Marine Flagellate Medium with B-Vitamins 1015 sterile CaCl 2 ·2H 2 O 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 Composition per 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 Composition per liter: Agar 15.0g Pancreatic digest of casein 2.0g Beef extract 0.5g Yeast extract 0.5g Sodium acetate 0.2g Seawater 500.0mL 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 Vibrio ordalii. Marine Cytophaga Medium C Composition per liter: Agar 15.0g Pancreatic digest of casein 2.0g 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 Composition per liter: Solution A 980.0mL Solution B 10.0mL Solution C 10.0mL pH 7.8 ± 0.2 at 25°C Solution A: Composition per 980.0mL: NaCl 25.0g DL-Sodium lactate 2.0g MgSO 4 ·7H 2 O 2.0g Na 2 SO 4 1.0g NH 4 Cl 1.0g Yeast extract 1.0g K 2 HPO 4 0.5g CaCl 2 ·2H 2 O 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% N 2 . Solution B: Composition per 10.0mL: FeSO 4 ·7H 2 O 0.5g Preparation of Solution B: Add FeSO 4 ·7H 2 O to distilled/deion- ized water and bring volume to 10.0mL. Mix thoroughly. Solution C: Composition per 10.0mL: 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 Composition per 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 Composition per liter: Seawater 990.0mL Vitamin solution 10.0mL Vitamin Solution: Composition per 100.0mL: Thiamine·HCl 0.15g Calcium D-(+)-pantothenate 0.05g Nicotinamide 0.05g © 2010 by Taylor and Francis Group, LLC 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) Composition per liter: Agar 8.0g Glucose 2.0g Pancreatic digest of casein 1.0g Yeast extract 1.0g L-Cysteine·HCl·H 2 O 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 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 under 97% N 2 + 3% H 2 . 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) Composition per liter: Glucose 2.0g Pancreatic digest of casein 1.0g Yeast extract 1.0g L-Cysteine·HCl·H 2 O 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 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 under 97% N 2 + 3% H 2 . 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 Composition per 1003.0mL: NaCl 21.0g MgCl 2 ·6H 2 O 3.0g NaCl 1.0g KCl 0.5g MgCl 2 ·6H 2 O 0.5g NH 4 Cl 0.4g Sodium acetate·3H 2 O 0.4g KH 2 PO 4 0.2g CaCl 2 ·2H 2 O 0.1g NaHCO 3 solution 60.0mL 2-Propanol 5.0mL Na 2 S·9H 2 O 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: Composition per 100.0mL: FeSO 4 ·7H 2 O 1400.0mg ZnSO 4 ·7H 2 O 145.0mg CoCl 2 ·6H 2 O 120.0mg MnCl 2 ·4H 2 O 100.0mg NiCl 2 ·6H 2 O 50.0mg H 3 BO 3 6.0mg CuSO 4 ·5H 2 O 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% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Selenite-Molybdate-Tungstate Solution: Composition per liter: NaOH 0.2g Na 2 MoO 4 ·2H 2 O 40.0mg Na 2 WO 4 ·2H 2 O 33.0mg Na 2 SeO 3 ·2H 2 O 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% N 2 . Autoclave for 15 min at 15 psi pres- sure–121°C. NaHCO 3 Solution: Composition per liter: NaHCO 3 84.0g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Na 2 S·9H 2 O Solution: Composition per 100.0mL: Na 2 S·9H 2 O 2.5g NaOH 1 pellet Preparation of Na 2 S·9H 2 O Solution: Bring 100.0mL of dis- tilled/deionized water to boiling. Cool to room temperature while sparging with 100%N 2 . Dissolve 1 pellet of NaOH in the anaerobic wa- ter. Weigh out a little more than 2.5g of Na 2 S·9H 2 O. Briefly rinse the crystals in distilled/deionized water. Dry the crystals by blotting on pa- per towels or filter paper. Add 2.5g of washed Na 2 S·9H 2 O crystals to 100.0mL of anaerobic NaOH solution. Distribute into serum bottles fit- ted with butyl rubber stoppers and aluminum seals. Do not grease stop- pers. Pressurize to 60kPa with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Store at room temperature in an anaerobic chamber. Preparation of 2-Propanol: Filter sterilize 10.0mL of 2-propanol. Sparge with 100% N 2 . © 2010 by Taylor and Francis Group, LLC Marine Methylotroph Broth 1017 Vitamin Solution: Composition per liter: 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% N 2 . Thiamine Solution: Composition per liter: 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% N 2 . Cyanocobalamin Solution: Composition per liter: 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% N 2 . Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 . Add components, except NaHCO 3 solution, 2-propa- nol, Na 2 S·9H 2 O 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% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 60.0mL of sterile NaHCO 3 solution, 5.0mL of sterile 2-propanol, 3.0mL of sterile Na 2 S·9H 2 O 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 anaerobically distribute into sterile tubes or bottles. Use: For the cultivation of marine Methanogenium species. Marine Methanol Medium Composition per liter: NaCl 20.0g (NH 4 ) 2 SO 4 2.0g K 2 HPO 4 2.0g KH 2 PO 4 1.0g MgSO 4 ·7H 2 O 0.3g Methanol 10.0mL Vitamin B 12 solution 10.0mL Trace metals solution 1.0mL pH 7.0 ± 0.2 at 25°C Vitamin B 12 Solution: Composition per 100.0mL: Vitamin B 12 10.0μg Preparation of Vitamin B 12 Solution: Add the vitamin B 12 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: Composition per liter: ZnSO 4 ·7H 2 O 1.4g MnSO 4 ·H 2 O 0.84g FeSO 4 ·7H 2 O 0.28g CuSO 4 ·5H 2 O 0.25g Na 2 MoO 4 ·2H 2 O 0.24g CoCl 2 ·6H 2 O 0.24g CaCl 2 ·2H 2 O 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 B 12 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 B 12 solution and filter-sterilized methanol. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Methylophaga thalassica. Marine Methylotroph Agar Composition per 1003.0mL: Agarose 12.0g Bis (2-hydroxyethyl) aminotris (hydroxy- methyl) methane 2.0g KH 2 PO 4 0.14g Ferric ammonium citrate 0.06g Methanol 2.0mL Vitamin B 12 solution 1.0mL pH 7.4 ± 0.2 at 25°C Vitamin B 12 Solution: Composition per 100.0mL: Vitamin B 12 0.1mg Preparation of Vitamin Solution: Add vitamin B 12 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 B 12 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 B 12 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 Composition per 1003.0mL: Bis (2-hydroxyethyl) aminotris (hydroxy- methyl) methane 2.0g KH 2 PO 4 0.14g Ferric ammonium citrate 0.06g Methanol 2.0mL Vitamin B 12 solution 1.0mL pH 7.4 ± 0.2 at 25°C Vitamin B 12 Solution: Composition per 100.0mL: Vitamin B 12 0.1mg © 2010 by Taylor and Francis Group, LLC 1018 Marine Oxidation Fermentation HiVeg Medium Preparation of Vitamin Solution: Add vitamin B 12 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 B 12 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 B 12 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) Composition per liter: NaCl 9.7g MnCl 2 4.4g Agar 3.0g Na 2 SO 4 1.6g Plant hydrolysate 1.0g CaCl 2 0.9g (NH 4 ) 2 SO 4 0.5g Tris hydroxymethyl aminomethane 0.5g KCl 0.275g Yeast extract 0.1g NaHCO 3 0.08g KBr 0.04g SrCl 2 0.017g H 3 BO 3 0.011g Phenol Red 0.01g Na 2 HPO 4 4.0mg Sodium silicate 2.0mg NaFl 1.2mg NH 4 NO 3 0.8mg pH 7.3 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Hi- Media. 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. 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) Composition per liter: Peptone 10.0g Succinic acid 1.0g (NH 4 ) 2 SO 4 1.0g MgSO 4 ·7H 2 O 1.0g FeCl 3 ·6H 2 O 2.0mg MnSO 4 ·H 2 O 2.0mg Synthetic seawater 1.0L pH 6.8 ± 0.2 at 25°C Synthetic Seawater: Composition per liter: NaCl 27.5g MgCl 2 5.0g MgSO 4 ·7H 2 O 2.0g KCl 1.0g CaCl 2 0.5g FeSO 4 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 Composition per liter: NaCl 20.0g Peptone 10.0g MgSO 4 ·7H 2 O 2.0g (NH 4 ) 2 SO 4 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 Composition per liter: Agar 15.0g Nutrient broth 8.0g Yeast extract 5.0g Salt solution 1.0L Salt Solution: Composition per liter: NaCl 12.86g MgCl 2 2.48g KCl 0.75g CaCl 2 0.56g Fe(SO 4 ) 2 (NH 4 ) 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 Composition per liter: Yeast extract 10.0g Malt extract 4.0g Glucose 4.0g Seawater 750.0mL © 2010 by Taylor and Francis Group, LLC Marine Salts Medium for Sporosarcina halophila 1019 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. Use: For the cultivation and maintenance of Rhodococcus marinona- scens. Marine Rhodopseudomonas Medium Composition per liter: NaCl 30.4g Yeast extract 1.0g Disodium succinate 1.0g KH 2 PO 4 0.5g MgSO 4 ·7H 2 O 0.4g NH 4 Cl 0.4g CaCl 2 ·2H 2 O 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: Composition per liter: H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g MnCl 2 ·4H 2 O 0.03g Na 2 MoO 4 ·H 2 O 0.03g NiCl 2 ·6H 2 O 0.02g CuCl 2 ·2H 2 O 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. 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. Use: For the cultivation and maintenance of Rhodopseudomonas marina. Marine Rhodopseudomonas Medium Composition per liter: NaCl 30.0g Peptone 2.5g Yeast extract 2.5g pH 7.0 ± 0.4 at 25°C Preparation of Medium: Add components to distilled/deionized 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 O 2 , and screw down the cap tightly. Use: For the cultivation of Rhodopseudomonas marina. Marine Salts Medium Composition per liter: NaCl 81.0g Yeast extract 10.0g MgSO 4 9.6g MgCl 2 7.0g Proteose peptone No.3 5.0g KCl 2.0g Glucose 1.0g CaCl 2 0.36g NaHCO 3 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: Composition per 100.0mL: 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% N 2 . 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% N 2 . Add the thioglyco- late. Adjust pH to 7.5 with 10N NaOH. Dispense into tubes or bottles under 100% N 2 . 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 Composition per liter: 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. © 2010 by Taylor and Francis Group, LLC 1020 Marine Spirochete Medium Marine Spirochete Medium Composition per liter: 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% N 2 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) Composition per liter: NaCl 19.45g MgCl 2 8.8g Sulfur 5.0g Peptone 5.0g Na 2 SO 3 3.24g CaCl 2 1.8g Yeast extract 1.0g KCl 0.55g NaHCO 3 0.16g Ferric citrate 0.1g KBr 0.08g SrCl 2 0.03g H 3 BO 3 0.02g Na 2 HPO 4 8.0mg Na 2 SiO 3 4.0mg NaF 2.4mg NH 4 NO 3 1.6mg Na 2 S·9H 2 O solution 0.5mL pH 6.0 ± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 1.0g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. Preparation of Medium: Add components, except sulfur and Na 2 S·9H 2 O 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 N 2 . 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 Na 2 S·9H 2 O 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. Marine Thermococcus Medium (DSMZ Medium 760) Composition per liter: NaCl 19.45g MgCl 2 8.8g Sulfur 5.0g Peptone 5.0g Na 2 SO 3 3.24g CaCl 2 1.8g Yeast extract 1.0g KCl 0.55g NaHCO 3 0.16g Ferric citrate 0.1g KBr 0.08g SrCl 2 0.03g H 3 BO 3 0.02g Na 2 HPO 4 8.0mg Na 2 SiO 3 4.0mg NaF 2.4mg NH 4 NO 3 1.6mg Na 2 S·9H 2 O solution 0.5mL pH 6.5 ± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 1.0g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. Preparation of Medium: Add components, except sulfur and Na 2 S·9H 2 O 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 N 2 . 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 Na 2 S·9H 2 O 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. Marine Thermococcus Medium (DSMZ Medium 760) Composition per liter: NaCl 19.45g MgCl 2 8.8g Sulfur 5.0g Peptone 5.0g Na 2 SO 3 3.24g CaCl 2 1.8g Yeast extract 1.0g © 2010 by Taylor and Francis Group, LLC Marine Thermococcus Medium 1021 KCl 0.55g NaHCO 3 0.16g Ferric citrate 0.1g KBr 0.08g SrCl 2 0.03g H 3 BO 3 0.02g Na 2 HPO 4 8.0mg Na 2 SiO 3 4.0mg NaF 2.4mg NH 4 NO 3 1.6mg Na 2 S·9H 2 O solution 0.5mL pH 6.5 ± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 1.0g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. Preparation of Medium: Add components, except sulfur and Na 2 S·9H 2 O 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 80% N 2 + 20% CO 2 . 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 Na 2 S·9H 2 O 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. Marine Thermococcus Medium (DSMZ Medium 760) Composition per liter: NaCl 19.45g MgCl 2 8.8g Sulfur 5.0g Peptone 5.0g Na 2 SO 3 3.24g CaCl 2 1.8g Yeast extract 1.0g KCl 0.55g NaHCO 3 0.16g Ferric citrate 0.1g KBr 0.08g SrCl 2 0.03g H 3 BO 3 0.02g Na 2 HPO 4 8.0mg Na 2 SiO 3 4.0mg NaF 2.4mg NH 4 NO 3 1.6mg Na 2 S·9H 2 O solution 0.5mL pH 5.8 ± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 1.0g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. Preparation of Medium: Add components, except sulfur and Na 2 S·9H 2 O 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 N 2 . 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 Na 2 S·9H 2 O 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. Marine Thermococcus Medium (DSMZ Medium 760) Composition per liter: NaCl 19.45g MgCl 2 8.8g Sulfur 5.0g Peptone 5.0g Na 2 SO 3 3.24g CaCl 2 1.8g Yeast extract 1.0g KCl 0.55g NaHCO 3 0.16g Ferric citrate 0.1g KBr 0.08g SrCl 2 0.03g H 3 BO 3 0.02g Na 2 HPO 4 8.0mg Na 2 SiO 3 4.0mg NaF 2.4mg NH 4 NO 3 1.6mg Na 2 S·9H 2 O solution 0.5mL pH 7.2 ± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 1.0g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. Preparation of Medium: Add components, except sulfur and Na 2 S·9H 2 O 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 N 2 . Ad- just pH to 7.2. Distribute the medium into Hungate tubes or serum bot- © 2010 by Taylor and Francis Group, LLC 1022 Marine Thermococcus Medium 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 Na 2 S·9H 2 O 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 profundus DSM 9503, Thermococcus peptonophilus DSM 10343, Thermococcus guaymasensis 11113, and Thermococcus aggregans DSM 12819. Marine Thermococcus Medium (DSMZ Medium 760) Composition per liter: NaCl 19.45g MgCl 2 8.8g Sulfur 5.0g Peptone 5.0g Na 2 SO 3 3.24g CaCl 2 1.8g Yeast extract 1.0g KCl 0.55g NaHCO 3 0.16g Ferric citrate 0.1g KBr 0.08g SrCl 2 0.03g H 3 BO 3 0.02g Na 2 HPO 4 8.0mg Na 2 SiO 3 4.0mg NaF 2.4mg NH 4 NO 3 1.6mg Na 2 S·9H 2 O solution 0.5mL pH 7.5 ± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 1.0g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. Preparation of Medium: Add components, except sulfur and Na 2 S·9H 2 O 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 N 2 . 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 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 Na 2 S·9H 2 O 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 litoralis DSM 5473, Thermococcus litoralis 5474, Thermococcus fumicolans DSM 12820, and Thermococcus sibiricus DSM 12597. Marinithermus hydrothermalis Medium (DSMZ Medium 973) Composition per liter: NaCl 30.0g MgCl 2 ·6H 2 O 4.18g MgSO 4 ·7H 2 O 3.4g Yeast extract 1.0g Tryptone 1.0g KCl 0.33g NH 4 Cl 0.25g K 2 HPO 4 0.14g CaCl 2 ·2H 2 O 0.14g Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 10.0mg NiCl 2 ·6H 2 O 0.5mg Na2Se 3 ·5H 2 O 0.5mg Trace elements solution 10.0mL pH 7.0 ± 0.2 at 25°C Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g H 3 BO 3 0.01g Na 2 MoO 4 ·4H 2 O 0.01g CuSO 4 ·5H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Preparation of Medium: 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 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 Na 2 S·9H 2 O solution 10.0mL L-Cysteine solution 10.0mL pH 7.0 ± 0.2 at 25°C Glucose Solution: Composition per 25.0mL: Glucose 2.5g © 2010 by Taylor and Francis Group, LLC 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% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. L-Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.5g Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.5g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components, except glucose solution, L-cysteine-HCl·H 2 O so- lution, and Na 2 S·9H 2 O 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·H 2 O solution, and 10.0mL Na 2 S·9H 2 O. Mix thoroughly. The final pH should be 7.0. Use: For the cultivation of Marinitoga camini and Caloranaerobacter azorensis. Marinitoga piezophila Medium (DSMZ Medium 945) Composition per liter: NaCl 30.0g Yeast extract 5.0g Trypticase™ 5.0g MES 1.95g NH 4 Cl 1.0g Na-acetate 0.83g K 2 HPO 4 0.3g KH 2 PO 4 0.3g MgCl 2 ·6H 2 O 0.2g CaCl 2 ·2H 2 O 0.1g KCl 0.1g Resazurin 0.5mg Maltose solution 100.0mL Na 2 S·9H 2 O solution 10.0mL Cysteine solution 10.0mL pH 6.0 ± 0.2 at 25°C Maltose Solution: Composition per 100.0mL: 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% N 2 . Filter sterilize. Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.3g Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Sparge with N 2 . 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, NaHCO 3 solution, and Na 2 S·9H 2 O solution, to 880.0mL distilled/deion- ized water. Mix thoroughly. Sparge for 30 min with 100% N 2 . Adjust pH to 6.0 with concentrated NaOH. Distribute under 100% N 2 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 Na 2 S·9H 2 O solution, and 10.0mL sterile cysteine solution per liter medium. Mix thoroughly. Use: For the cultivation of Marinitoga piezophila. Marinitoga piezophila Medium (DSMZ Medium 945) Composition per liter: NaCl 30.0g Sulfur 10.0g Yeast extract 5.0g Trypticase™ 5.0g MES 1.95g NH 4 Cl 1.0g Na-acetate 0.83g K 2 HPO 4 0.3g KH 2 PO 4 0.3g MgCl 2 ·6H 2 O 0.2g CaCl 2 ·2H 2 O 0.1g KCl 0.1g Resazurin 0.5mg Na 2 S·9H 2 O solution 10.0mL Cysteine solution 10.0mL pH 6.0 ± 0.2 at 25°C Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.3g Preparation of L-Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Sparge with N 2 . 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 Na 2 S·9H 2 O solution, to 980.0mL distilled/deionized wa- ter. Mix thoroughly. Sparge for 30 min with 100% N 2 . Adjust pH to 6.0 © 2010 by Taylor and Francis Group, LLC 1024 Marinobacter lutaoensis Medium with concentrated NaOH. Distribute under 100% N 2 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 Na 2 S·9H 2 O 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 MgCl 2 ·6H 2 O 2.0g pH 7.0 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized 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 (DSMZ Medium 941) Composition per liter: NaCl 6.0g NH 4 Cl 1.0g Na-acetate 1.0g MgSO 4 ·7H 2 O 0.2g KCl 0.1g KH 2 PO 4 0.1g Peptone 0.1g CaCl 2 ·2H 2 O 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: Composition per liter: FeCl 2 ·7H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 62.0mg CuCl 2 ·2H 2 O 17.0mg HCl (25% solution) 6.5mL Preparation of Trace Elements Solution SL-7: Add FeCl 2 ·7H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Vitamin Solution, Concentrated: Composition per 100.0mL: Pyridoxine-HCl 10.0mg Thiamine-HCl·2H 2 O 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg D-Ca-pantothenate 5.0mg p-Aminobenzoic acid 5.0mg Lipoic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg Preparation of Vitamin Solution, Concentrated: Add compo- nents to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. 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 of Marionobacter sp. Marinobacter Medium (DSMZ Medium 970) Composition per liter: NaCl 11.7g MgSO 4 7.85g TRIS 6.0g Yeast extract 5.0g Peptone 5.0g NH 4 Cl 3.0g CaCl 2 1.47g KCl 0.74g pH 7.8 ± 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 of Marinobacter sp. Marinococcus albus Agar (LMG Medium 212) Composition per liter: NaCl 81.0g Agar 15.0g Yeast 10.0g MgSO 4 ·7H 2 O 9.6g MgCl 2 ·6H 2 O 7.0g Proteose peptone 5.0g KCl 2.0g Glucose 1.0g CaCl 2 0.36g NaB 226.0mg NaHCO 3 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 Composition per liter: NaCl 81.0g Yeast extract 10.0g MgSO 4 ·7H 2 O 9.6g MgCl 2 ·6H 2 O 7.0g Proteose peptone No. 3 5.0g © 2010 by Taylor and Francis Group, LLC . anaerobically add 60.0mL of sterile NaHCO 3 solution, 5.0mL of sterile 2-propanol, 3.0mL of sterile Na 2 S·9H 2 O solution, 1.0mL of sterile cyanocobala- min solution, 1.0mL of sterile selenite-molybdate-tungstate. 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. 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%N 2 . Dissolve 1 pellet of NaOH in the anaerobic