Handbook of Microbiological Media, Fourth Edition part 116 ppt

Methylophaga alcalica Agar 1145 Preparation of Carbonate Solution: Add Na 2 CO 3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Thiosulfate Solution: Composition per 10.0mL: NaS 2 O 3 ·5H 2 O 18.0g Preparation of Thiosulfate Solution: Add NaS 2 O 3 ·5H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Acetate Solution: Composition per 10.0mL: Sodium acetate 0.2g Preparation of Acetate Solution: Add sodium acetate to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Preparation of Medium: Add components, except acetate, vitamin, carbonate, thiosulfate, and magnesium sulfate solutions, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Distribute into closed vessels with a medium to headspace ratio of 1:5 to 1:10. Au- toclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add the acetate, vitamin, carbonate, thiosulfate, and magnesium sulfate solutions. Use: For the cultivation with faster growth rates of Methylonatrum spp. Methylophaga Agar Composition per 103.0mL: Agar solution 50.0mL Mineral base, 2X 50.0mL Solution T 2.0mL Vitamin B 12 solution 1.0mL Methanol 0.3mL pH 7.3 ± 0.2 at 25°C Agar Solution: Composition per 500.0mL: Agar 15.0g Preparation of Agar Solution: Add agar to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Mineral Base, 2X: Composition per 500.0mL: NaCl 24.0g MgCl 2 ·6H 2 O 3.0g MgSO 4 ·7H 2 O 2.0g CaCl 2 ·2H 2 O 1.0g KCl 0.5g Bis-Tris buffer (bis[2-hydroxyethyl]amino- tris[hydroxymethyl]-methane) 0.5g Wolfe’s mineral solution 10.0mL Preparation of Mineral Base, 2X: Add components to distilled/ deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Wolfe’s Mineral Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·H 2 O 0.5g FeSO 4 ·7H 2 O 0.1g CoCl 2 ·6H 2 O 0.1g CaCl 2 0.1g ZnSO 4 ·7H 2 O 0.1g CuSO 4 ·5H 2 O 0.01g AlK(SO 4 ) 2 ·12H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Solution T: Composition per 100.0mL: NH 4 Cl 10.0g Bis-Tris buffer (bis[2-hydroxyethyl]amino- tris[hydroxymethyl]-methane) 10.0g KH 2 PO 4 0.7g Ferric ammonium citrate 0.3g Preparation of Solution T: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure–121°C. Vitamin B 12 Solution: Composition per 10.0mL: Vitamin B 12 1.0μg Preparation of Vitamin B 12 Solution: Add vitamin B 12 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Preparation of Medium: Aseptically mix 50.0mL of the sterile agar solution with 50.0mL of the sterile mineral base, 2X. Aseptically combine sterile solution T and sterile vitamin B 12 solution with the sterile mineral base. Filter sterilize methanol and add to basal medium. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Methylophaga marina. Methylophaga alcalica Agar (DSMZ Medium 976) Composition per liter: NaCl 30.0g Agar 20.0g KH 2 PO 4 1.0g KNO 3 1.0g MgSO 4 ·7H 2 O 0.22g Na 2 CO 3 solution 50.0mL Methanol solution 50.0mL Trace elements solution 1.0mL pH 9.5 ± 0.2 at 25°C Methanol Solution: Composition per 50.0mL: Methanol 10.0mL Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. © 2010 by Taylor and Francis Group, LLC 1146 Methylophaga alcalica Medium Na 2 CO 3 Solution: Composition per 50.0mL: Na 2 CO 3 5.0g Preparation of Na 2 CO 3 Solution: Add Na 2 CO 3 to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution: Composition per liter: Ferric citrate 30.0mg CaCl 2 ·2H 2 O 30.0mg MgCl 2 ·4H 2 O 5.0mg ZnSO 4 ·7H 2 O 5.0mg CuSO 4 ·5H 2 O 0.5mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components, except methanol solution and Na 2 CO 3 solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 55°C. Asepti- cally add 50.0mL warm sterile Na 2 CO 3 solution and 50.0mL warm sterile methanol solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the cultivation and maintenance of Methylophaga alcalica. Methylophaga alcalica Medium (DSMZ Medium 976) Composition per liter: NaCl 30.0g KH 2 PO 4 1.0g KNO 3 1.0g MgSO 4 ·7H 2 O 0.22g Na 2 CO 3 solution 50.0mL Methanol solution 50.0mL Trace elements solution 1.0mL pH 9.5 ± 0.2 at 25°C Methanol Solution: Composition per 50.0mL: Methanol 10.0mL Preparation of Methanol Solution: Add methanol to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Na 2 CO 3 Solution: Composition per 50.0mL: Na 2 CO 3 5.0g Preparation of Na 2 CO 3 Solution: Add Na 2 CO 3 to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Trace Elements Solution: Composition per liter: Ferric citrate 30.0mg CaCl 2 ·2H 2 O 30.0mg MgCl 2 ·4H 2 O 5.0mg ZnSO 4 ·7H 2 O 5.0mg CuSO 4 ·5H 2 O 0.5mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components, except methanol solution and Na 2 CO 3 solution, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 50.0mL sterile Na 2 CO 3 solution and 50.0mL sterile methanol solution. Mix thoroughly. Asep- tically distribute into sterile tubes or flasks. Use: For the cultivation of Methylophaga alcalica. Methylophaga Broth Composition per 103.0mL: Mineral base 100.0mL Solution T 2.0mL Vitamin B 12 solution 1.0mL Methanol 0.3mL pH 7.3 ± 0.2 at 25°C Mineral Base: Composition per liter: NaCl 24.0g MgCl 2 ·6H 2 O 3.0g MgSO 4 ·7H 2 O 2.0g CaCl 2 ·2H 2 O 1.0g KCl 0.5g Bis-Tris buffer (bis[2-hydroxyethyl]amino- tris[hydroxymethyl]-methane) 0.5g Wolfe’s mineral solution 10.0mL Preparation of Mineral Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure–121°C. Wolfe’s Mineral Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·H 2 O 0.5g FeSO 4 ·7H 2 O 0.1g CoCl 2 ·6H 2 O 0.1g CaCl 2 0.1g ZnSO 4 ·7H 2 O 0.1g CuSO 4 ·5H 2 O 0.01g AlK(SO 4 ) 2 ·12H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Solution T: Composition per 100.0mL: NH 4 Cl 10.0g Bis-Tris buffer (bis[2-hydroxyethyl]amino- tris[hydroxymethyl]-methane) 10.0g KH 2 PO 4 0.7g Ferric ammonium citrate 0.3g Preparation of Solution T: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.3. Autoclave for 15 min at 15 psi pressure–121°C. © 2010 by Taylor and Francis Group, LLC Methylosarcina quisquillarum/ Methylosarcina fibrata Medium 1147 Vitamin B 12 Solution: Composition per 10.0mL: Vitamin B 12 1.0μg Preparation of Vitamin B 12 Solution: Add vitamin B 12 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Preparation of Medium: Aseptically combine sterile solution T and sterile vitamin B 12 solution with the sterile mineral base. Filter sterilize methanol and add to basal medium. Aseptically distribute into sterile tubes or sterile flasks. Use: For the cultivation and maintenance of Methylophaga marina. Methylophaga Medium Composition per liter: NaCl 25.0g Agar 20.0g Peptone 10.0g Beef extract 7.0g K 2 HPO 4 1.0g (NH 4 ) 2 SO 4 1.0g Methanol, filter sterilized 10.0mL pH 7.0 ± 0.2 at 25°C Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL. 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 methanol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Methylophaga marina and Methylophaga thalassica. Methylophaga sulfidovorans Medium (DSMZ Medium 951) Composition per liter: NaCl 15.0g Na 2 CO 3 2.0g MgSO 4 ·7H 2 O 1.0g (NH 4 ) 2 SO 4 0.5g CaCl 2 ·6H 2 O 0.33g KCl 0.2g KH 2 PO 4 0.02g DMS (Dimethylsulphide) 62.0mg FeSO 4 ·7H 2 O 1.0mg Trace elements solution SL-10 1.0mL Vitamin solution 1.0mL pH 7.5 ± 0.3 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 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 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Vitamin Solution: Composition per liter: Pyridoxine-HCl 500.0mg Nicotinic acid 200.0mg Thiamine 100.0mg p-Aminobenzoic acid 100.0mg Pantothenate 50.0mg Biotin 20.0mg Riboflavin 10.0mg Vitamin B 12 10.0mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Methylophaga sulfidovorans. Methylophaga thalassica Agar (LMG Medium 73) Composition per liter: NaCl 25.0g Agar 20.0g Peptone 10.0g Lab Lemco beef extract 7.0g K 2 HPO 4 1.0g (NH 4 ) 2 SO 4 1.0g pH 7.0 ± 0.2 at 25°C Preparation of Medium: Add components, except methanol, to 990.0mL distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°C. Aseptically add 10.0mL sterile methanol. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Methylophaga thalassica. Methylosarcina quisquillarum/ Methylosarcina fibrata Medium (DSMZ Medium 921) Composition per 1012.1mL: Solution 1 100.0mL Phosphate buffer 10.0mL Solution 3 1.0mL Trace elements 1.0mL Solution 2 0.1mL pH 7.0 ± 0.2 at 25°C Solution 1 (10X NMS Salts): Composition per liter: KNO 3 10.0g MgSO 4 ·6H 2 O 10.0g CaCl 2 ·2H 2 O 2.0g Preparation of Solution 1 (10X NMS Salts): Add components to 700.0mL distilled/deionized water. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. © 2010 by Taylor and Francis Group, LLC 1148 Methylotrophic Arthrobacter Hyphomicrobium Medium Solution 2 (Fe EDTA): Composition per liter: Fe EDTA 3.8g Preparation of Solution 2 (Fe EDTA): Add Fe EDTA to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Solution 3 (Sodium Molybdate): Composition per liter: Na 2 MoO 4 ·4H 2 O 0.26g Preparation of Solution 3 (Sodium Molybdate): Add Na 2 MoO 4 ·4H 2 O to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Trace Elements: Composition per 100.0mL: CuSO 4 ·5H 2 O 100.0mg FeSO 4 ·7H 2 O 50.0mg ZnSO 4 ·7H 2 O 40.0mg EDTA disodium salt 25.0mg CoCl 2 ·6H 2 O 5.0mg MnCl 2 ·4H 2 O 2.0mg H 3 BO 3 1.5mg NiCl 2 ·6H 2 O 1.0mg Preparation of Trace Elements: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Phosphate Buffer: Composition per liter: Na 2 HPO 4 ·2H 2 O 71.6g KH 2 PO 4 26.0g Preparation of Phosphate Buffer: Add components to 800.0mL distilled/deionized water. Mix thoroughly. Adjust pH to 6.8. Bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 55°C. Preparation of Medium: Add 100.0mL solution 1 to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Add 1.0mL of solution 3, 1.0mL of the trace elements, and 0.1mL of solution 2. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 55°C. Asepti- cally add 10.0mL phosphate buffer. Mix thoroughly. Aseptically distribute to sterile tubes or bottles. Use: For the cultivation of Methylosarcina fibrata and Methylosarcina quisquiliarum. Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939) Composition per liter: Na 2 HPO 4 ·2H 2 O 7.9g Dimethylsulfone 1.9g KH 2 PO 4 1.5g NH 4 Cl 0.8g MgSO 4 ·7H 2 O 0.1g Trace elements solution 10.0mL pH 7.2-7.5 at 25°C Trace Elements Solution: Composition per liter: EDTA disodium salt 50.0g NaOH 9.0g CaCl 2 ·2H 2 O 7.34g FeSO 4 ·7H 2 O 5.0g MnCl 2 ·4H 2 O 2.5g ZnSO 4 ·7H 2 O 1.0g CoCl 2 ·6H 2 O 0.5g NH 4 (MoO 4 ) 0.5g CuSO 4 ·5H 2 O 0.2g Preparation of Trace Elements Solution: Add Na 2 -EDTA to 400.0mL distilled/deionized water. Mix thoroughly. Add 9.0g NaOH. Mix thoroughly. Individually dissolve each of the other components in 40.0mL distilled/deionized water. Add each of the other dissolved components to the EDTA solution and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Adjust the pH to 6.0 with 1M NaOH. 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 Hyphomicrobium sulfonivorans. Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939) Composition per liter: Na 2 HPO 4 ·2H 2 O 7.9g KH 2 PO 4 1.5g NH 4 Cl 0.8g MgSO 4 ·7H 2 O 0.1g Trace elements solution 10.0mL Methanol 10.0mL pH 7.2–7.5 at 25°C Trace Elements Solution: Composition per liter: EDTA disodium salt 50.0g NaOH 9.0g CaCl 2 ·2H 2 O 7.34g FeSO 4 ·7H 2 O 5.0g MnCl 2 ·4H 2 O 2.5g ZnSO 4 ·7H 2 O 1.0g CoCl 2 ·6H 2 O 0.5g NH 4 (MoO 4 ) 0.5g CuSO 4 ·5H 2 O 0.2g Preparation of Trace Elements Solution: Add Na 2 -EDTA to 400.0mL distilled/deionized water. Mix thoroughly. Add 9.0g NaOH. Mix thoroughly. Individually dissolve each of the other components in 40.0mL distilled/deionized water. Add each of the other dissolved components to the EDTA solution and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Adjust the pH to 6.0 with 1M NaOH. 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 Hyphomicrobium sulfonivorans. Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939) Composition per liter: Na 2 HPO 4 ·2H 2 O 7.9g KH 2 PO 4 1.5g Methylamine 1.0g NH 4 Cl 0.8g MgSO 4 ·7H 2 O 0.1g Trace elements solution 10.0mL pH 7.2–7.5 at 25°C © 2010 by Taylor and Francis Group, LLC Methylpyridine Medium 1149 Trace Elements Solution: Composition per liter: EDTA disodium salt 50.0g NaOH 9.0g CaCl 2 ·2H 2 O 7.34g FeSO 4 ·7H 2 O 5.0g MnCl 2 ·4H 2 O 2.5g ZnSO 4 ·7H 2 O 1.0g CoCl 2 ·6H 2 O 0.5g NH 4 (MoO 4 ) 0.5g CuSO 4 ·5H 2 O 0.2g Preparation of Trace Elements Solution: Add Na 2 -EDTA to 400.0mL distilled/deionized water. Mix thoroughly. Add 9.0g NaOH. Mix thoroughly. Individually dissolve each of the other components in 40.0mL distilled/deionized water. Add each of the other dissolved components to the EDTA solution and bring volume to 1.0L with distilled/ deionized water. Mix thoroughly. Adjust the pH to 6.0 with 1M NaOH. 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 Hyphomicrobium sulfonivorans. Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939) Composition per liter: Na 2 HPO 4 ·2H 2 O 7.9g KH 2 PO 4 1.5g Glucosel 1.0g NH 4 Cl 0.8g MgSO 4 ·7H 2 O 0.1g Trace elements solution 10.0mL pH 7.2–7.5 at 25°C Trace Elements Solution: Composition per liter: EDTA disodium salt 50.0g NaOH 9.0g CaCl 2 ·2H 2 O 7.34g FeSO 4 ·7H 2 O 5.0g MnCl 2 ·4H 2 O 2.5g ZnSO 4 ·7H 2 O 1.0g CoCl 2 ·6H 2 O 0.5g NH 4 (MoO 4 ) 0.5g CuSO 4 ·5H 2 O 0.2g Preparation of Trace Elements Solution: Add Na 2 -EDTA to 400.0mL distilled/deionized water. Mix thoroughly. Add 9.0g NaOH. Mix thoroughly. Individually dissolve each of the other components in 40.0mL distilled/deionized water. Add each of the other dissolved components to the EDTA solution and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Adjust the pH to 6.0 with 1M NaOH. 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 Hyphomicrobium sulfonivorans. Methylotrophic Arthrobacter Hyphomicrobium Medium (DSMZ Medium 939) Composition per liter: Na 2 HPO 4 ·2H 2 O 7.9g KH 2 PO 4 1.5g Fructose 1.0g NH 4 Cl 0.8g MgSO 4 ·7H 2 O 0.1g Trace elements solution 10.0mL pH 7.2–7.5 at 25°C Trace Elements Solution: Composition per liter: EDTA disodium salt 50.0g NaOH 9.0g CaCl 2 ·2H 2 O 7.34g FeSO 4 ·7H 2 O 5.0g MnCl 2 ·4H 2 O 2.5g ZnSO 4 ·7H 2 O 1.0g CoCl 2 ·6H 2 O 0.5g NH 4 (MoO 4 ) 0.5g CuSO 4 ·5H 2 O 0.2g Preparation of Trace Elements Solution: Add Na 2 -EDTA to 400.0mL distilled/deionized water. Mix thoroughly. Add 9.0g NaOH. Mix thoroughly. Individually dissolve each of the other components in 40.0mL distilled/deionized water. Add each of the other dissolved components to the EDTA solution and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Adjust the pH to 6.0 with 1M NaOH. 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 Hyphomicrobium sulfonivorans. Methylpyridine Medium Composition per 1002.0mL: K 2 HPO 4 0.61g KH 2 PO 4 0.39g KCl 0.25g Yeast extract 0.1g Wolfe’s mineral solution 10.0mL 2-Methylpyridine 1.0mL Wolfe’s Mineral 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 CoCl 2 ·6H 2 O 0.1g ZnSO 4 ·7H 2 O 0.1g 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 CuSO 4 ·5H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with © 2010 by Taylor and Francis Group, LLC 1150 M-FC HiVeg Agar Base with Rosalic Acid KOH. Add remaining components one at a time. Add distilled/deionized water to 1.0L. Adjust pH to 6.8. Preparation of Medium: Add components, except 2-methylpyridine, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. In a fume hood, aseptically add 1.0mL of 2-methylpyridine. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use polyurethane foam closures to eliminate odors caused by volatil- ization of 2-methylpyridine. Use: For the cultivation of Arthrobacter species. M-FC Agar See: FC Agar M-FC Broth See: FC Broth M-FC HiVeg Agar Base with Rosalic Acid Composition per liter: Agar 15.0g Lactose 12.5g Plant hydrolysate No. 1 10.0g Plant peptone No. 3 5.0g NaCl 5.0g Yeast extract 3.0g Synthetic detergent No. I 1.5g Aniline Blue 0.1g Rosolic acid solution 10.0mL pH 7.4 ± 0.2 at 25°C Source: This medium, without rosolic acid, is available as a premixed powder from HiMedia. Rosolic Acid Solution: Composition per 100.0mL: Rosolic acid 1.0g Preparation of Rosolic Acid Solution: Add rosolic acid to 0.2N NaOH and bring volume to 100.0L. Mix thoroughly. Preparation of Medium: Add 10.0mL rosolic acid solution to 950.0mL of distilled/deionized water. Mix thoroughly. Add other components and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling with frequent mixing. Do not autoclave. Pour into sterile Petri dishes or leave in tubes. Use: For the detection and enumeration of fecal coliforms using the membrane filter technique. M-FC HiVeg Agar Base, Modified with Rosalic Acid Composition per liter: Agar 15.0g Plant hydrolysate No. 1 10.0g Inositol 10.0g Plant peptone No. 3 5.0g NaCl 5.0g Yeast extract 3.0g Synthetic detergent No. I 1.5g Aniline blue 0.1g Rosolic acid solution 10.0mL pH 7.4 ± 0.2 at 25°C Source: This medium, without rosolic acid, is available as a premixed powder from HiMedia. Rosolic Acid Solution: Composition per 100.0mL: Rosolic acid 1.0g Preparation of Rosolic Acid Solution: Add rosolic acid to 0.2N NaOH and bring volume to 100.0L. Mix thoroughly. Preparation of Medium: Add 10.0mL rosolic acid solution to 950.0mL of distilled/deionized water. Mix thoroughly. Add other components and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling with frequent mixing. Do not autoclave. Pour into sterile Petri dishes or leave in tubes. Use: For the detection and enumeration of fecal coliforms using membrane filter technique. M-FC HiVeg Broth Base with Rosalic Acid Composition per liter: Lactose 12.5g Plant hydrolysate No. 1 10.0g Plant peptone No. 3 5.0g NaCl 5.0g Yeast extract 3.0g Synthetic detergent No. I 1.5g Aniline blue 0.1g Rosolic acid solution 10.0mL pH 7.4 ± 0.2 at 25°C Source: This medium, without rosolic acid, is available as a premixed powder from HiMedia. Rosolic Acid Solution: Composition per 100.0mL: Rosolic acid 1.0g Preparation of Rosolic Acid Solution: Add rosolic acid to 0.2N NaOH and bring volume to 100.0L. Mix thoroughly. Preparation of Medium: Add 10.0mL of rosolic acid solution to 950.0mL of distilled/deionized water. Mix thoroughly. Add other components and bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Gently heat and bring to boiling with frequent mixing. Do not autoclave. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of fecal coliform bacteria from waters and the enumeration of coliform bacteria using the membrane filtration method. M-FC Agar Composition per liter: Agar 15.0g Tryptose 10.0g Inositol 10.0g Proteose peptone 5.0g NaCl 5.0g Yeast extract 3.0g Bile salts mixture 1.5g Aniline Blue 0.1g Selective supplement solution 10.0mL Rosolic acid solution 10.0mL pH 7.4 ± 0.2 at 25°C Source: This medium is available from HiMedia. Rosolic Acid Solution: Composition per 10.0mL: Rosolic acid 0.1g © 2010 by Taylor and Francis Group, LLC MG Medium 1151 Preparation of Rosolic Acid Solution: Add rosoloic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Selective Supplement Solution: Composition per 10.0mL: Carbenicillin 0.05g Preparation of Selective Supplement Solution: Add carbenicillin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except selective supplement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add selective supplement solution. Mix thoroughly. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the rapid enumeration of Klebsiella using the membrane filter technique. M-FC Agar Base Composition per liter: Agar 15.0g Lactose 12.5g Tryptose 10.0g Proteose peptone 5.0g NaCl 5.0g Yeast extract 3.0g Bile salts mixture 1.5g Aniline Blue 0.1g Rosolic acid solution 10.0mL pH 7.4 ± 0.2 at 25°C Source: This medium is available from HiMedia. Rosolic Acid Solution: Composition per 10.0mL: Rosolic acid 0.1g Preparation of Rosolic Acid Solution: Add rosolic acidto distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat to dissolve components. Do not autoclave. Cool to 50°C. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the detection and enumeration of fecal coliforms using the membrane filter technique at 44.5°C. m-Fecal Coliform Agar See: FC Agar m-Fecal Coliform Agar, Modified See: Fecal Coliform Agar, Modified m-Fecal Coliform Broth See: FC Broth MG Medium Composition per liter: NaCl 100.0g MgSO 4 ·7H 2 O 3.45g MgCl 2 ·6H 2 O 2.75g Sodium acetate 1.0g KCl 0.335g NH 4 Cl 0.25g CaCl 2 ·2H 2 O 0.14g K 2 HPO 4 ·3H 2 O 0.14g Resazurin 1.0mg NaHCO 3 solution 80.0mL Trimethylamine·HCl solution 20.0mL Na 2 CO 3 solution 10.0mL Trace elements solution 10.0mL Vitamin solution 10.0mL L-Cysteine·HCl solution 10.0mL Na 2 S·9H 2 O solution 10.0mL pH 6.9 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 100.0mL: NaHCO 3 5.0g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Trimethylamine·HCl Solution: Composition per 20.0mL: Trimethylamine·HCl 5.0g Preparation of Trimethylamine·HCl Solution: Add trimethylamine·HCl to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Na 2 CO 3 Solution: Composition per 10.0mL: Na 2 CO 3 0.5g Preparation of Na 2 CO 3 Solution: Add Na 2 CO 3 to distilled/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. 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 CuSO 4 ·5H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500.0mL distilled/deionized water. Dissolve by adding KOH and adjust pH to 6.5. Add remaining components. Bring volume to 1.0L with additional distilled/deionized water. Adjust pH to 7.0 with KOH. Vitamin Solution: Composition per liter: Pyridoxine·HCl 10.0mg Calcium DL-pantothenate 5.0mg Lipoic acid 5.0mg © 2010 by Taylor and Francis Group, LLC 1152 MG Medium Nicotinic acid 5.0mg p-Aminobenzoic acid 5.0mg Riboflavin 5.0mg Thiamine·HCl 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N 2 . L-Cysteine·HCl Solution: Composition per 10.0mL: L-Cysteine·HCl 0.5g Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N 2 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 distilled/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: Add components, except NaHCO 3 solution, trimethylamine·HCl solution, Na 2 CO 3 solution, vitamin solution, L-cysteine·HCl solution, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 860.0mL. Mix thoroughly. Sparge with 100% N 2 for 20 min. Then sparge with 80% N 2 + 20% CO 2 for 10 min. Anaerobically distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 80.0mL of sterile NaHCO 3 solution, 20.0mL of sterile trimethylamine·HCl solution, 10.0mL of sterile Na 2 CO 3 solution, 10.0mL of sterile vitamin solution, 10.0mL of sterile L-cysteine·HCl solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. Use: For the cultivation and maintenance of Methanohalobium species, Methanohalophilus halophilus, and Methanohalophilus species. MG Medium Composition per liter: NaCl 150.0g MgSO 4 ·7H 2 O 3.45g MgCl 2 ·6H 2 O 2.75g Sodium acetate 1.0g KCl 0.335g NH 4 Cl 0.25g CaCl 2 ·2H 2 O 0.14g K 2 HPO 4 ·3H 2 O 0.14g Resazurin 1.0mg NaHCO 3 solution 80.0mL Trimethylamine·HCl solution 20.0mL Na 2 CO 3 solution 10.0mL Trace elements solution 10.0mL Vitamin solution 10.0mL L-Cysteine·HCl solution 10.0mL Na 2 S·9H 2 O solution 10.0mL pH 6.9 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 100.0mL: NaHCO 3 5.0g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Trimethylamine·HCl Solution: Composition per 20.0mL: Trimethylamine·HCl 5.0g Preparation of Trimethylamine·HCl Solution: Add trimethylamine·HCl to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Na 2 CO 3 Solution: Composition per 10.0mL: Na 2 CO 3 0.5g Preparation of Na 2 CO 3 Solution: Add Na 2 CO 3 to distilled/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. 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 CuSO 4 ·5H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to approximately 500.0mL of distilled/deionized water. Dissolve by adding KOH and adjust pH to 6.5. Add remaining components. Bring volume to 1.0L with additional distilled/deionized water. Adjust pH to 7.0 with KOH. Vitamin Solution: Composition per liter: Pyridoxine·HCl 10.0mg Calcium DL-pantothenate 5.0mg Lipoic acid 5.0mg Nicotinic acid 5.0mg p-Aminobenzoic acid 5.0mg Riboflavin 5.0mg Thiamine·HCl 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B 12 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N 2 . L-Cysteine·HCl Solution: Composition per 10.0mL: L-Cysteine·HCl 0.5g Preparation of L-Cysteine·HCl Solution: Add L-cysteine·HCl to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. © 2010 by Taylor and Francis Group, LLC MH IH Agar 1153 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 distilled/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: Add components, except NaHCO 3 solution, trimethylamine·HCl solution, Na 2 CO 3 solution, vitamin solution, L- cysteine·HCl solution, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 860.0mL. Mix thoroughly. Sparge with 100% N 2 for 20 min. Then sparge with 80% N 2 + 20% CO 2 for 10 min. Anaerobi- cally distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaerobically add 80.0mL of sterile NaHCO 3 solution, 20.0mL of sterile trimethylamine·HCl solution, 10.0mL of sterile Na 2 CO 3 solution, 10.0mL of sterile vitamin solution, 10.0mL of sterile L- cysteine·HCl solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. Use: For the cultivation and maintenance of Methanohalophilus species. MGA Agar Composition per liter: Agar 20.0g Glucose 2.0g L-Asparagine 1.0g K 2 HPO 4 0.5g MgSO 4 ·7H 2 O 0.5g Trace salts solution 1.0mL pH 7.4 ± 0.2 at 25°C Trace Salts Solution: Composition per liter: FeSO 4 ·7H 2 O 10.0g CuSO 4 ·5H 2 O 1.0g MnSO 4 ·7H 2 O 1.0g ZnSO 4 ·7H 2 O 1.0g Preparation of Trace Salts Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 8.0. 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 isolation and cultivation of Actinomadura species, Acti- nopolyspora species, Excellospora species, and Microspora species. m-Green Yeast and Mold Broth See: Green Yeast and Mold Broth MGTY Agar See: Marine Glucose Trypticase™ Yeast Extract Agar MGTY Broth See: Marine Glucose Trypticase™ Yeast Extract Broth MH Agar 15% (LMG Medium 258) Composition per liter: NaCl 121.5g Agar 20.0g MgSO 4 ·7H 2 O 14.4g MgCl 2 10.5g Yeast extract 10.0g Proteose peptone no.3 5.0g KCl 3.0g Glucose 1.0g CaCl 2 0.54g NaBr 39.0mg NaHCO 3 solution 10.0mL pH 7.5 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 10.0mL: NaHCO 3 0.09g Preparation of NaHCO 3 Solution: Add NaHCO 3 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except NaHCO 3 solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptical- ly add 10.0mL sterile NaHCO 3 solution. Pour into sterile Petri dishes or aseptically distribute into sterile tubes. Use: For cultivation and maintenance of Bacillus halophilus. MH IH Agar Composition per liter: Solution A 490.0mL Solution B 490.0mL Supplement solution 20.0mL pH 6.9 ± 0.2 at 25°C Solution A: Composition per 490.0mL: Beef infusion 300.0g Acid hydrolysate of casein 17.5g Agar 17.0g Starch 1.5g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 490.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Solution B: Composition per 490.0mL: Hemoglobin 10.0g Preparation of Solution B: Add hemoglobin to distilled/deionized water and bring volume to 490.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Supplement Solution: Composition per liter: Glucose 100.0g L-Cysteine·HCl 25.9g L-Glutamine 10.0g L-Cystine 1.1g Adenine 1.0g Nicotinamide adenine dinucleotide 0.25g Vitamin B 12 0.1g Thiamine pyrophosphate 0.1g Guanine·HCl 0.03g Fe(NO 3 ) 3 ·6H 2 O 0.02g © 2010 by Taylor and Francis Group, LLC 1154 MH Medium p-Aminobenzoic acid 0.013g Thiamine·HCl 3.0mg Source: The supplement solution IsoVitaleX ® enrichment is available from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems. Preparation of Supplement Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Aseptically combine cooled, sterile solution A and cooled, sterile solution B. Mix thoroughly. Adjust pH to 6.9 with sterile 1N HCl or sterile 1N KOH. Aseptically add 20.0mL of sterile supplement solution. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and differentiation of Legionella species. MH Medium Composition per liter: NaCl 60.7g Agar 20.0g MgCl 2 ·6H 2 O 15.0g Yeast extract 10.0g MgSO 4 ·7H 2 O 7.4g Proteose peptone No. 3 5.0g KCl 1.5g Glucose 1.0g CaCl 2 0.27g NaHCO 3 0.45g NaBr 0.19g 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 Deleya salina and Volca- niella eurihalina. MH Medium Composition per liter: NaCl 60.7g MgCl 2 ·6H 2 O 15.0g Yeast extract 10.0g MgSO 4 ·7H 2 O 7.4g Proteose peptone No. 3 5.0g KCl 1.5g Glucose 1.0g CaCl 2 0.27g NaHCO 3 0.045g NaBr 0.019g 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. Adjust pH to 7.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Halomonas eurihalina. MH Medium, 2% Composition per liter: KH 2 PO 4 2.0g (NH 4 ) 2 SO 4 2.0g NaCl 0.5g MgSO 4 ·7H 2 O 0.025g FeSO 4 ·7H 2 O 2.0mg Glucose solution 20.0mL Methanol, filter sterilized 20.0mL pH 7.0–7.5 at 25°C Glucose Solution: Composition per 20.0mL: Glucose 1.5g Preparation of Glucose Solution: Add glucose to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except glucose solution and methanol, to distilled/deionized water and bring volume to 960.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 20.0mL of sterile glucose solution and 20.0mL of filter-sterilized methanol. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Methylobacillus fructoseoxidans and Meth- ylophilus glucoseoxidans. MH Medium, 10% (LMG Medium 270) Composition per liter: NaCl 81.0g Agar 15.0g Yeast extract 10.0g MgSO 4 ·7H 2 O 9.6g MgCl 2 ·6H 2 O 7.0g Proteose peptone No.3 5.0g KCl 2.0g Glucose 1.0g CaCl 2 0.54g NaBr 26.0mg NaHCO 3 solution 10.0mL pH 7.5 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 10.0mL: NaHCO 3 0.06g Preparation of NaHCO 3 Solution: Add NaHCO 3 to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except NaHCO 3 solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°C. Aseptically add 10.0mL sterile NaHCO 3 solution. Pour into sterile Petri dishes or aseptically distribute into sterile tubes. Use: For cultivation and maintenance of Chromohalobacter israelensis and Chromohalobacter canadensis. MH Medium, 10% 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 KCl 2.0g Glucose 1.0g © 2010 by Taylor and Francis Group, LLC . anaerobically add 80.0mL of sterile NaHCO 3 solution, 20.0mL of sterile trimethylamine·HCl solution, 10.0mL of sterile Na 2 CO 3 solution, 10.0mL of sterile vitamin solution, 10.0mL of sterile L-cysteine·HCl. anaerobically add 80.0mL of sterile NaHCO 3 solution, 20.0mL of sterile trimethylamine·HCl solution, 10.0mL of sterile Na 2 CO 3 solution, 10.0mL of sterile vitamin solution, 10.0mL of sterile L- cysteine·HCl. 1.0g Preparation of Rosolic Acid Solution: Add rosolic acid to 0.2N NaOH and bring volume to 100.0L. Mix thoroughly. Preparation of Medium: Add 10.0mL of rosolic acid solution to 950.0mL of distilled/deionized