 6.0g

NH4Cl 1.0g

Na-acetate 1.0g

MgSO4·7H2O 0.2g

KCl 0.1g

KH2PO4 0.1g

Peptone 0.1g

CaCl2·2H2O 0.04g

Trace elements solution SL-7 1.0mL

Vitamin solution, concentrated 1.0mL

pH 7.2 ± 0.2 at 25°C

Trace Elements Solution SL-7:

FeCl2·7H2O 1.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

Na2MoO4·2H2O 36.0mg

NiCl2·6H2O 24.0mg

H3BO3 62.0mg

CuCl2·2H2O 17.0mg

HCl (25% solution) 6.5mL

Preparation of Trace Elements Solution SL-7: Add FeCl2·7H2O

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

Vitamin Solution, Concentrated:

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, Concentrated: Add compo-nents to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Filter sterilize

Use: For the cultivation of Marionobacter sp.

Marinobacter Medium

NaCl 11.7g MgSO4 7.85g TRIS 6.0g Yeast extract 5.0g Peptone 5.0g

NH4Cl 3.0g CaCl2 1.47g KCl 0.74g

pH 7.8 ± 0.2 at 25°C

Use: For the cultivation of Marinobacter sp.

Marinococcus albus Agar

(LMG Medium 212) Compositionper liter:

NaCl 81.0g Agar 15.0g Yeast 10.0g MgSO4·7H2O 9.6g MgCl2·6H2O 7.0g Proteose peptone 5.0g KCl 2.0g Glucose 1.0g CaCl2 0.36g NaB 226.0mg NaHCO3 60.0mg

pH 7.2 ± 0.2 at 25°C

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

Use: For the cultivation and maintenance of Marinococcus albus.

Marinococcus albus Medium

NaCl 81.0g Yeast extract 10.0g MgSO4·7H2O 9.6g MgCl2·6H2O 7.0g Proteose peptone No 3 5.0g

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