Pfennig’s Medium 1, Modified for Marine Purple Sulfur Bacteria 1375 KCl 1.7g CaCl 2 ·2H 2 O 1.25g Preparation of Solution A: Add components to 4.0L distilled wa- ter. Mix thoroughly. Autoclave for 45 min at 15 psi pressure–121°C in 5-liter special bottle or flask with four openings at the top, together with a teflon-coated magnetic bar. In this 5-liter bottle, two openings for tubes are in the central, silicon rubber stopper: a short, gas-inlet tube with a sterile cotton filter and an outlet tube for medium, which reaches the bottom of the vessel at one end and has, at the other end, a silicon rubber tube with a pinch cock and a bell for aseptic dispensing of the medium into bottles. The other two openings have gas-tight screw caps; one of these openings is for the addition of sterile solutions and the other serves as a gas outlet. After autoclaving, cool to room temperature under a N 2 atmosphere with a positive pressure of 0.05– 0.1 atm (a manometer for low pressure will be required). Saturate the cold medium with CO 2 by magnetic stirring for 30 min under a CO 2 atmosphere of 0.05– 0.1 atm. Solution B: Distilled water 860.0mL Preparation of Solution B: Autoclave distilled water for 15 min at 15 psi pressure–121°C in a cotton-stoppered Erlenmeyer flask. Cool to room temperature under an atmosphere of N 2 in an anaerobic jar. Solution C: Composition per 100.0mL: Vitamin B 12 2.0mg Preparation of Solution C: Add vitamin B 12 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge under 100% N 2 gas for 3 min. Filter sterilize. Store under N 2 gas. Solution D: Composition per liter: Disodium ethylendiamine-tetraacetate (Disodium EDTA) 3.0g FeSO 4 ·7H 2 O 1.1g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·2H 2 O 50.0mg ZnCl 2 42.0mg NiCl 2 ·6H 2 O 24.0mg Na 2 MoO 4 ·2H 2 O 18.0mg CuCl 2 ·2H 2 O 2.0mg Preparation of Solution D: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution E: Composition per 100.0mL: NaHCO 3 7.5g Preparation of Solution E: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster- ile, gas-tight 100.0mL screw-capped bottle. Solution F: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Solution F: Add Na 2 S·9H 2 O to distilled/deionized water in a 250.0mL screw-capped bottle fitted with a butyl rubber sep- tum and bring volume to 100.0mL. Mix thoroughly. Sparge under 100% N 2 gas for 3 min. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Preparation of Medium: Add solutions B, C, D, E, and F to solu- tion A through one of the screw-cap openings against a stream of either N 2 gas or, better, a mixture of 95% N 2 and 5% CO 2 while the medium is magnetically stirred. Adjust the pH of the medium with sterile HCl or Na 2 CO 3 solution (2M solution) to pH 7.3. Distribute the medium aseptically through the medium outlet tube into sterile, 100mL bottles (with metal caps and autoclavable rubber seals) using the positive gas pressure (0.05–0.1 atm) of the N 2 /CO 2 gas mixture. Leave a small air bubble in each bottle to meet possible pressure changes. The tightly sealed, screw-cap bottles can be stored for several weeks or months in the dark. During the first 24 hr, the iron of the medium precipitates in the form of black flocks. No other sediment should arise in the other- wise clear medium. Incubate in the light using a tungsten lamp. Feed periodically with neutralized solution of sodium sulfide to replenish sulfide and with other supplement solutions. Use: For the cultivation of marine purple sulfur bacteria. Pfennig’s Medium 1, Modified for Marine Purple Sulfur Bacteria (DSMZ Medium 28) Composition per 4990.0mL: Solution A 4000.0mL Solution B 860.0mL Solution E 100.0mL Solution F 20.0mL Solution C (Vitamin B 12 solution) 5.0mL Solution D (Trace elements solution SL-12B) 5.0mL Neutralized sulfide solution As needed pH 7.3 ± 0.2 at 25°C Solution A: Composition per 4000.0mL: NaCl 100.0g MgSO 4 ·7H 2 O 15.0g CaCl 2 ·2H 2 O 1.25g KH 2 PO 4 1.7g NH 4 Cl 1.7g KCl 1.7g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 4.0L. Mix thoroughly. Adjust pH to 6.0. Dispense into a 5L flask with four openings at the top (two openings are in a central silicon rubber stopper and two openings are gas-tight screw caps). Add a teflon-coated magnetic stir bar to the flask. Auto- clave for 45 min at 15 psi pressure–121°C. Cool to room temperature under 100% N 2 at 0.05–0.1 atm pressure (use a manometer to measure low pressure). Solution B: Composition per 860.0mL: Distilled/deionized water 860.0mL Preparation of Solution B: Add 860.0mL of distilled/deionized water to a cotton-stoppered flask. Autoclave for 20 min at 15 psi pres- sure–121°C. Cool to room temperature under 100% N 2 in an anaerobic jar. Solution C (Vitamin B 12 Solution): Composition per 5.0mL: Vitamin B 12 1.0mg © 2010 by Taylor and Francis Group, LLC 1376 Pfennig’s Medium 1 with 1% Salt Preparation of Solution C (Vitamin B 12 Solution): Add vita- min B 12 to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize. Solution D (Trace Elements Solution SL-12B): Composition per liter: Disodium ethylendiamine-tetraacetate (Na 2 -EDTA) 3.0g FeSO 4 ·7H 2 O 1.1g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·2H 2 O 50.0mg ZnCl 2 42.0mg NiCl 2 ·6H 2 O 24.0mg Na 2 MoO 4 ·2H 2 O 18.0mg CuCl 2 ·2H 2 O 2.0mg Preparation of Solution D (Trace Elements Solution SL-12B): Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution E: Composition per 100.0mL: NaHCO 3 7.5g Preparation of Solution E: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster- ile, gas-tight 100.0mL screw-capped bottle. Solution F: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Solution F: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Dispense into a screw-capped bottle. Sparge with 100% N 2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C. Neutralized Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 1.5g Preparation of Neutralized Sulfide Solution: Add Na 2 S·9H 2 O to distilled/deionized water in a 250.0mL screw-capped bottle fitted with a butyl rubber septum and bring volume to 100.0mL. Add a mag- netic stir bar. Mix thoroughly. Sparge under 100% N 2 gas for 3 min. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temper- ature. Adjust pH to about 7.3 with sterile 2M H 2 SO 4 . Do not open the bottle to add H 2 SO 4 ; use a sterile syringe. Stir the solution continuously to avoid precipitation of elemental sulfur. The final solution should be clear and yellow in color. Preparation of Medium: Saturate cooled solution A under 100% CO 2 at 0.05–0.1 atm pressure for 30 min with magnetic stirring. Add 860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution D, 100.0mL of solution E, and 20.0mL of solution F through one of the screw-cappped openings under 95% N 2 and 5% CO 2 with magnetic stirring. Adjust pH to 7.3 with sterile 2M HCl or sterile 2M Na 2 CO 3 so- lution. Aseptically and anaerobically distribute the medium through the medium outlet tube into sterile 100.0mL bottles under 95% N 2 and 5% CO 2 at 0.05–0.1 atm pressure. Leave a small gas bubble in each bottle to accommodate pressure changes. After 24 hr, the iron in the medium will precipitate out of solution as black flocs. No other sedi- ment should arise in the otherwise clear medium. Incubate in the light using a tungsten lamp. Feed periodically with neutralized solution of sodium sulfide to replenish sulfide and with other supplement solu- tions. Use: For the cultivation of purple sulfur bacteria. For the cultivation of purple sulfur bacteria.For the cultivation and maintenance of Amoe- bobacter pendens, Amoebobacter roseus, Chromatium minus, Chroma- tium minutissimum, Chromatium okenii, Chromatium species, Chroma- tium vinosum, Chromatium violascens, Chromatium weissei, Lamprocystis roseopersicina, and Streptomyces venezuelae. Pfennig’s Medium 1 with 1% Salt Composition per 4990.0mL: Solution A 4000.0mL Solution B 860.0mL Solution E 100.0mL Solution F 20.0mL Solution C (Vitamin B 12 solution) 5.0mL Solution D (Trace elements solution SL-12B) 5.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 4000.0mL: NaCl 50.0g CaCl 2 ·2H 2 O 1.25g KH 2 PO 4 1.7g NH 4 Cl 1.7g KCl 1.7g MgSO 4 2.5g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 4.0L. Mix thoroughly. Adjust pH to 6.0. Dispense into a 5L flask with four openings at the top (two openings are in a central silicon rubber stopper and two openings are gas-tight screw caps). Add a teflon-coated magnetic stir bar to the flask. Auto- clave for 45 min at 15 psi pressure–121°C. Cool to room temperature under 100% N 2 at 0.05–0.1 atm pressure (use a manometer to measure low pressure). Solution B: Composition per 860.0mL: Distilled/deionized water 860.0mL Preparation of Solution B: Add 860.0mL of distilled/deionized water to a cotton-stoppered flask. Autoclave for 20 min at 15 psi pres- sure–121°C. Cool to room temperature under 100% N 2 in an anaerobic jar. Solution C (Vitamin B 12 Solution): Composition per 5.0mL: Vitamin B 12 1.0mg Preparation of Solution C (Vitamin B 12 Solution): Add vita- min B 12 to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize. Solution D (Trace Elements Solution SL-12B): Composition per liter: Disodium ethylendiamine-tetraacetate (Na 2 -EDTA) 3.0g FeSO 4 ·7H 2 O 1.1g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·2H 2 O 50.0mg ZnCl 2 42.0mg NiCl 2 ·6H 2 O 24.0mg Na 2 MoO 4 ·2H 2 O 18.0mg CuCl 2 ·2H 2 O 2.0mg © 2010 by Taylor and Francis Group, LLC Pfennig’s Medium 1 with 3% Salt 1377 Preparation of Solution D (Trace Elements Solution SL-12B): Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution E: Composition per 100.0mL: NaHCO 3 7.5g Preparation of Solution E: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster- ile, gas-tight 100.0mL screw-capped bottle. Solution F: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Solution F: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Dispense into a screw-capped bottle. Sparge with 100% N 2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Saturate cooled solution A under 100% CO 2 at 0.05–0.1 atm pressure for 30 min with magnetic stirring. Add 860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution D, 100.0mL of solution E, and 20.0mL of solution F through one of the screw-cappped openings under 95% N 2 and 5% CO 2 with magnetic stirring. Adjust pH to 7.3 with sterile 2M HCl or sterile 2M Na 2 CO 3 so- lution. Aseptically and anaerobically distribute the medium through the medium outlet tube into sterile 100.0mL bottles under 95% N 2 and 5% CO 2 at 0.05–0.1 atm pressure. Leave a small gas bubble in each bottle to accommodate pressure changes. After 24 hr, the iron in the medium will precipitate out of solution as black flocs. Use: For the cultivation and maintenance of Ectothiorhodospira mobi- lis. Pfennig’s Medium 1 with 3% Salt Composition per 4990.0mL: Solution A 4000.0mL Solution B 860.0mL Solution E 100.0mL Solution F 20.0mL Solution C (Vitamin B 12 solution) 5.0mL Solution D (Trace elements solution SL-12B) 5.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 4000.0mL: NaCl 150.0g CaCl 2 ·2H 2 O 1.25g KH 2 PO 4 1.7g NH 4 Cl 1.7g KCl 1.7g MgSO 4 2.5g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 4.0L. Mix thoroughly. Adjust pH to 6.0. Dispense into a 5-liter flask with four openings at the top (two openings are in a central silicon rubber stopper and two openings are gas-tight screw caps). Add a teflon-coated magnetic stir bar to the flask. Auto- clave for 45 min at 15 psi pressure–121°C. Cool to room temperature under 100% N 2 at 0.05–0.1 atm pressure (use a manometer to measure low pressure). Solution B: Composition per 860.0mL: Distilled/deionized water 860.0mL Preparation of Solution B: Add 860.0mL of distilled/deionized wa- ter to a cotton-stoppered flask. Autoclave for 20 min at 15 psi pressure– 121°C. Cool to room temperature under 100% N 2 in an anaerobic jar. Solution C (Vitamin B 12 Solution): Composition per 5.0mL: Vitamin B 12 1.0mg Preparation of Solution C (Vitamin B 12 Solution): Add vita- min B 12 to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize. Solution D (Trace Elements Solution SL-12B): Composition per liter: Disodium ethylendiamine-tetraacetate (Na 2 -EDTA) 3.0g FeSO 4 ·7H 2 O 1.1g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·2H 2 O 50.0mg ZnCl 2 42.0mg NiCl 2 ·6H 2 O 24.0mg Na 2 MoO 4 ·2H 2 O 18.0mg CuCl 2 ·2H 2 O 2.0mg Preparation of Solution D (Trace Elements Solution SL-12B): Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution E: Composition per 100.0mL: NaHCO 3 7.5g Preparation of Solution E: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster- ile, gas-tight 100.0mL screw-capped bottle. Solution F: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Solution F: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Dispense into a screw-capped bottle. Sparge with 100% N 2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Saturate cooled solution A under 100% CO 2 at 0.05–0.1 atm pressure for 30 min with magnetic stirring. Add 860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution D, 100.0mL of solution E, and 20.0mL of solution F through one of the screw-cappped openings under 95% N 2 and 5% CO 2 with magnetic stirring. Adjust pH to 7.3 with sterile 2M HCl or sterile 2M Na 2 CO 3 so- lution. Aseptically and anaerobically distribute the medium through the medium outlet tube into sterile 100.0mL bottles under 95% N 2 and 5% CO 2 at 0.05–0.1 atm pressure. Leave a small gas bubble in each bottle to accommodate pressure changes. After 24 hr, the iron in the medium will precipitate out of solution as black flocs. Use: For the cultivation and maintenance of Chromatium gracile, Thi- ocystis gelatinosa, and Thiocystis violacea. © 2010 by Taylor and Francis Group, LLC 1378 Pfennig’s Medium 1 with Yeast Extract Pfennig’s Medium 1 with Yeast Extract Composition per 4990.0mL: Solution A 4000.0mL Solution B 860.0mL Solution E 100.0mL Solution F 20.0mL Solution C (Vitamin B 12 solution) 5.0mL Solution D (Trace elements solution SL-12B) 5.0mL pH 7.3 ± 0.2 at 25°C Solution A: Composition per 4000.0mL: CaCl 2 ·2H 2 O 1.25g KH 2 PO 4 1.7g NH 4 Cl 1.7g KCl 1.7g MgSO 4 2.5g Yeast extract 2.5g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 4.0L. Mix thoroughly. Adjust pH to 6.0. Dispense into a 5L flask with four openings at the top (two openings are in a central silicon rubber stopper and two openings are gas-tight screw caps). Add a teflon-coated magnetic stir bar to the flask. Auto- clave for 45 min at 15 psi pressure–121°C. Cool to room temperature under 100% N 2 at 0.05–0.1 atm pressure. Solution B: Composition per 860.0mL: Distilled/deionized water 860.0mL Preparation of Solution B: Add 860.0mL of distilled/deionized wa- ter to a cotton-stoppered flask. Autoclave for 20 min at 15 psi pressure– 121°C. Cool to room temperature under 100% N 2 in an anaerobic jar. Solution C (Vitamin B 12 Solution): Composition per 5.0mL: Vitamin B 12 1.0mg Preparation of Solution C (Vitamin B 12 Solution): Add vita- min B 12 to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize. Solution D (Trace Elements Solution SL-12B): Composition per liter: Disodium ethylendiamine-tetraacetate (Na 2 -EDTA) 3.0g FeSO 4 ·7H 2 O 1.1g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·2H 2 O 50.0mg ZnCl 2 42.0mg NiCl 2 ·6H 2 O 24.0mg Na 2 MoO 4 ·2H 2 O 18.0mg CuCl 2 ·2H 2 O 2.0mg Preparation of Solution D (Trace Elements Solution SL-12B): Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution E: Composition per 100.0mL: NaHCO 3 7.5g Preparation of Solution E: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster- ile, gas-tight 100.0mL screw-capped bottle. Solution F: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Solution F: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Dispense into a screw-capped bottle. Sparge with 100% N 2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Saturate cooled solution A under 100% CO 2 at 0.05–0.1 atm pressure for 30 min with magnetic stirring. Add 860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution D, 100.0mL of solution E, and 20.0mL of solution F through one of the screw-cappped openings under 95% N 2 and 5% CO 2 with magnetic stirring. Adjust pH to 7.3 with sterile 2M HCl or sterile 2M Na 2 CO 3 so- lution. Aseptically and anaerobically distribute the medium through the medium outlet tube into sterile 100.0mL bottles under 95% N 2 and 5% CO 2 at 0.05–0.1 atm pressure. Leave a small gas bubble in each bottle to accommodate pressure changes. After 24 hr, the iron in the medium will precipitate out of solution as black flocs. Use: For the cultivation and maintenance of Amoebobacter pendens. Pfennig’s Medium 2, Modified for Green Sulfur Bacteria Composition per 5000.0mL: Solution A 4000.0mL Solution B 860.0mL Solution E 100.0mL Solution F 30.0mL Solution C (Vitamin B 12 solution) 5.0mL Solution D (Trace elements solution SL-10B) 5.0mL pH 6.8 ± 0.2 at 25°C Solution A: Composition per 4000.0mL: CaCl 2 ·2H 2 O 1.25g KH 2 PO 4 1.7g NH 4 Cl 1.7g KCl 1.7g MgSO 4 2.5g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 4.0L. Mix thoroughly. Adjust pH to 6.0. Dispense into a 5L flask with four openings at the top (two openings are in a central silicon rubber stopper and two openings are gas-tight screw caps). Add a teflon-coated magnetic stir bar to the flask. Auto- clave for 45 min at 15 psi pressure–121°C. Cool to room temperature under 100% N 2 at 0.05–0.1 atm pressure (use a manometer to measure low pressure). Solution B: Composition per 860.0mL: Distilled/deionized water 860.0mL Preparation of Solution B: Add 860.0mL of distilled/deionized wa- ter to a cotton-stoppered flask. Autoclave for 20 min at 15 psi pressure– 121°C. Cool to room temperature under 100% N 2 in an anaerobic jar. Solution C (Vitamin B 12 Solution): Composition per 5.0mL: Vitamin B 12 1.0mg Preparation of Solution C (Vitamin B 12 Solution): Add vita- min B 12 to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize. © 2010 by Taylor and Francis Group, LLC Pfennig's Medium 2 Modified for Green Sulfur Bacteria 1379 Solution D (Trace Elements Solution SL-10B): Composition per liter: FeCl 2 ·4H 2 O 1.5g H 3 BO 3 300.0mg 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 CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Solution D (Trace Elements Solution SL-10B): Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add dis- tilled/deionized water and bring volume to 1.0L. Add remaining com- ponents. Mix thoroughly. Solution E: Composition per 100.0mL: NaHCO 3 7.5g Preparation of Solution E: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster- ile, gas-tight 100.0mL screw-capped bottle. Solution F: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Solution F: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Dispense into a screw-capped bottle. Sparge with 100% N 2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Saturate cooled solution A under 100% CO 2 at 0.05–0.1 atm pressure for 30 min with magnetic stirring. Add 860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution D, 100.0mL of solution E, and 20.0mL of solution F through one of the screw-cappped openings under 95% N 2 and 5% CO 2 with magnetic stirring. Adjust pH to 6.8 with sterile 2M HCl or sterile 2M Na 2 CO 3 so- lution. Aseptically and anaerobically distribute the medium through the medium outlet tube into sterile 100.0mL bottles under 95% N 2 and 5% CO 2 at 0.05–0.1 atm pressure. Leave a small gas bubble in each bottle to accommodate pressure changes. After 24 hr, the iron in the medium will precipitate out of solution as black flocs. Use: For the cultivation and maintenance of Chlorobium limicola and Chlorobium phaeovibrioides. Pfennig's Medium 2 Modified for Green Sulfur Bacteria (DSMZ Medium 29) Composition per 5.0L: Solution A 4.0L Solution B 860.0mL Solution E 100.0mL Solution F 30.0mL Solution C 5.0mL Solution D 5.0mL Neutralized sulfide solution As needed pH 6.8 at 25°C Solution A: Composition per 4.0L: MgSO 4 2.5g KH 2 PO 4 1.7g NH 4 Cl 1.7g KCl 1.7g CaCl 2 ·2H 2 O 1.25g Preparation of Solution A: Add components to 4.0L distilled wa- ter. Mix thoroughly. Autoclave for 45 min at 15 psi pressure–121°C in 5-liter special bottle or flask with four openings at the top, together with a teflon-coated magnetic bar. In this 5-liter bottle, two openings for tubes are in the central, silicon rubber stopper: a short, gas-inlet tube with a sterile cotton filter; and an outlet tube for medium, which reaches the bottom of the vessel at one end and has, at the other end, a silicon rubber tube with a pinch cock and a bell for aseptic dispensing of the medium into bottles. The other two openings have gas-tight screw caps; one of these openings is for the addition of sterile solutions and the other serves as a gas outlet. After autoclaving, cool to room temperature under a N 2 atmosphere with a positive pressure of 0.05– 0.1 atm (a manometer for low pressure will be required). Saturate the cold medium with CO 2 by magnetic stirring for 30 min under a CO 2 atmosphere of 0.05–0.1 atm. Solution B: Distilled water 860.0mL Preparation of Solution B: Autoclave distilled water for 15 min at 15 psi pressure–121°C in a cotton-stoppered Erlenmeyer flask. Cool to room temperature under an atmosphere of N 2 in an anaerobic jar. Solution C: Composition per 100.0mL: Vitamin B 12 2.0mg Preparation of Solution C: Add vitamin B 12 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge under 100% N 2 gas for 3 min. Filter sterilize. Store under N 2 gas. Solution D: 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 3 300.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 7.7mL Preparation of Solution D: Add FeCl 2 ·4H 2 O to 10.0mL of HCl so- lution. Mix thoroughly. Add distilled/deionized water and bring vol- ume to 1.0L. Add remaining components. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Solution E: Composition per 100.0mL: NaHCO 3 7.5g Preparation of Solution E: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster- ile, gas-tight 100.0mL screw-capped bottle. Solution F: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Solution F: Add Na 2 S·9H 2 O to distilled/deionized water in a 250.0mL screw-capped bottle fitted with a butyl rubber sep- tum and bring volume to 100.0mL. Mix thoroughly. Sparge under © 2010 by Taylor and Francis Group, LLC 1380 Pfennig’s Medium 2 with Salt 100% N 2 gas for 3 min. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Neutralized Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 1.5g Preparation of Neutralized Sulfide Solution: Add Na 2 S·9H 2 O to distilled/deionized water in a 250.0mL screw-capped bottle fitted with a butyl rubber septum and bring volume to 100.0mL. Add a mag- netic stir bar. Mix thoroughly. Sparge under 100% N 2 gas for 3 min. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temper- ature. Adjust pH to about 6.8 with sterile 2M H 2 SO 4 . Do not open the bottle to add H 2 SO 4 ; use a sterile syringe. Stir the solution continuously to avoid precipitation of elemental sulfur. The final solution should be clear and yellow in color. Preparation of Medium: Add solution B, C, D, E, and F to solution A through one of the screw-cap openings against a stream of either N 2 gas or, better, a mixture of 95% N 2 and 5% CO 2 while the medium is magnetically stirred. Adjust the pH of the medium with sterile HCl or Na 2 CO 3 solution (2M solution) to pH 6.8. Distribute the medium asep- tically through the medium outlet tube into sterile, 100mL bottles (with metal caps and autoclavable rubber seals) using the positive gas pres- sure (0.05–0.1 atm) of the N 2 /CO 2 gas mixture. Leave a small air bub- ble in each bottle to meet possible pressure changes. The tightly sealed, screw-cap bottles can be stored for several weeks or months in the dark. During the first 24 hr, the iron of the medium precipitates in the form of black flocs. No other sediment should arise in the otherwise clear medium. Incubate in the light using a tungsten lamp. Feed peri- odically with neutralized solution of sodium sulfide to replenish sulfide and with other supplement solutions. Use: For the cultivation of green sulfur bacteria. Pfennig’s Medium 2 with Salt Composition per 5000.0mL: Solution A 4000.0mL Solution B 860.0mL Solution E 100.0mL Solution F 30.0mL Solution C (Vitamin B 12 solution) 5.0mL Solution D (Trace elements solution SL-10B) 5.0mL pH 6.8 ± 0.2 at 25°C Solution A: Composition per 4000.0mL: NaCl 50.0g CaCl 2 ·2H 2 O 1.25g KH 2 PO 4 1.7g NH 4 Cl 1.7g KCl 1.7g MgSO 4 2.5g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 4.0L. Mix thoroughly. Adjust pH to 6.0. Dispense into a 5L flask with four openings at the top (two openings are in a central silicon rubber stopper and two openings are gas-tight screw caps). Add a teflon-coated magnetic stir bar to the flask. Auto- clave for 45 min at 15 psi pressure–121°C. Cool to room temperature under 100% N 2 at 0.05–0.1 atm pressure (use a manometer to measure low pressure). Solution B: Composition per 860.0mL: Distilled/deionized water 860.0mL Preparation of Solution B: Add 860.0mL of distilled/deionized wa- ter to a cotton-stoppered flask. Autoclave for 20 min at 15 psi pressure– 121°C. Cool to room temperature under 100% N 2 in an anaerobic jar. Solution C (Vitamin B 12 Solution): Composition per 5.0mL: Vitamin B 12 1.0mg Preparation of Solution C (Vitamin B 12 Solution): Add vita- min B 12 to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize. Solution D (Trace Elements Solution SL-10B): Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 0.19g 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 Solution D (Trace Elements Solution SL-10B): Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add dis- tilled/deionized water and bring volume to 1.0L. Add remaining com- ponents. Mix thoroughly. Solution E: Composition per 100.0mL: NaHCO 3 7.5g Preparation of Solution E: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 until saturated. Filter sterilize under 100% CO 2 into a ster- ile, gas-tight 100.0mL screw-capped bottle. Solution F: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Solution F: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Dispense into a screw-capped bottle. Sparge with 100% N 2 for 3–4 min. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Saturate cooled solution A under 100% CO 2 at 0.05–0.1 atm pressure for 30 min with magnetic stirring. Add 860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution D, 100.0mL of solution E, and 20.0mL of solution F through one of the screw-cappped openings under 95% N 2 and 5% CO 2 with magnetic stirring. Adjust pH to 6.8 with sterile 2M HCl or sterile 2M Na 2 CO 3 so- lution. Aseptically and anaerobically distribute the medium through the medium outlet tube into sterile 100.0mL bottles under 95% N 2 and 5% CO 2 at 0.05–0.1 atm pressure. Leave a small gas bubble in each bottle to accommodate pressure changes. After 24 hr, the iron in the medium will precipitate out of solution as black flocs. Use: For the cultivation and maintenance of Chlorobium phaeobacte- roides, Chlorobium vibrioforme, Pelodictyon luteolum, Pelodictyon phaeum, and Prosthecochloris aestuarii. Pfizer Selective Enterococcus Agar (PSE Agar) Peptone C 17.0g Agar 15.0g © 2010 by Taylor and Francis Group, LLC PFS Medium 1381 Bile 10.0g NaCl 5.0g Yeast extract 5.0g Peptone B 3.0g Esculin 1.0g Sodium citrate 1.0g Ferric ammonium citrate 0.5g NaN 3 0.25g pH 7.1 ± 0.2 at 25°C Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted. 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 selective isolation, cultivation, and enumeration of Enterococcus species by the multiple tube technique. Pfizer Selective Enterococcus HiVeg Agar Composition per liter: Plant hydrolysate 21.0g Agar 15.0g Plant peptone 6.0g NaCl 5.0g Yeast extract 5.0g Synthetic detergent 3.0g Esculin 1.0g Sodium citrate 1.0g Ferric ammonium citrate 0.5g NaN 3 0.25g pH 7.1 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Hi- Media. Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted. 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 selective isolation, cultivation, and enumeration of Enterococcus species by the multiple tube technique. Pfizer TB Medium Base with Glycerol, Egg Yolk, Glucose, and Malachite Green Composition per liter: Agar 15.0g Potato starch 15.0g Casein acid hydrolysate 10.0g K 2 HPO 4 3.5g Beef extract 3.0g L-Asparagine 3.0g Citric acid 0.1g Ferric ammonium citrate 0.1g MgSO 4 0.015g Egg yolk emulsion 100.0mL Glycerol 40.0mL Malachite Green solution 13.0mL Glucose solution 1.0mL pH 7.0 ± 0.2 at 35°C Source: This medium, without glycerol, glucose solution, Malachite Green solution, and egg yolk emulsion, is available as a premixed pow- der from HiMedia. Egg Yolk Emulsion: Composition per 100.0mL: Chicken egg yolks 9 Whole chicken egg 1 NaCl (0.9% solution) 25.0mL Preparation of Egg Yolk Emulsion: Soak eggs with 1:100 dilu- tion of saturated mercuric chloride solution for 1 min. Crack eggs and separate yolks from whites. Mix egg yolks with 1 chicken egg. Beat to form emulsion. Measure 50.0mL of egg yolk emulsion and add to 50.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Glucose Solution: Composition per 100.0mL: Glucose 20.0g Preparation of Glucose Solution: Add glucose to distilled/deion- ized water and bring volume to 100.0mL. Mix thoroughly. Filter ster- ilize. Malachite Green Solution: Composition per 20.0mL: Malachite Green 0.2g Preparation of Malachite Green Solution: Add Malachite Green to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add glycerol and the other components, except glucose solution, egg yolk emulsion, and Malachite Green solu- tion, to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 12 psi pressure–118°C. Cool to 55°C. Asepti- cally add 100.0mL egg yolk emulsion, 1.0mL glucose solution, and 13.0mL Malachite Green solution. Mix thoroughly. Aseptically dis- pense into sterile tubes. Allow to solidity as slants. Use: For the cultivation of Mycobacterium tuberculosis. PFS Medium (Peptone Fumarate Sulfate Medium) Composition per liter: Peptone 10.0g Fumaric acid 2.0g (NH 4 ) 2 SO 4 1.0g MgSO 4 ·7H 2 O 0.5g FeCl 3 ·6H 2 O 0.2mg MnSO 4 ·H 2 O 0.2mg 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 with KOH. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Aquaspirillum fasciculus. © 2010 by Taylor and Francis Group, LLC 1382 PGA PGA (Potato Glucose Agar) Composition per liter: Potatoes 500.0g Glucose 20.0g Agar 15.0g Yeast extract 5.0g Preparation of Medium: Slice potatoes with skin. Place 500.0g of potatoes in 1.0L of distilled/deionized water. Gently heat and bring to boiling. Allow to boil for 20 min. Filter through Whatman filter paper. Add agar and other components to filtrate. Bring volume to 1.0L with distilled/deionized water. 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 numerous filamentous fungi. PGLE Medium (Peptone Glucose Liver Extract Medium) Composition per liter: Agar 25.0g Glucose 20.0g Peptone 5.0g Yeast extract 5.0g K 2 HPO 4 1.0g Liver extract 0.5g pH 9.0 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat 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 Enterobacter cloacae. PGP Broth (Peptone Glycerol Phosphate Broth) Composition per liter: Peptone 5.0g K 2 HPO 4 2.0g Glycerol 10.0mL 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 Serratia marcescens. PGS Agar (Peptone Glucose Salt Agar) Composition per liter: Agar 15.0g Glucose 10.0g NaCl 10.0g Peptone 10.0g 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 Rhodococcus australis. PGT Medium Composition per liter: Casamino acids 30.0g L-Glutamic acid 0.5g MgSO 4 ·7H 2 O 0.45g Maltose 0.2g L-Cystine 0.2g DL-Tryptophan 0.1g Solution 3 100.0mL Solution 2 2.0mL Calcium pantothenate (0.1% solution) 0.5mL pH 6.8 ± 0.2 at 25°C Solution 3: Composition per 500.0mL: Maltose 200.0g CaCl 2 1.5g Calcium pantothenate (0.1% solution) 3.0mL FeSO 4 (1% in 1N HCl) 0.2mL Preparation of Solution 3: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Autoclave for 15 min at 7 psi pressure–111°C. Cool to 45°–50°C. Solution 2: Composition per 100.0mL: β-Alanine 0.115g Nicotinic acid 0.115g CuSO 4 ·5H 2 O 0.05g ZnSO 4 ·7H 2 O 0.045g MnCl 2 ·4H 2 O 0.015g Pimelic acid 7.5mg HCl, concentrated 3.0mL Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Preparation of Medium: Add components, except solution 3, to dis- tilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Ad- just pH to 6.8 with 50% KOH. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile solution 3. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Corynebacterium diphtheriae. PGY Agar (Peptone Glucose Yeast Extract Agar) Composition per liter: Agar 15.0g Peptone 10.0g Yeast extract 5.0g Glucose 1.0g 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 Micrococcus luteus. PH Medium (DSMZ Medium 1077) Composition per 1118.0mL: Pancreatic digest of casein 7.0g NaCl 5.0g © 2010 by Taylor and Francis Group, LLC PHB/Pyruvate Medium 1383 Beef extract 3.0g Yeast extract 3.0g Beef heart, solids from infusion 2.0g Supplement solution 200.0mL pH 7.8 ± 0.2 at 25°C Supplement Solution: Composition per liter: Horse serum, inactivated 187.0mL Yeast extract solution 93.6mL Fish sperm DNA solution, filter sterilized 18.7mL Yeast Extract Solution: Composition per 100.0mL: Yeast extract 25.0g Preparation of Yeast Extract Solution: Add yeast extract to dis- tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Preparation of Supplement Solution: Aseptically add compo- nents to sterile distilled/deionized water and bring volume to 318.0mL. Mix thoroughly. Preparation of Medium: Add components, except supplement so- lution, to distilled/deionized water and bring volume to 800.0L. Mix thoroughly. Adjust pH to 6.5. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 25°C. Aseptically add 318.0mL of supplement solu- tion. Adjust pH to 7.8. Mix thoroughly. Aseptically distribute into cul- ture vessels. Use: For the cultivation of Mycoplasma orale. PHB Delafield Agar (LMG Medium 123) Composition per liter: Agar 20.0g NH 4 Cl 1.0g MgSO 4 ·7H 2 O 0.5g Casamino acids 0.1g Ferric ammonium citrate 0.05g Yeast extract 0.05g Phosphate buffer solution 1.0L Solution A/B 264.0mL pH 6.8 ± 0.2 at 25°C Phosphate Buffer Solution: Composition per liter: KH 2 PO 4 4.5g Na 2 HPO 4 ·2H 2 O 5.8g Preparation of Phosphate Buffer Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.8. Solution A/B: Composition per 700.0mL: Solution A 140.0mL Solution B 560.0mL Solution A: Composition per 140.0mL: Poly-β-hydroxybutyrate, powdered 1.75g Preparation of Solution A: Add poly-β-hydroxybutyrate to distilled/ deionized water and bring volume to 140.0mL. Mix thoroughly. Sonicate for 30 min. Autoclave for 15 min at 15 psi pressure–121°C. Sonicate again for 30 min in the sterile bottle. Warm to 50°C. Solution B: Composition per 560.0mL: Agar 14.0g Preparation of Solution B: Add agar to distilled/deionized water and bring volume to 560.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Preparation of Solution A/B: Aseptically combine 140.0mL of solution A with 560.0mL of solution B. Warm to 50°C. Preparation of Medium: Combine components except solution A/ B. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes. Allow agar to solidify. Bring the Petri dishes to 65°C. Aseptically add 4.0mL of sterile solution A/B onto the surface of each agar plate. Use: For the cultivation and maintenance of Pseudomonas lemoignei Delafield. PHB Medium See: Poly-β-Hydroxybutyrate Medium PHB/Pyruvate Medium (DSMZ Medium 193a) Composition per 991.0mL: Solution A 870.0mL Solution C 100.0mL Solution D 10.0mL Solution E (Vitamin solution) 10.0mL Solution B (Trace elements solution SL-10) 1.0mL pH 7.1–7.4 at 25°C Solution A: Composition per 870.0mL: Na-pyruvate 5.0g NaCl 7.0g Na 2 SO 4 3.0g MgCl 2 ·6H 2 O 1.3g Poly-hydroxybutyrate (PHB) 1.0g KH 2 PO 4 0.2g NH 4 Cl 0.3g KCl 0.5g CaCl 2 ·2H 2 O 0.15g Resazurin 1.0mg Preparation of Solution A: Add components to distilled/deionized water and bring volume to 870.0mL Mix thoroughly. Solution B (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 Solution B (Trace Elements Solution SL-10): Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add dis- tilled/deionized water and bring volume to 1.0L. Add remaining com- ponents. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. © 2010 by Taylor and Francis Group, LLC 1384 PHC Medium Solution C: Composition per 100.0mL: NaHCO 3 5.0g Preparation of Solution C: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Flush with 80% N 2 + 20% CO 2 to remove dissolved oxygen. Solution D: Composition per 10.0mL: Na-acetate·3H 2 O 2.5g Preparation of Solution D: Add Na-acetate·3H 2 O to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Solution E (Vitamin Solution): Composition per liter: 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 0.10mg Solution E (Vitamin Solution): Add components to distilled/de- ionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Solution F: Composition per 10.0mL: Na 2 S·9H 2 O 0.4g Preparation of Solution F: 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: Gently heat solution A and bring to boil- ing. Boil solution A for a few minutes. Cool to room temperature. Gas with 80% N 2 + 20% CO 2 gas mixture to reach a pH below 6. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Se- quentially add 1.0mL solution B, 100.0mL solution C, 10.0mL solu- tion D, 10.0mL solution E, and 10.0mL solution F. Distribute anaerobically under 80% N 2 + 20% CO 2 into appropriate vessels. Ad- dition of 10–20mg sodium dithionite per liter from a 5% (w/v) solution, freshly prepared under N 2 and filter sterilized, may stimulate growth. After inoculation, pressurize vessels up to 1 bar H 2 + CO 2 (80% + 20%) overpressure. Use: For the cultivation of unclassified bacterium DSM 6754. PHC Medium (Polyhexamethylene Carbonate Medium) (DSMZ Medium 885) K 2 HPO 4 1.6g Polyhexamethylene carbonate, emulsified 1.0g (NH 4 ) 2 SO 4 1.0g YKH 2 PO 4 0.2g Yeast extract 0.1g Surfactant, Plysurf A210G 60.0mg CaCl 2 ·2H 2 O 20.0mg NaCl 10.0mg FeSO 4 ·7H 2 O 10.0mg Na 2 MoO 4 ·4H 2 O 0.5mg Na 2 WO 4 ·2H 2 O 0.5mg MnSO 4 0.5mg 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 of Roseateles depolymerans. Phenethyl Alcohol Agar (Phenylethanol Agar) (Phenylethyl Alcohol Agar) Composition per liter: Agar 15.0g Pancreatic digest of casein 15.0g NaCl 5.0g Papaic digest of soybean meal 5.0g β-Phenethyl alcohol 2.5g Blood 50.0mL pH 7.3 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Preparation of Medium: Add components, except blood, to dis- tilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 13 psi pres- sure–118°C. Cool to 45°–50°C. Aseptically add sterile defibrinated blood. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the selective isolation of Gram-positive bacteria, particularly Gram-positive cocci, from specimens with a mixed flora. Do not use for the observation of hemolytic reactions. Phenol Nutrient-Supplemented Broth Composition per 1000.05mL: (NH 4 ) 2 SO 4 2.0g Na 2 HPO 4 1.0g Nutrient broth 20.0mL Phenol (90% solution) 1.05mL pH 6.8 ± 0.2 at 25°C Nutrient Broth: Composition per liter: Peptone 5.0g Beef extract 3.0g Preparation of Nutrient Broth: Add components to tap water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boil- ing. Preparation of Medium: Add components, except phenol, to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Au- toclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add phenol. Mix thoroughly. Use: For the cultivation of ATCC strain 1413. Phenol Red Adonitol Broth Composition per liter: Proteose peptone 10.0g NaCl 5.0g © 2010 by Taylor and Francis Group, LLC . min with magnetic stirring. Add 860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution D, 100.0mL of solution E, and 20.0mL of solution F through one of the screw-cappped openings under 95%. temperature. Preparation of Medium: Add solutions B, C, D, E, and F to solu- tion A through one of the screw-cap openings against a stream of either N 2 gas or, better, a mixture of 95% N 2 and 5%. pressure–121°C. Preparation of Medium: Saturate cooled solution A under 100% CO 2 at 0.05–0.1 atm pressure for 30 min with magnetic stirring. Add 860.0mL of solution B, 5.0mL of solution C, 5.0mL of solution