Thioglycolate Medium, USP 1775 Thioglycolate Medium without Indicator Composition per liter: Pancreatic digest of casein 17.0g Glucose 6.0g Papaic digest of soybean meal 3.0g NaCl 2.5g Agar 0.7g Sodium thioglycolate 0.5g L-Cystine 0.25g Na 2 SO 3 0.1g pH 7.0 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath. 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. Prepare freshly or boil and cool the medium just before use. Use: For the growth of aerobic and anaerobic microorganisms in diag- nostic bacteriology. Thioglycolate Medium without Indicator-135C Composition per liter: Pancreatic digest of casein 17.0g Glucose 6.0g Papaic digest of soybean meal 3.0g NaCl 2.5g Agar 0.7g Sodium thioglycolate 0.5g Na 2 SO 3 0.1g L-Cystine 0.25g pH 7.0 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic 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, filling them half full. For maintenance of cultures, a small quantity of CaCO 3 may be added to tubes before adding medium. Autoclave for 15 min at 13 psi pressure–118°C. Prepare freshly or boil and cool the medium just before use. Store prepared medium at 2°–8°C in the dark. Use: For the isolation and cultivation of a wide variety of microorgan- isms, particularly obligate anaerobes, from clinical specimens and other materials. Thioglycolate Medium without Indicator with Hemin (Thioglycolate Medium, Supplemented) Composition per liter: Pancreatic digest of casein 17.0g CaCO 3 , chips or powder 10.0g Glucose 6.0g Papaic digest of soybean meal 3.0g NaCl 2.5g Agar 0.7g Sodium thioglycolate 0.5g L-Cystine 0.25g Na 2 SO 3 0.1g Hemin 5.0mg Na 2 CO 3 solution 10.0mL Vitamin K 1 solution 10.0mL pH 7.2 ± 0.2 at 25°C Na 2 CO 3 Solution: Composition per 10.0mL: Na 2 CO 3 1.0g Preparation of Na 2 CO 3 Solution: Add Na 2 CO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Filter ster- ilize. Vitamin K 1 Solution: Composition per 100.0mL: Vitamin K 1 1.0g Ethanol, absolute 99.0mL Preparation of Vitamin K 1 Solution: Add vitamin K 1 to 99.0mL of absolute ethanol. Mix thoroughly. Preparation of Medium: Add components, except CaCO 3 , Na 2 CO 3 solution, and vitamin K 1 solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Add 0.1g of CaCO 3 chips or powder to each of 100 test tubes. Distribute broth into the same tubes in 10.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 0.1mL of sterile Na 2 CO 3 solution and 0.1mL of sterile vitamin K 1 so- lution to each tube. Mix thoroughly. Use: For the cultivation of a wide variety of obligate anaerobes. Thioglycolate Medium, Supplemented See: Thioglycolate Medium without Indicator with Hemin Thioglycolate Medium, USP Composition per liter: Pancreatic digest of casein 15.0g Glucose 5.5g Yeast extract 5.0g NaCl 2.5g Agar 0.5g L-cystine 0.5g Sodium thioglycolate 0.5g Resazurin 1.0mg pH 7.1 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath. 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. Use: For the cultivation of both aerobic and anaerobic organisms in the performance of sterility tests. Thioglycolate Medium with Vitamin K 1 and Hemin See: Thioglycolate Medium, Enriched Thioglycolate Peptone Glucose Yeast Extract Medium See: TPGY Medium Thioglycolate Potato Liver Medium See: TPL Medium © 2010 by Taylor and Francis Group, LLC 1776 Thiol Broth Thiol Broth Composition per liter: Proteose peptone, No. 3 10.0g Thiol complex 8.0g Yeast extract 5.0g NaCl 5.0g Glucose 1.0g p-Aminobenzoic acid 0.05g pH 7.1 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. 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. For neutralization of penicil- lin, distribute medium into tubes to a depth of 60.0mm. For neutraliza- tion of streptomycin, distribute medium into tubes in shallow layers. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of bacteria from body fluids and other mate- rials containing penicillin, streptomycin, or sulfonamides. Also used for the cultivation and maintenance of Bifidobacterium species. Thiohalophilus Medium (DSMZ Medium 1058) Composition per liter: NaCl 120.0g K 2 HPO 4 1.5g NH 4 Cl 0.5g Bicarbonate solution 10.0mL Calcium chloride solution 10.0mL Magnesium chloride solution 10.0mL Seven vitamin solution 1.0mL Trace elements solution SL-10 with EDTA 1.0mL Carbonate solution variable pH 7.6 ± 0.2 at 25°C Thiosulfate Solution: Composition per 10.0mL: Na 2 S 2 O 3 ·5H 2 O 5.0g Preparation of Thiosulfate Solution: Add components to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Bicarbonate Solution: Composition per 10.0mL: NaHCO 3 5.0g Preparation of Bicarbonate Solution: Add NaHCO 3 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. Carbonate Solution: Composition per 10.0mL: Na 2 CO 3 2.0g Preparation of Carbonate Solution: Add Na 2 CO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. Magnesium Chloride Solution: Composition per 10.0mL: MgCl 2 ·6H 2 O 0.4g Preparation of Magnesium Chloride Solution: Add 0.4g of MgCl 2 ·6H 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. Sparge with 100% N 2 . Calcium Chloride Solution: Composition per 10.0mL: CaCl 2 ·2H 2 O 0.05g Preparation of Calcium Chloride Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room tem- perature. Sparge with 100% N 2 gas. Seven Vitamin Solution: Composition per liter: Pyridoxine·HCl 300.0mg Nicotinic acid 200.0mg Thiamine·HCl 200.0mg Calcium DL-pantothenate 100.0mg Cyanocobalamine 100.0mg p-Aminobenzoic acid 80.0mg D(+)-Biotin 20.0mg Preparation of Seven Vitamin Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N 2 . Trace Elements Solution SL-10 with EDTA: Composition per liter: FeCl 2 ·4H 2 O 1.5g Na 2 -EDTA 0.5g 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 with EDTA: 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. Adjust pH to 7.0. Preparation of Medium: Add components, except magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions, to distilled/ deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 gas mixture for at least 30 min to remove dis- solved oxygen and to saturate the solution with CO 2 . Dispense into an- aerobic culture vessels (e.g., Balch tubes) to 1/2 volume under air atmosphere. Close vials with butyl rubber septa to prevent free ex- change of oxygen with the external atmosphere. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions. Mix thoroughly. Adjust pH to 7.5–7.8 using a sterile solution of sodium carbonate (5% w/v). Use: For the cultivation of Thiohalomonas denitrificans, Thiohalo- philus thiocyanatoxydans, and Thiomicrospira halophila. Thiohalophilus Medium (DSMZ Medium 1058) Composition per liter: NaCl 233.0g K 2 HPO 4 1.5g © 2010 by Taylor and Francis Group, LLC Thiohalophilus Medium 1777 NH 4 Cl 0.5g Bicarbonate solution 10.0mL Calcium chloride solution 10.0mL Magnesium chloride solution 10.0mL Seven vitamins solution 1.0mL Trace elements solution SL-10 with EDTA 1.0mL Carbonate solution variable pH 7.2 ± 0.2 at 25°C Thiosulfate Solution: Composition per 10.0mL: Na 2 S 2 O 3 ·5H 2 O 5.0g Preparation of Thiosulfate Solution: Add Na 2 S 2 O 3 ·5H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Bicarbonate Solution: Composition per 10.0mL: NaHCO 3 5.0g Preparation of Bicarbonate Solution: Add NaHCO 3 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. Carbonate Solution: Composition per 10.0mL: Na 2 CO 3 2.0g Preparation of Carbonate Solution: Add Na 2 CO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. Magnesium Chloride Solution: Composition per 10.0mL: MgCl 2 ·6H 2 O 0.4g Preparation of Magnesium Chloride Solution: Add 0.4g of MgCl 2 ·6H 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. Sparge with 100% N 2 . Calcium Chloride Solution: Composition per 10.0mL: CaCl 2 ·2H 2 O 0.05g Preparation of Calcium Chloride Solution: Add CaCl 2 ·2H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Sparge with 100% N 2 . Seven Vitamin Solution: Composition per liter: Pyridoxine·HCl 300.0mg Nicotinic acid 200.0mg Thiamine·HCl 200.0mg Calcium DL-pantothenate 100.0mg Cyanocobalamine 100.0mg p-Aminobenzoic acid 80.0mg D(+)-Biotin 20.0mg Preparation of Seven Vitamin Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N 2 . Trace Elements Solution SL-10 with EDTA: Composition per liter: FeCl 2 ·4H 2 O 1.5g Na 2 -EDTA 0.5g 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 with EDTA: 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. Adjust pH to 7.0. Preparation of Medium: Add components, except magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions, to distilled/ deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 gas mixture for at least 30 min to remove dis- solved oxygen and to saturate the solution with CO 2 . Dispense into an- aerobic culture vessels (e.g., Balch tubes) to 1/2 volume under air atmosphere. Close vials with butyl rubber septa to prevent free ex- change of oxygen with the external atmosphere. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions. Mix thoroughly. Adjust pH to 7.2 using a sterile solution of sodium car- bonate (5% w/v). Use: For the cultivation of Thiomicrospira halophila DSM 15071. Thiohalophilus Medium (DSMZ Medium 1058) Composition per liter: NaCl 180.0g K 2 HPO 4 1.5g KNO 3 1.0g NH 4 Cl 0.5g Bicarbonate solution 10.0mL Calcium chloride solution 10.0mL Magnesium chloride solution 10.0mL Seven vitamin solution 1.0mL Trace elements solution SL-10 with EDTA 1.0mL Carbonate solution Variable pH 7.6 ± 0.2 at 25°C Thiosulfate Solution: Composition per 10.0mL: Na 2 S 2 O 3 ·5H 2 O 5.0g Preparation of Thiosulfate Solution: Add Na 2 S 2 O 3 ·5H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Bicarbonate Solution: Composition per 10.0mL: NaHCO 3 5.0g Preparation of Bicarbonate Solution: Add NaHCO 3 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. Carbonate Solution: Composition per 10.0mL: Na 2 CO 3 2.0g Preparation of Carbonate Solution: Add Na 2 CO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. © 2010 by Taylor and Francis Group, LLC 1778 Thiohalophilus Medium Magnesium Chloride Solution: Composition per 10.0mL: MgCl 2 ·6H 2 O 0.4g Preparation of Magnesium Chloride Solution: Add 0.4g of MgCl 2 ·6H 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. Sparge with 100% N 2 . Calcium Chloride Solution: Composition per 10.0mL: CaCl 2 ·2H 2 O 0.05g Preparation of Calcium Chloride Solution: Add CaCl 2 ·2H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Sparge with 100% N 2 . Seven Vitamin Solution: Composition per liter: Pyridoxine·HCl 300.0mg Nicotinic acid 200.0mg Thiamine·HCl 200.0mg Calcium DL-pantothenate 100.0mg Cyanocobalamine 100.0mg p-Aminobenzoic acid 80.0mg D(+)-Biotin 20.0mg Preparation of Seven Vitamin Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N 2 . Trace Elements Solution SL-10 with EDTA: Composition per liter: FeCl 2 ·4H 2 O 1.5g Na 2 -EDTA 0.5g 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 with EDTA: 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. Adjust pH to 7.0. Preparation of Medium: Add components, except magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions, to distilled/ deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 gas mixture for at least 30 min. to remove dis- solved oxygen and to saturate the solution with CO 2 . Dispense into an- aerobic culture vessels (e.g., Balch tubes) to 1/2 volume under air atmosphere. Close vials with butyl rubber septa to prevent free ex- change of oxygen with the external atmosphere. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions. Mix thoroughly. Adjust pH to 7.5–7.8 using a sterile solution of sodium carbonate (5% w/v). Use: For the cultivation of Thiohalomonas denitrificans. Thiohalophilus Medium (DSMZ Medium 1058) Composition per liter: NaCl 180.0g K 2 HPO 4 1.5g KNO 3 2.0g NH 4 Cl 0.5g Bicarbonate solution 10.0mL Calcium chloride solution 10.0mL Magnesium chloride solution 10.0mL Seven vitamin solution 1.0mL Trace elements solution SL-10 with EDTA 1.0mL Carbonate solution Variable pH 7.6 ± 0.2 at 25°C Thiosulfate Solution: Composition per 10.0mL: Na 2 S 2 O 3 ·5H 2 O 5.0g Preparation of Thiosulfate Solution: Add Na 2 S 2 O 3 ·5H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Bicarbonate Solution: Composition per 10.0mL: NaHCO 3 5.0g Preparation of Bicarbonate Solution: Add NaHCO 3 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. Carbonate Solution: Composition per 10.0mL: Na 2 CO 3 2.0g Preparation of Carbonate Solution: Add Na 2 CO 3 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. Magnesium Chloride Solution: Composition per 10.0mL: MgCl 2 ·6H 2 O 0.4g Preparation of Magnesium Chloride Solution: Add 0.4g of MgCl 2 ·6H 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. Sparge with 100% N 2 . Calcium Chloride Solution: Composition per 10.0mL: CaCl 2 ·2H 2 O 0.05g Preparation of Calcium Chloride Solution: Add CaCl 2 ·2H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Sparge with 100% N 2 . Seven Vitamin Solution: Composition per liter: Pyridoxine·HCl 300.0mg Nicotinic acid 200.0mg Thiamine·HCl 200.0mg Calcium DL-pantothenate 100.0mg Cyanocobalamine 100.0mg p-Aminobenzoic acid 80.0mg D(+)-Biotin 20.0mg © 2010 by Taylor and Francis Group, LLC Thiomicrospira denitrificans Agar 1779 Preparation of Seven Vitamin Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Sparge with 100% N 2 . Trace Elements Solution SL-10 with EDTA: Composition per liter: FeCl 2 ·4H 2 O 1.5g Na 2 -EDTA 0.5g 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 with EDTA: 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. Adjust pH to 7.0. Preparation of Medium: Add components, except magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions, to distilled/ deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 gas mixture for at least 30 min. to remove dis- solved oxygen and to saturate the solution with CO 2 . Dispense into an- aerobic culture vessels (e.g., Balch tubes) to 1/2 volume under air atmosphere. Close vials with butyl rubber septa to prevent free ex- change of oxygen with the external atmosphere. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add magnesium chloride, thiosulfate, bicarbonate, and vitamin solutions. Mix thoroughly. Adjust pH to 7.5–7.8 using a sterile solution of sodium carbonate (5% w/v). Use: For the cultivation of Thiohalomonas nitratireducens. Thiol HiVeg Broth Composition per liter: Plant peptone No. 3 10.0g Thiol compound 8.0g NaCl 5.0g Yeast extract 5.0g Glucose 1.0g p-Aminobenzoic acid (PABA) 0.05g pH 7.1 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Hi- Media. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. For neutralization of penicil- lin, distribute medium into tubes to a depth of 60.0mm. For neutraliza- tion of streptomycin, distribute medium into tubes in shallow layers. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of bacteria from body fluids and other mate- rials containing penicillin, streptomycin, or sulfonamides. Also used for the cultivation and maintenance of Bifidobacterium species. Thiol HiVeg Medium Composition per liter: Plant peptone No. 3 10.0g Thiol compound 8.0g Yeast extract 5.0g NaCl 5.0g Agar 1.0g Glucose 1.0g p-Aminobenzoic acid (PABA) 0.05g pH 7.1 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Hi- Media. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. For neutralization of penicil- lin, distribute medium into tubes to a depth of 60 mm. For neutraliza- tion of streptomycin, distribute medium into tubes in shallow layers. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of bacteria from body fluids and other mate- rials containing penicillin, streptomycin, or sulfonamides. Also used for the cultivation and maintenance of Bifidobacterium species. Thiol Medium Composition per liter: Proteose peptone No. 3 10.0g Thiol complex 8.0g Yeast extract 5.0g NaCl 5.0g Glucose 1.0g Agar 1.0g p-Aminobenzoic acid 0.05g pH 7.1 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. 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. For neutralization of penicil- lin, distribute medium into tubes to a depth of 60mm. For neutraliza- tion of streptomycin, distribute medium into tubes in shallow layers. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of bacteria from body fluids and other mate- rials containing penicillin, streptomycin, or sulfonamides. Also used for the cultivation and maintenance of Bifidobacterium species. Thiomicrospira denitrificans Agar Composition per 1001.0mL: Solution A 940.0mL Solution B 40.0mL Solution C 20.0mL Solution D 1.0mL pH 7.0 ± 0.2 at 25°C Solution A: Composition per 940.0mL: Agar 15.0g KH 2 PO 4 2.0g KNO 3 2.0g NH 4 Cl 1.0g MgSO 4 ·7H 2 O 0.8g Trace elements solution SL-4 2.0mL Preparation of Solution A: Add components to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Gently heat and © 2010 by Taylor and Francis Group, LLC 1780 Thiomicrospira denitrificans Broth bring to boiling. Adjust pH to 7.0 with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Trace Elements Solution SL-4: Composition per liter: EDTA 0.5g FeSO 4 ·7H 2 O 0.2g Trace elements solution SL-6 100.0 Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Trace Elements Solution SL-6: Composition per liter: H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g MnCl 2 ·4H 2 O 0.03g Na 2 MoO 4 ·H 2 O 0.03g NiCl 2 ·6H 2 O 0.02g CuCl 2· ·2H 2 O 0.01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4. Solution B: Composition per 40.0mL: Na 2 S 2 O 3 ·5H 2 O 5.0g Preparation of Solution B: Add Na 2 S 2 O 3 ·5H 2 O to distilled/deion- ized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Solution C: Composition per 20.0mL: NaHCO 3 1.0g Preparation of Solution C: Add NaHCO 3 to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Solution D: Composition per liter: FeSO 4 ·7H 2 O 2.0mg H 2 SO 4 (0.1N solution) 1.0mL Preparation of Solution D: Add FeSO 4 ·7H 2 O to 1.0mL of 0.1N H 2 SO 4 solution. Mix thoroughly. Autoclave for 15 min at 15 psi pres- sure–121°C. Cool to 45°–50°C. Preparation of Medium: Aseptically add 40.0mL of sterile solu- tion B, 20.0mL of sterile solution C, and 1.0mL of sterile solution D to 940.0mL of sterile solution A. Mix thoroughly. Aseptically and anaer- obically distribute into sterile tubes under 100% N 2 . Use: For the cultivation and maintenance of Thiomicrospira denitrifi- cans. Thiomicrospira denitrificans Broth Composition per 1001.0mL: Solution A 940.0mL Solution B 40.0mL Solution C 20.0mL Solution D 1.0mL pH 7.0 ± 0.2 at 25°C Solution A: Composition per 940.0mL: KH 2 PO 4 2.0g KNO 3 2.0g NH 4 Cl 1.0g MgSO 4 ·7H 2 O 0.8g Trace elements solution SL-4 2.0mL Preparation of Solution A: Add components to distilled/deionized water and bring volume to 940.0mL. Mix thoroughly. Adjust pH to 7.0 with NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Solution B: Composition per 40.0mL: Na 2 S 2 O 3 ·5H 2 O 5.0g Preparation of Solution B: Add Na 2 S 2 O 3 ·5H 2 O to distilled/deion- ized water and bring volume to 40.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Solution C: Composition per 20.0mL: NaHCO 3 1.0g Preparation of Solution C: Add NaHCO 3 to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Solution D: Composition per liter: FeSO 4 ·7H 2 O 2.0mg H 2 SO 4 (0.1N solution) 1.0mL Preparation of Solution D: Add FeSO 4 ·7H 2 O to 1.0mL of 0.1N H 2 SO 4 solution. Mix thoroughly. Autoclave for 15 min at 15 psi pres- sure–121°C. Cool to 45°–50°C. Trace Elements Solution SL-4: Composition per liter: EDTA 0.5g FeSO 4 ·7H 2 O 0.2g Trace elements solution SL-6 100.0mL Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Trace Elements Solution SL-6: Composition per liter: H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g MnCl 2 ·4H 2 O 0.03g Na 2 MoO 4 ·H 2 O 0.03g NiCl 2 ·6H 2 O 0.02g CuCl 2· 2H 2 O 0.01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 3.4. Preparation of Medium: Aseptically add 40.0mL of sterile solution B, 20.0mL of sterile solution C, and 1.0mL of sterile solution D to 940.0mL of sterile solution A. Mix thoroughly. Aseptically and anaero- bically distribute into sterile tubes under 100% N 2 . Use: For the cultivation and maintenance of Thiomicrospira denitrifi- cans. © 2010 by Taylor and Francis Group, LLC Thiomicrospira Medium 1781 Thiomicrospira denitrificans Medium Composition per liter: Part I 500.0mL Part II 500.0mL pH 7.0–8.0 at 25°C Part I: Composition per liter: NaCl 20.0g KNO 3 4.0g (NH 4 ) 2 SO 4 2.0g MgSO 4 ·7H 2 O 1.5g K 2 HPO 4 0.6g KH 2 PO 4 0.4g FeSO 4 solution 2.0mL Trace metals solution 2.0mL HCl, concentrated 1.0mL Preparation of Part I: Add components to distilled/deionized wa- ter and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. FeSO 4 Solution: Composition per 100.0mL: FeSO 4 ·7H 2 O 0.5g HCl (1N solution) 100.0mL Preparation of FeSO 4 Solution: Combine the FeSO 4 ·7H 2 O and 100.0mL of HCl solution. Mix thoroughly. Trace Metals Solution: Composition per liter: Disodium EDTA 50.0g NaOH 11.0g CaCl 2 ·2H 2 O 7.34g MnCl 2 ·2H 2 O 2.5g ZnSO 4 ·7H 2 O 2.2g CoCl 2 ·6H 2 O 0.5g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.5g CuSO 4 ·5H 2 O 0.2g Preparation of Trace Metals Solution: Add EDTA to distilled/ deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust pH to 6.0 with NaOH. Add remaining components, one by one. Main- tain the pH at 6.0. After dissolution of all the salts, adjust the pH to 4.0 with HCl. Store at 4°C. Part II: Composition per liter: Na 2 S 2 O 3 ·5H 2 O 10.0g NaHCO 3 3.0g NaOH 0.05g Preparation of Part II: Add components to distilled/deionized wa- ter and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Aseptically combine 500.0mL of sterile part I and 500.0mL of sterile part II. Mix thoroughly. Aseptically dis- tribute into sterile tubes or flasks. Use: For the isolation and cultivation of Thiomicrospira denitrificans. Thiomicrospira Medium (ATCC Medium 1036) Composition per liter: NaCl 25.0g Na 2 S 2 O 3 ·5H 2 O 8.0g MgSO 4 ·7H 2 O 1.5g (NH 4 ) 2 SO 4 1.0g K 2 HPO 4 0.5g CaCl 2 0.3g Vitamin B 12 15.0μg Vishniac and Santer trace metals 0.2mL Bromcresol Purple (0.05% solution) 0.1mL pH 7.2 ± 0.2 at 25°C Vishniac and Santer Trace Metals: Composition per liter: Ethylenediamine tetraacetic acid (EDTA) 50.0g ZnSO 4 ·7H 2 O 22.0g CaCl 2 5.54g MnCl 2 ·4H 2 O 5.06g FeSO 4 ·7H 2 O 4.99g CoCl 2 ·6H 2 O 1.61g CuSO 4 ·5H 2 O 1.57g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g Preparation of Vishniac and Santer Trace Metals: Add com- ponents to distilled/deionized water and bring volume to 1.0L. Adjust pH to 6.0 with KOH. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2. Fil- ter sterilize. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Thiomicrospira species. Thiomicrospira Medium (ATCC Medium 1422) Composition per liter: NaCl 25.1g Tris·HCl 3.07g Na 2 S 2 O 3 ·5H 2 O 2.48g MgSO 4 ·7H 2 O 1.5g (NH 4 ) 2 SO 4 1.0g KH 2 PO 4 0.42g CaCl 2 ·2H 2 O 0.29g NaHCO 3 0.2g Phenol Red (0.5% solution) 1.0mL Vishniac and Santer trace metals 0.2mL pH 7.5 ± 0.2 at 25°C Vishniac and Santer Trace Metals: Composition per liter: Ethylenediamine tetraacetic acid (EDTA) 50.0g ZnSO 4 ·7H 2 O 22.0g CaCl 2 5.54g MnCl 2 ·4H 2 O 5.06g FeSO 4 ·7H 2 O 4.99g CoCl 2 ·6H 2 O 1.61g CuSO 4 ·5H 2 O 1.57g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g Preparation of Vishniac and Santer Trace Metals: Add com- ponents to distilled/deionized water and bring volume to 1.0L. Adjust pH to 6.0 with KOH. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5. Fil- ter sterilize. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Thiomicrospira species. © 2010 by Taylor and Francis Group, LLC 1782 Thiomicrospira pelophila Medium Thiomicrospira pelophila Medium Composition per liter: NaCl 25.0g Agar 10.0g Na 2 S 2 O 3 ·5H 2 O 5.0–8.0g MgSO 4 ·7H 2 O 1.5g (NH 4 ) 2 SO 4 1.0g K 2 HPO 4 0.5g CaCl 2 0.3g Vitamin B 12 0.15mg Trace metals solution 0.2mL Trace Metals Solution: Composition per liter: Disodium EDTA 50.0g NaOH 11.0g CaCl 2 ·2H 2 O 7.34g FeSO 4 ·7H 2 O 5.0g MnCl 2 ·2H 2 O 2.5g ZnSO 4 ·7H 2 O 2.2g CoCl 2 ·6H 2 O 0.5g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.5g CuSO 4 ·5H 2 O 0.2g Preparation of Trace Metals Solution: Add EDTA to distilled/ deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust pH to 6.0 with NaOH. Add remaining components, one by one. Main- tain the pH at 6.0. After dissolution of all the salts, adjust the pH to 4.0 with HCl. Store at 4°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 Thiomicrospira pelophila. Thiomicrospira pelophila Medium Composition per 1000.2mL: NaCl 25.0g MgSO 4 ·7H 2 O 1.5g (NH 4 ) 2 SO 4 1.0g CaCl 2 ·2H 2 O 0.42g Bromthymol Blue 4.0mg K 2 HPO 4 solution 100.0mL Na 2 S 2 O 3 ·5H 2 O solution 100.0mL Vitamin B 12 solution 10.0mL Trace elements solution 0.2mL Na 2 CO 3 solution variable pH 7.2 ± 0.2 at 25°C K 2 HPO 4 Solution: Composition per 100.0mL: K 2 HPO 4 0.5g Preparation of K 2 HPO 4 Solution: Add K 2 HPO 4 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Na 2 S 2 O 3 Solution: Composition per 10.0mL: Na 2 S 2 O 3 ·5H 2 O 5.0g Preparation of Na 2 S 2 O 3 Solution: Add Na 2 S 2 O 3 ·5H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Vitamin B 12 Solution: Composition per 10.0mL: Vitamin B 12 15.0mg Preparation of Vitamin B 12 Solution: Add vitamin B 12 to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Trace Elements Solution: Composition per liter: Disodium EDTA 50.0g ZnSO 4 ·7H 2 O 22.0g CaCl 2 ·2H 2 O 5.54g MnCl 2 ·4H 2 O 5.06g FeSO 4 ·7H 2 O 5.0g CoCl 2 ·6H 2 O 1.61g CuSO 4 ·5H 2 O 1.57g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Na 2 CO 3 Solution: Composition per 100.0mL: Na 2 CO 3 0.4g Preparation of Na 2 CO 3 Solution: Add Na 2 CO 3 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except K 2 HPO 4 solu- tion, Na 2 S 2 O 3 ·5H 2 O solution, vitamin B 12 solution, and Na 2 CO 3 solu- tion, to distilled/deionized water and bring volume to 790.0mL. Mix thoroughly. Adjust pH to 7.2. Autoclave for 15 min at 15 psi pressure– 121°C. Aseptically add 100.0mL of sterile K 2 HPO 4 solution, 100.0mL of sterile Na 2 S 2 O 3 ·5H 2 O solution, and 10.0mL of sterile vitamin B 12 solution. Mix thoroughly. Aseptically adjust pH to 7.2 with the appro- priate volume of sterile Na 2 CO 3 solution. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Thiomicrospira pelo- phila. Thiomonas delicata Medium (DSMZ Medium 1037) Composition per liter: Na 2 S 2 O 3 ·5H 2 O 5.0g Na 2 HPO 4 4.5g K 2 HPO 4 1.5g Yeast extract 1.0g Na-aspartate 1.0g NH 4 Cl 0.3g MgSO 4 ·7H 2 O 0.1g pH 6.0 ± 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 6.0. Dispense into culture vessels. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Thiomonas delicata. Thiomonas delicata Medium (DSMZ Medium 1037) Composition per liter: Na 2 S 2 O 3 ·5H 2 O 5.0g Na 2 HPO 4 4.5g © 2010 by Taylor and Francis Group, LLC Thiorhodococcus Medium 1783 K 2 HPO 4 1.5g Yeast extract 1.0g NH 4 Cl 0.3g MgSO 4 ·7H 2 O 0.1g pH 6.0 ± 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 6.0. Dispense into culture vessels. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Thiomonas perometabolis. Thiophaeococcus mangrovi Medium (DSMZ Medium 1162) Composition per liter: NaCl 20.0g Sodium pyruvate 3.0g MgSO 4 ·7H 2 O 2.0g NH 4 Cl 0.64g K 2 HPO 4 0.5g Yeast extract 0.4g CaCl 2 ·2H 2 O 0.15g Bicarbonate solution 10.0mL Sulfide solution 10.0mL Thiosulfate solution 10.0mL Trace element solution SL-8 1.0mL Vitamin solution 1.0mL pH 7.5 ± 0.2 at 25°C Sulfide Solution: Composition per 10.0mL: Na 2 S·9H 2 O 1.0g Preparation of Sulfide Solution: Add Na 2 S·9H 2 O to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Thiosulfate Solution: Composition per 10.0mL: Na 2 S 2 O 3 ·5H 2 O 1.5g Preparation of Thiosulfate Solution: Add Na 2 S 2 O 3 ·5H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Bicarbonate Solution: Composition per 10.0mL: NaHCO 3 1.0g Preparation of Bicarbonate Solution: Add NaHCO 3 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with a gas mixture of 80% N 2 + 20% CO 2 . Filter sterilize. Vitamin Solution: Composition per 10.0mL: Vitamin B 12 2.0mg Preparation of Vitamin Solution: Add Vitamin B 12 to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Trace Elements Solution SL-8: Composition per liter: Disodium EDTA 5.2g FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·4H 2 O 0.1g ZnCl 2 0.07g H 3 BO 3 0.06g NaMoO 4 ·2H 2 O 0.04g CuCl 2 ·2H 2 O 0.02g NiCl 2 ·6H 2 0 0.02g Preparation of Trace Elements Solution SL-8: Add compo- nents to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Preparation of Medium: Add components, except sulfide, bicar- bonate, thiosulfate, and vitamin solutions, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 gas mixture. Dispense into culture vessels. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add sulfide, thiosulfate, bicarbonate, and vitamin solutions. Mix thor- oughly. Adjust pH to 7.5. Use: For the cultivation of Thiophaeococcus mangrovi. Thiophene-2-Carboxylic Acid Hydrazide See: TCH Medium Thiorhodococcus Medium (DSMZ Medium 28a) Composition per 5.0L: Solution A 4.0L Solution B 860.0mL Solution E 100.0mL Solution F 20.0mL Solution C 5.0mL Solution D 5.0mL pH 7.3 at 25°C Solution A: Composition per 4.0L: NaCl 100.0g MgCl 2 ·6H 2 O 5.0g MgSO 4 2.5g Na 2 S 2 O 3 ·5H 2 O 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 a 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; one is a short, gas- inlet tube with a sterile cotton filter, and the other is 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 100% N 2 atmosphere with a positive pressure of 0.05– 0.1 atm (a manometer for low pressure will be re- quired). 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 © 2010 by Taylor and Francis Group, LLC 1784 Thiosphaera Agar 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. 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: 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 solutions) 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 flocs. No other sediment should arise in the otherwise clear medium. Incubate in the light at 500–1,000 lux intensity. Feed pe- riodically with neutralized solution of sodium sulfide to replenish sul- fide and with other supplement solutions. Use: For the cultivation of Thiorhodococcus minor. Thiosphaera Agar Composition per liter: Agar 15.0g Na 2 HPO 4 4.2g KH 2 PO 4 1.5g NH 4 Cl 0.3g MgSO 4 ·7H 2 O 0.1g KNO 3 0.1g Vishniac and Santer trace metals 2.0mL pH 8.0–8.2 at 25°C Vishniac and Santer Trace Metals: Composition per liter: Ethylenediamine tetraacetic acid (EDTA) 50.0g ZnSO 4 ·7H 2 O 22.0g CaCl 2 5.54g MnCl 2 ·4H 2 O 5.06g FeSO 4 ·7H 2 O 4.99g CoCl 2 ·6H 2 O 1.61g CuSO 4 ·5H 2 O 1.57g (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g Preparation of Vishniac and Santer Trace Metals: Add com- ponents to distilled/deionized water and bring volume to 1.0L. Adjust pH to 6.0 with KOH. Mix thoroughly. Preparation of Medium: Add components, except agar, to dis- tilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust pH to 8.0–8.2. Filter sterilize. Warm to 45°–50°C. Add agar to distilled/deionized water and bring volume to 500.0mL. Mix thorough- ly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically combine the two ster- ile solutions. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Thiosphaera pantotro- pha. Thiosphaera Broth Composition per liter: Na 2 HPO 4 4.2g KH 2 PO 4 1.5g NH 4 Cl 0.3g MgSO 4 ·7H 2 O 0.1g KNO 3 0.1g Vishniac and Santer trace metals 2.0mL pH 8.0–8.2 at 25°C Vishniac and Santer Trace Metals: Composition per liter: Ethylenediamine tetraacetic acid (EDTA) 50.0g ZnSO 4 ·7H 2 O 22.0g CaCl 2 5.54g MnCl 2 ·4H 2 O 5.06g © 2010 by Taylor and Francis Group, LLC . min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Aseptically combine 500.0mL of sterile part I and 500.0mL of sterile part II. Mix thoroughly. Aseptically dis- tribute into sterile. Preparation of Medium: Aseptically add 40.0mL of sterile solu- tion B, 20.0mL of sterile solution C, and 1.0mL of sterile solution D to 940.0mL of sterile solution A. Mix thoroughly. Aseptically. After dissolution of all the salts, adjust the pH to 4.0 with HCl. Store at 4°C. Part II: Composition per liter: Na 2 S 2 O 3 ·5H 2 O 10.0g NaHCO 3 3.0g NaOH 0.05g Preparation of Part II: Add components