Marine Agar (DSMZ Medium 123) Composition per liter: Agar ..................................................................15.0g Tryptone............................................................10.0g Peptone ...............................................................5.0g Yeast extract ........................................................1.0g Synthetic seawater ............................................. 1.0L pH 7.8 ± 0.2 at 25°C Synthetic Seawater Composition per liter: NaCl ..................................................................24.0g MgCl2•6H2O .....................................................11.0g Na2SO4................................................................4.0g CaCl2•6H2O.........................................................2.0g KCl......................................................................0.7g KBr......................................................................0.1g SrCl2•6H2O .......................................................0.04g H3BO3 ...............................................................0.03g NaSiO3•9H2O...................................................5.0mg NaF...................................................................3.0mg NH4NO3 ...........................................................2.0mg Fe3PO4•4H2O ...................................................1.0mg Preparation of Synthetic Seawater: Add components to distilled water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add agar, tryptone, peptone, and yeast extract to synthetic seawater and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.8. 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 Halobacillus halophilus, Halomonas spp., Vibrio harveyi,Cobetia marina, and Ruegeria atlantica.
Marine Agar 2216(DSMZ Medium 604) Composition per liter: NaCl ................................................................19.45g Agar ..................................................................15.0g MgCl2..................................................................8.8g 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 7.6 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from BD Diagnostic Systems. 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 Hyphomonas spp., Oceanospirillum spp., Hyphomicrobium indicum, Psychroflexus gondwanensis=Flavobacterium gondwanense, Salegentibacter salegens=Flavobacterium salegens, Psychromonas antarctica, Sulfitobacter mediterraneus, Thalassomonas viridans,Vibrio spp., Marinospirillum minutulum=Oceanospirillum minutulum, Terasakiella pusilla=Oceanospirillum pusillum, Pseudoalteromonas atlantica=Alteromonas atlantica, Pseudomonas atlantica,Roseobacter spp., Erythrobacter longus, Pseudospirillum japonicum=Oceanospirillum japonicum,Marinobacter hydrocarbonoclasticus (Pseudomonas nautica), Psychrobacter spp., and Moritella japonica. For the isolation, cultivation, and maintenance of a wide variety of heterotrophic marine bacteria.
Marine Agar with Biphenyl Composition per liter: NaCl ................................................................19.45g Agar ..................................................................15.0g MgCl2..................................................................8.8g 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 Biphenyl...........................................................1.0mg pH 7.6 ± 0.2 at 25°C Preparation of Medium: Add components, except biphenyl, 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. After agar solidifies, aseptically add a few crystals of biphenyl to each plate. Use: For the cultivation and maintenance of biphenyl-utilizing marine bacteria, such as Cycloclasticus pugetii. Marine Agar with ι-Carrageenan Composition per 1070.0mL: Solution A.......................................................... 1.0L Solution B ..................................................... 60.0mL Solution C ..................................................... 10.0mL pH 7.2 ± 0.2 at 25°C Solution A: Composition per liter: NaCl ..................................................................25.0g Agar ..................................................................15.0g MgSO4•7H2O......................................................5.0g Casamino acids ...................................................2.5g ι−Carrageenan.....................................................2.5g NaNO3.................................................................2.0g CaCl2•2H2O.........................................................0.2g KCl......................................................................0.1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Solution B: Composition per 100.0mL: Na2HPO4•2H2O.................................................3.56g Preparation of Solution B: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution C: Composition per 100.0mL: FeSO4•7H2O........................................................0.3g Preparation of Solution C: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of ATCC strain 43554. Marine Agar with κ- and λ-Carrageenan Composition per 1070.0mL: Solution A.......................................................... 1.0L Solution B..................................................... 60.0mL Solution C..................................................... 10.0mL pH 7.2 ± 0.2 at 25°C Solution A: Composition per liter: NaCl ..................................................................25.0g Agar ..................................................................15.0g MgSO4•7H2O......................................................5.0g Casamino acids ...................................................2.5g NaNO3 ................................................................2.0g κ-Carrageenan...................................................1.25g λ-Carrageenan...................................................1.25g CaCl2•2H2O ........................................................0.2g KCl......................................................................0.1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Solution B: Composition per 100.0mL: Na2HPO4•2H2O.................................................3.56g Preparation of Solution B: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution C: Composition per 100.0mL: FeSO4•7H2O........................................................0.3g Preparation of Solution C: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Pseudomonas carrageenovora. Marine Agar with Naphthalene Composition per liter: NaCl ................................................................19.45g Agar ..................................................................15.0g MgCl2..................................................................8.8g 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 Naphthalene ........................................................1mg pH 7.6 ± 0.2 at 25°C Preparation of Medium: Add components, except naphthalene, 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. After agar solidifies, aseptically add a few crystals of naphthalene to each plate. Use: For the cultivation and maintenance of naphthalene-utilizing marine bacteria Marine Agar with Sulfur (ATCC Medium 1922) Composition per liter: NaCl ................................................................19.45g Sulfur ................................................................10.0g MgCl2..................................................................8.8g 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 7.6 ± 0.2 at 25°C Preparation of Sulfur: Autoclave sulfur for 15 min at 0 psi pressure–100°C on three successive days. Preparation of Medium: Prepare anaerobically under a gas phase of 80% N2 + 10% CO2 + 10% H2. Add components, except sulfur, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50–55°C. Aseptically add 10.0g of sterile sulfur. Mix thoroughly. Aseptically and anaerobically, under a gas phase of 80% N2 + 10% CO2 + 10% H2, distribute into sterile tubes. Use: For the cultivation and maintenance of Thermococcus litoralis. Marine Ameba Medium Composition per liter: Agar ..................................................................10.0g Malt extract .........................................................0.1g Yeast extract........................................................0.1g Artificial seawater.............................................. 1.0L Artificial Seawater: Composition per liter: NaCl ..................................................................27.5g MgSO4 •7H2O...................................................6.78g MgCl2•6H2O .....................................................5.38g KCl....................................................................0.72g NaHCO3............................................................. 0.2g CaCL2•2H2O.......................................................1.4g Preparation of Artificial Seawater: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to artificial seawater 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 Cochliopodium clarum, Heteramoeba clara, Ling***oeba leei, Paramoeba pemaquidensis, and Vannella species. Marine Broth 2216 (LMG Medium 164) Composition per liter: NaCl ................................................................19.45g MgCl2..................................................................8.8g 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 7.6 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Diagnostic Systems. 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 of Vibrio liquefaciens and for the isolation, cultivation, and maintenance of a wide variety of heterotrophic marine bacteria. Marine Broth with Biphenyl Composition per liter: NaCl ................................................................19.45g MgCl2..................................................................8.8g 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 Biphenyl ...........................................................1.0mg pH 7.6 ± 0.2 at 25°C Preparation of Medium: Add components, except biphenyl, 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. Aseptically add a few crytals of biphenyl to each tube or flask. Use: For the cultivation of biphenyl-utilizing marine bacteria. Marine Broth with ι-Carrageenan Composition per 1070.0mL: Solution A.......................................................... 1.0L Solution B ..................................................... 60.0mL Solution C ..................................................... 10.0mL pH 7.2 ± 0.2 at 25°C Solution A: Composition per liter: NaCl ..................................................................25.0g MgSO4•7H2O......................................................5.0g Casamino acids ...................................................2.5g ι-Carrageenan .....................................................2.5g NaNO3 ................................................................2.0g CaCl2•2H2O ........................................................0.2g KCl......................................................................0.1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Solution B: Composition per 100.0mL: Na2HPO4•2H2O.................................................3.56g Preparation of Solution B: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution C: Composition per 100.0mL: FeSO4•7H2O........................................................0.3g Preparation of Solution C: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of ATCC strain 43554. Marine Broth with κ- and λ-Carrageenan Composition per 1070.0mL: Solution A.......................................................... 1.0L Solution B..................................................... 60.0mL Solution C..................................................... 10.0mL pH 7.2 ± 0.2 at 25°C Solution A: Composition per liter: NaCl ..................................................................25.0g MgSO4•7H2O......................................................5.0g Casamino acids ...................................................2.5g NaNO3 ................................................................2.0g κ-Carrageenan...................................................1.25g λ-Carrageenan...................................................1.25g CaCl2•2H2O ........................................................0.2g KCl......................................................................0.1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Solution B: Composition per 100.0mL: Na2HPO4•2H2O.................................................3.56g Preparation of Solution B: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution C: Composition per 100.0mL: FeSO4•7H2O........................................................0.3g Preparation of Solution C: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Pseudomonas carrageenovora. Marine Broth with Naphthalene Composition per liter: NaCl ................................................................19.45g MgCl2..................................................................8.8g 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 Naphthalene ........................................................1mg pH 7.6 ± 0.2 at 25°C Preparation of Medium: Add components, except biphenyl, 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. Aseptically add a few crytals of naphthalene to each tube or flask. Use: For the cultivation of naphthalene-utilizing marine bacteria. Marine Broth with Sulfur Composition per liter: NaCl ................................................................19.45g Sulfur ................................................................10.0g MgCl2..................................................................8.8g 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 7.6 ± 0.2 at 25°C Preparation of Sulfur: Autoclave for 15 min at 0 psi pressure–100°C on three successive days. Preparation of Medium: Prepare anaerobically under a gas phase of 80% N2 + 10% CO2 + 10% H2. Add components, except sulfur, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 10.0g of sulfur. Mix thoroughly. Aseptically and anaerobically, under a gas phase of 80% N2 + 10% CO2 + 10% H2, distribute into sterile tubes. Use: For the cultivation of Thermococcus litoralis. Marine Caulobacter Medium Composition per liter: Proteose peptone...............................................10.0g Yeast extract........................................................3.0g Artificial seawater.............................................. 1.0L pH 7.2–7.4 at 25°C Artificial Seawater: Composition per liter: Commercially available marine aquarium salts mixture ...........................variable Preparation of Artificial Seawater: Add commercially available marine aquarium salts mixture to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Combine components. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Caulobacter halobacteroides and Caulobacter maris. Marine Chlorobiaceae Medium 2 Composition per 1051.0mL: Solution 1.................................................... 950.0mL Na2S•9H2O solution...................................... 60.0mL NaHCO3 solution .......................................... 40.0mL Vitamin B12 solution ....................................... 1.0mL pH 6.8 ± 0.2 at 25°C Solution 1: Composition per 950.0mL: NaCl ..................................................................20.0g MgSO4•7H2O......................................................3.0g KH2PO4...............................................................1.0g NH4Cl .................................................................0.5g CaCl2•2H2O.......................................................0.05g Trace elements solution SL-8 ......................... 1.0mL Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Trace Elements Solution SL-8: Composition per liter: Disodium EDTA .................................................5.2g FeCl2•4H2O.........................................................1.5g CoCl2•6H2O ......................................................0.19g MnCl2•4H2O .......................................................0.1g ZnCl2.................................................................0.07g H3BO3 ...............................................................0.06g NaMoO4•2H2O..................................................0.04g CuCl2•2H2O ......................................................0.02g NiCl2•6H20........................................................0.02g Preparation of Trace Elements Solution SL8: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Na2S•9H2O Solution: Composition per 100.0mL: Na2S•9H2O..........................................................5.0g Preparation of Na2S•9H2O Solution: Add Na2S•9H2O to distilled/deionized water and bring volume to 100.0mL. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ..............................................................5.0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12 ......................................................2.0mg Preparation of Vitamin B12 Solution: Add vitamin B12 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: To 950.0mL of cooled, sterile solution 1, aseptically add 60.0mL of sterile Na2S•9H2O solution, 40.0mL of sterile NaHCO3 solution, and 1.0mL of sterile vitamin B12 solution. Mix thoroughly. Adjust pH to 6.8 with sterile H2SO4 or Na2CO3 . Aseptically distribute into sterile 50.0mL or 100.0mL bottles with metal screw-caps and rubber seals. Completely fill bottles with medium except for a pea-sized air bubble. Use: For the isolation and cultivation of marine members of the Chlorobiaceae. Marine Chromatiaceae Medium 2 Composition per 1051.0mL: Solution 1.................................................... 950.0mL Na2S•9H2O solution...................................... 60.0mL NaHCO3 solution.......................................... 40.0mL Vitamin B12 solution ....................................... 1.0mL pH 7.3 ± 0.2 at 25°C Solution 1: Composition per 950.0mL: NaCl ..................................................................20.0g MgSO4•7H2O......................................................3.0g KH2PO4...............................................................1.0g NH4Cl .................................................................0.5g CaCl2•2H2O ......................................................0.05g Trace elements solution SL-8 ......................... 1.0mL Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Trace Elements Solution SL-8: Composition per liter: Disodium EDTA.................................................5.2g FeCl2•4H2O.........................................................1.5g CoCl2•6H2O ......................................................0.19g MnCl2•4H2O .......................................................0.1g ZnCl2.................................................................0.07g H3BO3 ...............................................................0.06g NaMoO4•2H2O..................................................0.04g CuCl2•2H2O ......................................................0.02g NiCl2•6H20........................................................0.02g Preparation of Trace Elements Solution SL8: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Na2S•9H2O Solution: Composition per 100.0mL: Na2S•9H2O..........................................................5.0g Preparation of Na2S•9H2O Solution: Add Na2S•9H2O to distilled/deionized water and bring volume to 100.0mL. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ..............................................................5.0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12 ......................................................2.0mg Preparation of Vitamin B12 Solution: Add vitamin B12 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: To 950.0mL of cooled, sterile solution 1, aseptically add 60.0mL of sterile Na2S•9H2O solution, 40.0mL of sterile NaHCO3 solution, and 1.0mL of sterile vitamin B12 solution. Mix thoroughly. Adjust pH to 7.3 with sterile H2SO4 or Na2CO3 . Aseptically distribute into sterile 50.0mL or 100.0mL bottles with metal screw-caps and rubber seals. Completely fill bottles with medium except for a pea-sized air bubble. Use: For the isolation and cultivation of marine members of the Chromatiaceae. Marine Cytophaga Agar 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 MgCl2................................................................2.48g KCl....................................................................0.75g CaCl2.................................................................0.56g Fe(SO4)2(NH4)2...............................................0.048g Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add solid components to 1.0L of salt solution. 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 Cytophaga species. Marine Cytophaga Medium Composition per liter: NaCl ..................................................................24.7g Agar ..................................................................15.0g MgSO4•7H2O......................................................6.3g MgCl2•6H2O .......................................................4.6g Tryptic digest of casein.......................................1.0g Yeast extract........................................................1.0g KCl......................................................................0.7g NaHCO3 solution .......................................... 10.0mL CaCl2•2H2O solution..................................... 10.0mL pH 7.2 ± 0.2 at 25°C NaHCO3 Solution: Composition per 10.0mL: NaHCO3..............................................................0.2g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. CaCl2•2H2O Solution: Composition per 10.0mL: CaCl2•2H2O ........................................................1.2g Preparation of CaCl2•2H2O Solution: Add CaCl2•2H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except NaHCO3 solution and CaCl2•2H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°–55°C. Aseptically add 10.0mL of sterile NaHCO3 solution and 10.0mL of 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 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. 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 Cytophaga agarovorans, Cytophaga fermentans, 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 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: Composition per 10.0mL: FeSO4•7H2O........................................................0.5g Preparation of Solution B: Add FeSO4•7H2O to distilled/deionized 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, anaerobically add 10.0mL of solution B and 10.0mL of solution C. Mix thoroughly. Adjust pH to 7.8 with NaOH. Distribute into tubes or flasks. During distribution, swirl the medium to keep the precipitate in suspension. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Desulfovibrio desulfuricans, 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-sterilized 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, Pseudobodo tremulans, Rhynchomonas nasuta, Salpingoeca urceolata, Stephanoeca 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 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 solution. 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•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 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% N2 + 3% H2. Cap with rubber stoppers and place tubes in a press. Autoclave 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•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 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% N2 + 3% H2. Cap with rubber stoppers and place tubes in a press. Autoclave 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 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
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