Spirochaeta litoralis Medium 1605 Glucose Salts Solution: Composition per liter: NaCl 49.3g MgSO 4 ·7H 2 O 49.2g CaCl 2 ·2H 2 O 5.9g Glucose solution 100.0mL Sulfide solution 100.0mL Preparation of Glucose Salts Solution: Add components, except glucose solution and sulfide solution, to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add sterile glucose so- lution and sulfide solution. Mix thoroughly. Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 0.5g Preparation of Sulfide Solution: Add Na 2 S·9H 2 O to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Glucose Solution: Composition per 100.0mL: Glucose 5.0g Preparation of Glucose Solution: Add glucose to distilled/deion- ized water and bring volume to 100.0mL. Mix thoroughly. Filter ster- ilize. Yeast Extract Peptone Solution: Composition per 30.0mL: Yeast extract 4.0g Peptone 2.0g Preparation of Yeast Extract Peptone Solution: Add compo- nents to distilled/deionized water and bring volume to 30.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Aseptically combine 30.0mL of yeast ex- tract peptone solution with 970.0mL of glucose salts solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the isolation and cultivation of Spirochaeta halophila. Spirochaeta isovalerica Medium Composition per liter: Glucose 2.0g Pancreatic digest of casein 1.0g Yeast extract 0.5g L-Cysteine·HCl 0.05g Resazurin 0.001g Seawater 750.0mL Tris·HCl buffer (0.2M solution, pH 7.5) 250.0mL pH 7.5 ± 0.2 at 25°C Preparation of Medium: Prepare and dispense medium under 100% N 2 . Combine components. Mix thoroughly. Sparge with 100% N 2 . Adjust pH to 7.5 while continuing to sparge with 100% N 2 . Anaer- obically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Spirochaeta isovalerica. Spirochaeta litoralis Medium Composition per liter: Pancreatic digest of casein 3.0g NaCl 2.0g Yeast extract 0.5g Glucose solution 2.0mL Potasssium phosphate buffer (1M, pH 7.4) 2.0mL Sulfide solution 0.5mL Salts solution 0.2mL pH 7.3 ± 0.2 at 25°C Glucose Solution: Composition per 100.0mL: D-Glucose 25.0g Preparation of Glucose Solution: Add D-glucose to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Sulfide Solution: Add Na 2 S·9H 2 O to distilled/de- ionized water and bring volume to 100.0mL. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Salts Solution: Composition per 100.0mL: MgSO 4 ·7H 2 O 12.5g CaCl 2 ·2H 2 O 3.75g EDTA 1.0g FeSO 4 ·7H 2 O 0.5g Trace elements solution 25.0mL Preparation of Salts Solution: Add components to distilled/deion- ized water and bring volume to 100.0mL. Mix thoroughly. Trace Elements Solution: Composition per 1800.0mL: H 3 BO 3 5.5g MnCl 2 ·4H 2 O 3.5g AlCl 3 ·6H 2 O 0.5g CoCl 2 ·6H 2 O 0.5g CuCl 2 ·2H 2 O 0.5g NiCl 2 ·6H 2 O 0.5g ZnCl 2 0.5g KI 0.25g LiCl 0.25g Na 2 MoO 4 ·2H 2 O 0.25g BaCl 2 ·2H 2 O 0.15g SnCl 2 ·2H 2 O 0.15g NaVO 3 0.05g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1800.0mL. Mix thor- oughly. Adjust pH to 3–4 with HCl. Preparation of Medium: Add components, except glucose solu- tion and sulfide solution, to distilled/deionized water and bring volume to 997.5mL. Mix thoroughly. Gently heat and bring to boiling. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Asepti- cally add sterile glucose solution and sulfide solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the isolation of Spirochaeta litoralis from marine habitats. © 2010 by Taylor and Francis Group, LLC 1606 Spirochaeta litoralis Medium Spirochaeta litoralis Medium Composition per liter: Glucose 2.0g Pancreatic digest of casein 1.0g Yeast extract 1.0g L-Cysteine 0.5g Resazurin 1.0mg Seawater 750.0mL Tris·HCl buffer (1.0M solution, pH 7.5) 50.0mL pH 7.2 ± 0.2 at 25°C Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components to distilled/deionized water and bring vol- ume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Adjust pH to 7.5 while continuing to sparge with 100% N 2 . Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Final pH of medium after autoclaving should be 7.2. Use: For the cultivation and maintenance of Spirochaeta litoralis. Spirochaeta smaragdinae Medium (DSMZ Medium 819) Composition per liter: NaCl 50.0g Yeast extract 5.0g NH 4 Cl 1.0g Cysteine-HCl·H 2 O 0.5g K 2 HPO 4 0.3g KH 2 PO 4 0.3g MgCl 2 ·6H 2 O 0.2g KCl 0.2g CaCl 2 ·2H 2 O 0.1g Resazurin 0.5mg NaHCO 3 solution 80.0mL Na 2 S·9H 2 O solution 10.0mL Trace elements solution 10.0mL pH 7.8 ± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.2g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. NaHCO 3 Solution: Composition per 100.0mL: NaHCO 3 5.0g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Filter sterilize. Trace Elements Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·2H 2 O 0.5g CoSO 4 ·7H 2 O 0.18g ZnSO 4 ·7H 2 O 0.18g CaCl 2 ·2H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g NiCl 2 ·6H 2 O 0.025g KAl(SO 4 ) 2 ·12H 2 O 0.02g H 3 BO 3 0.01g Na 2 MoO 4 ·4H 2 O 0.01g CuSO 4 ·5H 2 O 0.01g Na 2 SeO 3 ·5H 2 O 0.3mg Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Dissolve by adjusting pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. Mix thoroughly. Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 gas atmosphere. Add components, except NaHCO 3 solu- tion and Na 2 S·9H 2 O solution, to distilled/deionized water and bring vol- ume to 910.0mL. Mix thoroughly. Adjust pH to 7.8. Gently heat and bring to boiling. Cool while sparging with 80% N 2 + 20% CO 2 . Auto- clave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically and anaerobically add 80.0mL NaHCO 3 solution and 10.0mL Na 2 S·9H 2 O solution. Aseptically and anaerobically distribute into sterile tubes or bottles. Use: For the cultivation of Spirochaeta smaragdinae. Spirochaeta stenostrepta Medium Composition per liter: Glucose 5.0g Peptone 2.0g Yeast extract 0.3g Vitamin B 12 0.01mg Salts solution 100.0mL Phosphate solution 15.0mL Sulfide solution 10.0mL pH 7.0 ± 0.2 at 25°C Phosphate Solution: Composition per liter: KH 2 PO 4 30.0g K 2 HPO 4 70.0g Preparation of Phosphate Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Salts Solution: Composition per liter: MgSO 4 ·7H 2 O 2.0g CaCl 2 ·2H 2 O 0.75g EDTA 0.2g FeSO 4 ·7H 2 O 0.1g Trace elements solution 5.0mL Preparation of Salts Solution: Add EDTA to approximately 800.0mL of distilled/deionized water. Gently heat until dissolved. Ad- just pH to 7.0 with 2.5% KOH. Add the remaining components. Mix thoroughly. Bring volume to 1.0L with distilled/deionized water. Trace Elements Solution: Composition per 1800.0mL: H 3 BO 3 5.5g MnCl 2 ·4H 2 O 3.5g AlCl 3 ·6H 2 O 0.5g CoCl 2 ·6H 2 O 0.5g CuCl 2 ·2H 2 O 0.5g NiCl 2 ·6H 2 O 0.5g ZnCl 2 0.5g © 2010 by Taylor and Francis Group, LLC Spirochete Medium 1607 KI 0.25g LiCl 0.25g Na 2 MoO 4 ·2H 2 O 0.25g BaCl 2 ·2H 2 O 0.15g SnCl 2 ·2H 2 O 0.15g NaVO 3 0.05g Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1800.0mL. Mix thor- oughly. Adjust pH to 3–4 with HCl. Sulfide Solution: Composition per 100.0mL: Na 2 S·9H 2 O 2.0g Preparation of Sulfide Solution: Add Na 2 S·9H 2 O to distilled/de- ionized water and bring volume to 100.0mL. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Prepare solution freshly. Preparation of Medium: Add components, except sulfide solution, to distilled/deionized water and bring volume to 990.0mL. Mix thor- oughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile sulfide solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the isolation of Spirochaeta stenostrepta. Spirochaeta stenostrepta Medium Composition per liter: Glucose 5.0g Peptone 2.0g Yeast extract 2.0g L-Cysteine 0.5g Resazurin 1.0mg pH 7.3–7.6 at 25°C Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components to distilled/deionized water and bring vol- ume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Adjust pH to 7.3– 7.6 while continuing to sparge with 100% N 2 . Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Spirochaeta stenostrepta. Spirochaeta zuelzerae Medium Composition per liter: Solution 1 480.0mL Solution 2 480.0mL Solution 3 20.0mL Solution 4 20.0mL pH 7.2 ± 0.2 at 25°C Solution 1: Composition per 480.0mL: KH 2 PO 4 0.75g L-Cysteine·HCl·H 2 O 0.5g NaH 2 PO 4 ·H 2 O 0.25g Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 480.0mL. Mix thoroughly. Adjust pH to 7.2 with 5.0% KOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Solution 2: Composition per 480.0mL: NH 4 Cl 1.0g MgSO 4 ·7H 2 O 0.5g Yeast extract 0.2g CaCl 2 0.02g Resazurin 1.0mg FeCl 3 ·6H 2 O solution (0.25g/L) 10.0mL Trace elements solution 2.0mL Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 480.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Trace Elements Solution: Composition per 100.0mL: Na 2 MoO 4 ·2H 2 O 0.075g H 3 BO 3 0.056g ZnSO 4 ·7H 2 O 0.044g CoCl 2 ·6H 2 O 0.02g CuSO 4 ·5H 2 O 2.0mg MnCl 2 2.0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thorough- ly. Solution 3: Composition per 20.0mL: NaHCO 3 1.0g Preparation of Solution 3: Add NaHCO 3 to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize un- der pressure. Solution 4: Composition per 20.0mL: Glucose 2.0g Preparation of Solution 4: Add glucose to distilled/deionized wa- ter and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Aseptically add 480.0 mL of sterile solu- tion 1 to 480.0mL of sterile solution 2 under 80% N 2 + 20% CO 2 . While gassing, add 20.0mL of sterile solution 3 and 20.0mL of sterile solution 4. Mix thoroughly. Adjust pH to 7.2. Aseptically and anaero- bically distribute into tubes. Cap with rubber stoppers. Use: For the cultivation and maintenance of Spirochaeta zuelzerae. Spirochete Enrichment Medium Composition per liter: Agar 10.0g Beef extract 1.0g Peptone 1.0g Yeast extract 1.0g Seawater 500.0mL 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 of spirochetes from muds. A well is cut into the agar plate and filled with mud samples. Spirochetes migrate out of the mud into the agar surrounding the well. Spirochete Medium (ATCC Medium 164) Composition per liter: Agar 15.0g KH 2 PO 4 1.0g © 2010 by Taylor and Francis Group, LLC 1608 Spirochete Medium NH 4 Cl 1.0g Yeast extract 1.0g MgSO 4 0.5g CaCl 2 0.04g FeCl 3 ·6H 2 O 1.25mg NaHCO 3 solution 20.0mL Glucose solution 10.0mL Na 2 S·9H 2 O solution 5.0mL Glucose Solution: Composition per 100.0mL: Glucose 10.0g Preparation of Glucose Solution: Add glucose to distilled/deion- ized water and bring volume to 100.0mL. Mix thoroughly. Filter ster- ilize. NaHCO 3 Solution: Composition per 100.0mL: NaHCO 3 5.0g Preparation of NaHCO 3 Solution: Add the NaHCO 3 to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Na 2 S·9H 2 O Solution: Composition per 100.0mL: Na 2 S·9H 2 O 10.0g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components—except NaHCO 3 solu- tion, glucose solution, and Na 2 S·9H 2 O solution—to distilled/deionized water and bring volume to 965.0mL. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 20.0mL of sterile NaHCO 3 solution, 10.0mL of sterile glucose solution, and 5.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of spirochetes. Spirochete Medium (ATCC Medium 1712) Composition per liter: Tris(hydroxymethyl)aminomethane buffer 7.52g Pancreatic digest of casein 1.0g Yeast extract 1.0g L-Cysteine·HCl·2H 2 O 0.5g Resazurin 1.0mg Seawater 750.0mL Glucose solution 20.0mL pH 7.2 ± 0.2 at 25°C Glucose Solution: Composition per 20.0mL: Glucose 2.0g Preparation of Glucose Solution: Add glucose to distilled/deion- ized water and bring volume to 20.0mL. Mix thoroughly. Filter steril- ize. Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components, except glucose solution, to distilled/deion- ized water and bring volume to 980.0mL. Mix thoroughly. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add sterile glucose solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Spirochaeta litoralis. Spirochete Thermophile Medium (DSMZ Medium 509) Composition per 1012.0mL: Solution A 920.0mL Solution D 50.0mL Solution E 20.0mL Solution F 10.0mL Solution G 10.0mL Solution B 1.0mL Solution C 1.0mL pH 7.0 ± 0.2 at 25°C Solution A: Composition per 920.0mL: NaCl 4.0g MgCl 2 ·6H 2 O 0.8g KCl 0.5g NH 4 Cl 0.3g KH 2 PO 4 0.2g CaCl 2 ·2H 2 O 0.03g Resazurin 1.0mg Preparation of Solution A: Add components to 920.0mL distilled/ deionized water. Mix thoroughly. Bring to boiling for a few minutes. Cool to room temperature while gassing with 80% N 2 + 20% CO 2 gas. Adjust pH to 6.0. Immediately distribute under N 2 into anaerobic tubes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution B: 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: 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 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution C: Composition per liter: NaOH 0.5g Na 2 WO 4 ·2H 2 O 4.0mg Na 2 SeO 3 ·5H 2 O 3.0mg Preparation of Solution C: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Solution D: Composition per 100.0mL: NaHCO 3 5.0g Preparation of Solution D: Add NaHCO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with © 2010 by Taylor and Francis Group, LLC Spiroplasma Agar MID 1609 100% N 2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution E: 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 0.1mg Preparation of Solution E: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Filter sterilize. Solution F: Composition per 10.0mL: Starch 1.0g Preparation of Solution F: Add starch to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 gas mixture. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Solution G: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Solution G: Add Na 2 S·9H 2 O 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. Cool to 25°C. Preparation of Medium: Solution A is distributed into anaerobic tubes with rubber stoppers prior to autoclaving. Using aseptic and anaer- obic conditions and syringes, appropriate volumes of sterile solutions B– G are injected into each tube to yield the specified concentrations. Use: For the cultivation of Spirochaeta thermophila. Spirolate Broth Composition per liter: Pancreatic digest of casein 15.0g Glucose 5.0g Yeast extract 5.0g NaCl 2.5g L-Cysteine·HCl·H 2 O 1.0g Sodium thioglycolate 0.5g Palmitic acid 0.05g Stearic acid 0.05g Oleic acid 0.05g Linoleic acid 0.05g Serum 100.0mL 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, except serum, to dis- tilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Distribute into screw-capped tubes in 20.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 2.0mL of serum to each tube. Heat-inactivated sheep, rabbit, or bovine serum may be used. Tighten caps. Mix thoroughly. Use: For the cultivation of Treponema phagedenis and other spiro- chetes. Spirolate HiVeg Broth, OMATA with Serum Composition per liter: Plant hydrolysate 15.0g Glucose 5.0g Yeast extract 5.0g NaCl 2.5g L-Cysteine·HCl 1.0g Na-thioglycolate 0.5g Sheep or rabbit blood serum, inactivated 10% 100.0mL pH 7.1 ± 0.2 at 25°C Source: This medium, without serum, is available as a premixed pow- der from HiMedia. Preparation of Medium: Add components, except serum, to dis- tilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Distribute into screw-capped tubes in 20.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 2.0mL of serum to each tube. Heat-inactivated sheep, rabbit, or bovine serum may be used. Tighten caps. Mix thoroughly. Use: For the cultivation of Treponema phagedenis and other spiro- chetes. Spiroplasma Agar MID Composition per 291.2mL: Schneider’s Drosophila medium 160.0mL Solution 1 80.0mL Fetal calf serum 50.0mL Phenol Red (0.5% solution) 1.2mL pH 7.4 ± 0.2 at 25°C Schneider’s Drosophila Medium: Composition per liter: MgSO 4 ·7H 2 O 3.7g NaCl 2.1g Yeast extract 2.0g Trehalose 2.0g D-Glucose 2.0g L-Glutamine 1.8g L-Lysine·HCl 1.7g L-Proline 1.7g KCl 1.6g Na 2 HPO 4 ·7H 2 O 1.3g L-Glutamic acid 0.8g L-Methionine 0.8g CaCl 2 , anhydrous 0.6g KH 2 PO 4 0.5g β-Alanine 0.5g L-Tyrosine 0.5g L-Arginine 0.4g L-Aspartic acid 0.4g L-Histidine 0.4g L-Threonine 0.4g NaHCO 3 0.4g Glycine 0.3g L-Serine 0.3g L-Valine 0.3g © 2010 by Taylor and Francis Group, LLC 1610 Spiroplasma Broth MID L-Isoleucine 0.2g L-Leucine 0.2g L-Phenylalanine 0.2g α-Ketoglutaric acid 0.2g Fumaric acid 0.1g Malic acid 0.1g Succinic acid 0.1g L-Cystine 0.1g L-Tryptophan 0.1g L-Cysteine 0.06g Preparation of Schneider’s Drosophila Medium: Add compo- nents to 1.0L of distilled/deionized water. Mix thoroughly. Filter ster- ilize. Solution 1: Composition per 80.0mL: Sorbitol 7.0g Noble agar 5.0g Beef heart, solids from infusion 5.0g Peptone 1.8g Sucrose 1.0g Pancreatic digest of casein 1.0g NaCl 0.5g D-Fructose 0.1g D-Glucose 0.1g Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 80.0mL. Mix thoroughly. Adjust pH to 7.8 with 1N NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Preparation of Medium: Bring fetal calf serum and Phenol Red so- lution to 56°C. Rapidly bring Schneider’s Drosophila medium to 37°C. Rapidly combine the components. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Spiroplasma kunkelii and Spiroplasma species. Spiroplasma Broth MID Composition per 291.2mL: Schneider’s Drosophila medium 160.0mL Solution 1 80.0mL Fetal calf serum 50.0mL Phenol Red (0.5% solution) 1.2mL pH 7.4 ± 0.2 at 25°C Schneider’s Drosophila Medium: Composition per liter: MgSO 4 ·7H 2 O 3.7g NaCl 2.1g Yeast extract 2.0g Trehalose 2.0g D-Glucose 2.0g L-Glutamine 1.8g L-Lysine·HCl 1.7g L-Proline 1.7g KCl 1.6g Na 2 HPO 4 ·7H 2 O 1.3g L-Glutamic acid 0.8g L-Methionine 0.8g CaCl 2 , anhydrous 0.6g KH 2 PO 4 0.5g β-Alanine 0.5g L-Tyrosine 0.5g L-Arginine 0.4g L-Aspartic acid 0.4g L-Histidine 0.4g L-Threonine 0.4g NaHCO 3 0.4g Glycine 0.3g L-Serine 0.3g L-Valine 0.3g L-Isoleucine 0.2g L-Leucine 0.2g L-Phenylalanine 0.2g α-Ketoglutaric acid 0.2g Fumaric acid 0.1g Malic acid 0.1g Succinic acid 0.1g L-Cystine 0.1g L-Tryptophan 0.1g L-Cysteine 0.06g Preparation of Schneider’s Drosophila Medium: Add compo- nents to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Filter sterilize. Solution 1: Composition per 80.0mL: Sorbitol 7.0g Beef heart, solids from infusion 5.0g Peptone 1.8g Sucrose 1.0g Pancreatic digest of casein 1.0g NaCl 0.5g D-Fructose 0.1g D-Glucose 0.1g Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 80.0mL. Mix thoroughly. Adjust pH to 7.8 with 1N NaOH. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Bring fetal calf serum and Phenol Red so- lution to 56°C. Rapidly bring Schneider’s Drosophila medium to 37°C. Rapidly combine the components. Mix thoroughly. Aseptically distrib- ute into sterile tubes or flasks. Use: For the cultivation and maintenance of Spiroplasma kunkelii and Spiroplasma species. Spiroplasma Medium Composition per liter: Sucrose 80.0g Beef heart, solids from infusion 34.7g Peptone 6.9g NaCl 3.5g Horse serum, heat inactivated 100.0mL pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 900.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Asep- tically add horse serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Spiroplasma species. © 2010 by Taylor and Francis Group, LLC Sporobacter Medium 1611 Spiroplasma Medium Composition per liter: Sorbitol 70.0g Pancreatic digest of casein 7.0g Yeast extract 5.0g NaCl 5.0g Beef extract 3.0g Yeast extract 3.0g Beef heart, solids from infusion 2.0g Fructose 1.0g Glucose 1.0g Phenol Red 20.0mg Horse serum 100.0mL pH 7.8 ± 0.2 at 25°C Preparation of Medium: Add components 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 50°–55°C. Aseptically add 100.0mL of sterile horse serum. Mix thoroughly. Use: For the cultivation of Spiroplasma citri. Spiroplasma Medium with 25 mg/L of Phenol Red Composition per liter: Sucrose 80.0g Beef heart, solids from infusion 34.7g Peptone 6.9g NaCl 3.5g Phenol Red 25.0mg Horse serum, heat inactivated 100.0mL pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 900.0mL. 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 add heat-inactivated horse serum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Spiroplasma floricola. Spizizen Potato Agar Composition per liter: Potatoes 200.0g Agar 15.0g MnSO 4 5.0mg pH 6.8 ± 0.2 at 25°C Preparation of Medium: Peel and dice potatoes. Add potatoes to 1.0L of tap water. Gently heat and bring to boiling. Continue boiling for 30 min. Filter through cheesecloth. Add MnSO 4 to filtrate and bring volume to 1.0L with tap water. Mix thoroughly. Adjust pH to 6.8. Add agar. 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 Bacillus amyloliquefa- ciens. SPMA See: Mineral Salt Peptonized Milk Agar Spore Strip Broth Composition per liter: Spore strip broth 9.0g Preparation of Medium: Add 9.0g of spore strip broth powder (a mixture of glucose, buffer salts, growth factors, and Bromthymol Blue) 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 recovery of spores of Bacillus stearothermophilus on spore strips used to determine the sterilization efficiency of autoclaves. Sporobacter Medium (DSMZ Medium 711) Composition per liter: NH 4 Cl 1.0g NaCl 0.6g Na-acetate·3H 2 O 0.5g Cysteine-HCl·H 2 O 0.5g K 2 HPO 4 0.3g KH 2 PO 4 0.3g Yeast extract 0.2g MgCl 2 ·6H 2 O 0.2g CaCl 2 ·2H 2 O 0.1g KCl 0.1g Resazurin 0.5mg NaHCO 3 solution 40.0mL Trimethoxycinnamate solution 10.0mL Na 2 S·9H 2 O solution 10.0mL Trace elements solution SL-10 1.5mL pH 7.1 ± 0.2 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 thor- oughly. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. NaHCO 3 Solution: Composition per 100.0mL: NaHCO 3 10.0g © 2010 by Taylor and Francis Group, LLC 1612 Sporocytophaga Medium Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Filter sterilize. Trimethoxycinnamate Solution: Composition per 10.0mL: Trans-3,4,5-trimethoxycinnamate 1.2g Preparation of Trimethoxycinnamate Solution: Add trans- 3,4,5-trimethoxycinnamate to distilled/deionized water and bring vol- ume to 10.0mL. Mix thoroughly. Neutralize with NaOH. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 gas atmosphere. Add components, except NaHCO 3 solu- tion, Na 2 S·9H 2 O solution, trimethoxycinnamate solution, and trace ele- ments solution SL-10, to distilled/deionized water and bring volume to 938.5mL. Mix thoroughly. Adjust pH to 7.1. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and an- aerobically add 40.0mL NaHCO 3 solution, 10.0mL Na 2 S·9H 2 O solution, 10.0mL trimethoxycinnamate solution, and 1.5mL trace elements solution SL-10. Mix thoroughly. Aseptically and anaerobically distribute into ster- ile tubes or bottles. Use: For the cultivation of Sporobacter termitidis. Sporocytophaga Medium Composition per liter: NaNO 3 2.0g K 2 HPO 4 1.2g MgSO 4 ·7H 2 O 1.0g KCl 0.5g KH 2 PO 4 0.14g Yeast extract 0.02g FeSO 4 ·7H 2 O 6.0mg Filter paper strips variable pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components, except filter paper strips, to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Distribute into tubes in 5.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add a sterile filter paper strip to each tube so that 1.0–2.0cm of the strip extends above the medium. Use: For the cultivation and maintenance of Sporocytophaga myxo- coccoides. Sporohalobacter lortetii Agar Composition per liter: NaCl 105.0g L-Glutamic acid 4.0g Agar 20.0g CaCO 2 5.0g Soluble starch 2.0g Casamino acids 2.0g Nutrient broth 2.0g Yeast extract 2.0g KCl 0.75g L-Cysteine 0.5g FeSO 4 ·7H 2 O 0.002g Resazurin 1.0mg MgCl 2 ·6H 2 O solution 40.0mL CaCl 2 ·2H 2 O solution 10.0mL Trace elements solution 10.0mL Vitamin solution 10.0mL pH 6.5 ± 0.2 at 25°C MgCl 2 ·6H 2 O Solution: Composition per 40.0mL: MgCl 2 ·6H 2 O 0.01g Preparation of MgCl 2 ·6H 2 O Solution: Add MgCl 2 ·6H 2 O to dis- tilled/deionized water and bring volume to 40.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. CaCl 2 ·2H 2 O Solution: Composition per 40.0mL: CaCl 2 ·2H 2 O 0.01g Preparation of CaCl 2 ·2H 2 O Solution: Add CaCl 2 ·2H 2 O to dis- tilled/deionized water and bring volume to 40.0mL. 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 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. 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. Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components, except MgCl 2 ·6H 2 O and CaCl 2 ·2H 2 O so- lutions, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 40.0mL of sterile MgCl 2 ·6H 2 O solution and 10.0mL of sterile CaCl 2 ·2H 2 O solution. Mix thoroughly. Aseptically and anaerobically pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Sporohalobacter lortetii. © 2010 by Taylor and Francis Group, LLC Sporomusa Medium 1613 Sporohalobacter lortetii Broth Composition per liter: NaCl 105.0g L-Glutamic acid 4.0g Casamino acids 2.0g Nutrient broth 2.0g Yeast extract 2.0g KCl 0.75g L-Cysteine 0.5g FeSO 4 ·7H 2 O 0.002g Resazurin 1.0mg MgCl 2 ·6H 2 O solution 40.0mL CaCl 2 ·2H 2 O solution 10.0mL Trace elements solution 10.0mL Vitamin solution 10.0mL pH 6.5 ± 0.2 at 25°C MgCl 2 ·6H 2 O Solution: Composition per 40.0mL: MgCl 2 ·6H 2 O 0.01g Preparation of MgCl 2 ·6H 2 O Solution: Add MgCl 2 ·6H 2 O to dis- tilled/deionized water and bring volume to 40.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. CaCl 2 ·2H 2 O Solution: Composition per 40.0mL: CaCl 2 ·2H 2 O 0.01g Preparation of CaCl 2 ·2H 2 O Solution: Add CaCl 2 ·2H 2 O to dis- tilled/deionized water and bring volume to 40.0mL. 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 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components. Add distilled/deionized water to 1.0L. 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. Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components, except MgCl 2 ·6H 2 O and CaCl 2 ·2H 2 O so- lutions, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pres- sure–121°C. Aseptically add 40.0mL of sterile MgCl 2 ·6H 2 O solution and 10.0mL of sterile CaCl 2 ·2H 2 O solution. Mix thoroughly. Asepti- cally and anaerobically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Sporohalobacter lortetii. Sporomusa Medium Composition per 877.0mL: NaCl 2.25g Pancreatic digest of casein 2.0g Yeast extract 2.0g MgSO 4 ·7H 2 O .0.5g NH 4 Cl 0.5g K 2 HPO 4 0.35g KH 2 PO 4 0.23g CaCl 2 ·2H 2 O 0.025g FeSO 4 ·7H 2 O 2.0mg Resazurin 1.0mg NaHSeO 3 15.0μg NaHCO 3 solution 50.0mL Glycine betaine solution 50.0mL Wolfe’s vitamin solution 10.0mL L-Cysteine·HCl·H 2 O solution 10.0mL Trace elements solution SL-6 3.0mL pH 7.0–7.2 at 25°C NaHCO 3 Solution: Composition per 50.0mL: NaHCO 3 4.0g Preparation of NaHCO 3 Solution: Add the NaHCO 3 to distilled/ deionized water and bring volume to 50.0mL. Mix thoroughly. Gas un- der 80% N 2 + 20% CO 2 for 20 min. Glycine Betaine Solution: Composition per 50.0mL: Glycine betaine 5.0g Preparation of Glycine Betaine Solution: Add the glycine be- taine to distilled/deionized water and bring volume to 50.0mL. Mix thoroughly. Filter sterilize. Aseptically gas under 80% N 2 + 20% CO 2 . Wolfe’s Vitamin Solution: Composition per liter: Pyridoxine·HCl 0.01g Thiamine·HCl 5.0mg Riboflavin 5.0mg Nicotinic acid 5.0mg Calcium pantothenate 5.0mg p-Aminobenzoic acid 5.0mg Thioctic acid 5.0mg Biotin 2.0mg Folic acid 2.0mg Cyanocobalamin 0.1mg Preparation of Wolfe’s Vitamin Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. © 2010 by Taylor and Francis Group, LLC 1614 Sporomusa Medium L-Cysteine·HCl·H 2 O Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.3g Preparation of L-Cysteine·HCl·H 2 O Solution: Add L-cyste- ine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Aseptically gas under 100% N 2 . Trace Elements Solution SL-6: Composition per liter: H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g MnCl 2 ·4H 2 O 0.03g Na 2 MoO 4 ·H 2 O 0.03g NiCl 2 ·6H 2 O 0.02g CuCl 2· 2H 2 O 0.01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 3.4. Preparation of Medium: Add components—except NaHCO 3 solu- tion, glycine betaine solution, and L-cysteine·HCl·H 2 O solution—to distilled/deionized water and bring volume to 890.0mL. Mix thorough- ly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool rapidly to 25°C under 80% N 2 + 20% CO 2 . Add 50.0mL of NaHCO 3 solution. Mix thoroughly. Autoclave anaerobically for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add sterile glycine betaine solution. Mix thoroughly. Immediately prior to inoculation, aseptically and anaerobically add L-cysteine·HCl·H 2 O solution. Use: For the cultivation and maintenance of Sporomusa ovata and Sporomusa sphaeroides. Sporomusa Medium Composition per 1010.0mL: Betaine·H 2 O 6.7g NaHCO 3 4.0g NaCl 2.25g Pancreatic digest of casein 2.0g Yeast extract 2.0g MgSO 4 ·7H 2 O 0.5g NH 4 Cl 0.5g K 2 HPO 4 0.348g CaCl 2 ·2H 2 O 0.25g KH 2 PO 4 0.227g FeSO 4 ·7H 2 O 2.0mg Resazurin 1.0mg NaHSeO 3 26.3μg Vitamin solution 10.0mL Reducing agent solution 10.0mL Trace elements solution SL-10 1.0mL pH 7.0 ± 0.2 at 25°C 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. Reducing Agent Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.3g Na 2 S·9H 2 O 0.3g Preparation of Reducing Agent Solution: Add 10.0mL of dis- tilled/deionized water to a flask. Gently heat and bring to boiling. Con- tinue to boil for 1 min while sparging with 100% N 2 . Cool to room temperature. Add L-cysteine·HCl·H 2 O. Mix thoroughly. Adjust pH to 9 with 5N NaOH. Add Na 2 S·9H 2 O. Mix thoroughly. Autoclave for 10 min at 15 psi pressure–121°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 thor- oughly. Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 . Add components, except reducing agent solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Using a sy- ringe, aseptically and anaerobically add sterile reducing agent to each tube (10.0mL per liter of medium). Use: For the cultivation and maintenance of Sporomusa species. Sporomusa Medium, Modified Composition per liter: NaHCO 3 4.0g NaCl 2.25g Pancreatic digest of casein 2.0g Yeast extract 2.0g MgSO 4 ·7H 2 O 0.5g NH 4 Cl 0.5g K 2 HPO 4 0.348g CaCl 2 ·2H 2 O 0.25g KH 2 PO 4 0.227g FeSO 4 ·7H 2 O 2.0mg Resazurin 1.0mg NaHSeO 3 26.3μg Fructose solution 50.0mL Reducing agent solution 10.0mL Vitamin solution 10.0mL Trace elements solution SL-10 1.0mL pH 7.0 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC . at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Aseptically combine 30.0mL of yeast ex- tract peptone solution with 970.0mL of glucose salts solution. Mix thoroughly. Aseptically. 2.0g Preparation of Solution 4: Add glucose to distilled/deionized wa- ter and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Aseptically add 480.0 mL of sterile solu- tion. 480.0 mL of sterile solu- tion 1 to 480.0mL of sterile solution 2 under 80% N 2 + 20% CO 2 . While gassing, add 20.0mL of sterile solution 3 and 20.0mL of sterile solution 4. Mix thoroughly. Adjust