Martin-Lewis Agar, Enriched 1025 KCl 2.0g Glucose 1.0g CaCl 2 0.36g NaHCO 3 0.06g NaBr 0.026g 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. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Marinococcus albus. Marinomonas vaga Medium See: Nutrient Agar with 3% NaCl Marinomonas vaga Medium (DSMZ Medium 617) Composition per liter: NaCl 30.0g Agar 15.0g Beef extract 10.0g Peptone 10.0g pH 7.1 ± 0.2 at 25°C Preparation of Medium: Add components to tap water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Dis- tribute 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 Marinomonas commu- nis=Alteromonas communis and Marinomonas vaga=Alteromonas vaga. Martin-Lewis Agar Composition per liter: Agar 12.0g Hemoglobin 10.0g Pancreatic digest of casein 7.5g Selected meat peptone 7.5g NaCl 5.0g K 2 HPO 4 4.0g Cornstarch 1.0g KH 2 PO 4 1.0g Supplement solution 10.0mL VCAT inhibitor 10.0mL pH 7.2 ± 0.22 at 25°C Source: Martin-Lewis agar is available as a prepared medium from BD Diagnostic Systems. Supplement Solution: Composition per liter: Glucose 100.0g L-Cysteine·HCl 25.9g L-Glutamine 10.0g L-Cystine 1.1g Adenine 1.0g Nicotinamide adenine dinucleotide 0.25g Vitamin B 12 0.1g Thiamine pyrophosphate 0.1g Guanine·HCl 0.03g Fe(NO 3 ) 3 ·6H 2 O 0.02g p-Aminobenzoic acid 0.013g Thiamine·HCl 3.0mg Source: The supplement solution IsoVitaleX ® enrichment is available from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems. Preparation of Supplement Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. VCAT Inhibitor: Composition per 10.0mL: Colistin 7.5mg Trimethoprim lactate 5.0mg Vancomycin 4.0mg Anisomycin 0.02g Preparation of VCAT Inhibitor: Add components to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Filter ster- ilize. Preparation of Medium: Add components, except supplement so- lution and VCAT inhibitor, to distilled/deionized water and bring vol- ume to 980.0mL. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile supplement solution and sterile VCAT inhibitor. Mix thoroughly. Pour into sterile Petri dishes. Use: For the isolation and cultivation of pathogenic Neisseria from specimens containing mixed flora of bacteria and fungi. Martin-Lewis Agar, Enriched Composition per liter: Agar 12.0g Pancreatic digest of casein 7.5g Selected meat peptone 7.5g NaCl 5.0g K 2 HPO 4 4.0g Cornstarch 1.0g KH 2 PO 4 1.0g Sarcina lutea suspension 20.0mL Horse serum, inactivated 20.0mL Supplement solution 10.0mL PCAT inhibitor 10.0mL pH 7.2 ± 0.22 at 25°C Source: The supplement solution (IsoVitaleX ® enrichment) is avail- able from BD Diagnostic Systems. This enrichment may be replaced by supplement VX from BD Diagnostic Systems. Sarcina lutea Suspension: Composition per 20.0mL: Sarcina lutea FDA 1001 10 6 –10 7 cells Preparation of Sarcina lutea Suspension: Aseptically wash the growth of 24-hr cultures of Sarcina lutea FDA 1001 cells from Thayer- Martin plates with sterile soybean casein digest broth. Standardize the suspension by adding additional sterile tryptic soy broth to yield 40% light transmission at 530nm wavelength. Soybean Casein Digest Broth: Composition per liter: Pancreatic digest of casein 17.0g NaCl 5.0g Papaic digest of soybean meal 3.0g K 2 HPO 4 2.5g Glucose 2.5g pH 7.3 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC 1026 Mating Agar Preparation of Soybean Casein Digest Broth: 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. Supplement Solution: Composition per liter: Glucose 100.0g L-Cysteine·HCl 25.9g L-Glutamine 10.0g L-Cystine 1.1g Adenine 1.0g Nicotinamide adenine dinucleotide 0.25g Vitamin B 12 0.1g Thiamine pyrophosphate 0.1g Guanine·HCl 0.03g Fe(NO 3 ) 3 ·6H 2 O 0.02g p-Aminobenzoic acid 0.013g Thiamine·HCl 3.0mg Preparation of Supplement Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. PCAT Inhibitor: Composition per 10.0mL: Anisomycin 0.02g Colistin 7.5mg Trimethoprim lactate 5.0mg Penicillin G 25,000U Preparation of PCAT Inhibitor: Add components to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Filter ster- ilize. Preparation of Medium: Add components—except Sarcina lutea suspension, horse serum, supplement solution, and PCAT inhibitor—to distilled/deionized water and bring volume to 940.0mL. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pres- sure–121°C. Cool to 45°–50°C. Aseptically add 20.0mL of sterile Sarci- na lutea suspension, 20.0mL of sterile horse serum, 10.0mL of supplement solution, and 10.0mL of sterile PCAT inhibitor. Mix thor- oughly. Pour into sterile Petri dishes. Use: For the isolation and cultivation of pathogenic Neisseria, espe- cially penicillinase-producing strains, from specimens containing mixed flora of bacteria and fungi. Mating Agar Composition per liter: Agar 40.0g Sucrose 10.0g Xylose 2.0g KH 2 PO 4 1.0g MgSO 4 0.5g Yeast extract 0.5g CaCl 2 0.1g NaCl 0.1g Biotin 5.0μg pH 5.7 ± 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. Adjust pH to 5.7. 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 Filobasidiella neofor- mans. Maximum Recovery Diluent Composition per liter: NaCl 8.5g Peptone 1.0g 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. Use: This diluent is a physiologically isotonic and protective medium for maximal recovery of microorganisms from a variety of sources. M-Azide HiVeg Broth Base with Triphenyltetrazolium Chloride Composition per liter: Saccharose 100.0g Plant hydrolysate No. 1 40.0g Yeast extract 10.0g K 2 HPO 4 4.0g Glucose 2.0g NaN 3 0.4g Triphenyltetrazolium chloride solution 5.0mL pH 7.2 ± 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. Triphenyltetrazolium Choride Solution: Composition per 5.0mL: Triphenyltetrazolium chloride 0.1g Preparation of Triphenyltetrazolium Choride Solution: Add triphenyltetrazolium chloride to distilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except triphenyltetrazo- lium chloride solution, 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. Cool to 45°–50°C. Aseptically add 5.0mL triphenyltetrazolium chloride so- lution. Mix thoroughly. Aseptically distribute into tubes. Use: For the detection and enrichment of fecal streptococci in water and sewage by the membrane filtration method. MB Medium (DSMZ Medium 924) Composition per liter: NaCl 10.0g NaHCO 3 4.0g Yeast extract 2.0g Trypticase™ 2.0g NH 4 Cl 1.0g MgCl 2 ·6H 2 O 1.0g KCl 0.5g CaCl 2 ·2H 2 O 0.4g © 2010 by Taylor and Francis Group, LLC MB Medium 1027 K 2 HPO 4 0.4g Resazurin 0.5mg Sodium formate solution 50.0mL Na 2 S·9H 2 O solution 10.0mL Cysteine-HCl·H 2 O solution 10.0mL Vitamin solution 10.0mL Trace elements solution 10.0mL Selenite-tungstate solution 1.0mL pH 7.2 ± 0.2 at 25°C Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.25g Preparation of Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Sodium Formate Solution: Composition per 50.0mL: Na-formate 6.8g Preparation of Sodium Formate Solution: Add sodium formate to distilled/deionized water and bring volume to 50.0mL. Mix thor- oughly. Sparge with 100% N 2 . Filter sterilize. Selenite-Tungstate Solution 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 Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.25g 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. Sparge with N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an- aerobically. 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. 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 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Filter sterilize. Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% N 2 + 20% CO 2 gas mixture. Add components, except sodium formate solution, NaHCO 3 , cysteine solution, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to 25°C while sparging with 80% N 2 + 20% CO 2 . Add solid NaHCO 3 . Mix thoroughly. Adjust pH to 6.8–7.0. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaer- obically add per liter 50.0mL sterile sodium formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. The final pH should be 7.2. Use: For the cultivation of Methanocalculus taiwanensis, Methanococ- cus voltae (Methanococcus voltaei), and Methanofollis aquaemaris. MB Medium (DSMZ Medium 924) Composition per liter: NaCl 5.0g NaHCO 3 4.0g Yeast extract 2.0g Trypticase™ 2.0g NH 4 Cl 1.0g MgCl 2 ·6H 2 O 1.0g KCl 0.5g CaCl 2 ·2H 2 O 0.4g K 2 HPO 4 0.4g Resazurin 0.5mg Sodium formate solution 50.0mL Na 2 S·9H 2 O solution 10.0mL Cysteine-HCl·H 2 O solution 10.0mL Vitamin solution 10.0mL Trace elements solution 10.0mL Selenite-tungstate solution 1.0mL pH 6.5 ± 0.2 at 25°C Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.25g Preparation of Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Sodium Formate Solution: Composition per 50.0mL: Na-formate 6.8g © 2010 by Taylor and Francis Group, LLC 1028 MB Medium Preparation of Sodium Formate Solution: Add sodium formate to distilled/deionized water and bring volume to 50.0mL. Mix thor- oughly. Sparge with 100% N 2 . Filter sterilize. Selenite-Tungstate Solution 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 Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.25g 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. Sparge with N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an- aerobically. 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. 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 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Filter sterilize. Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% N 2 + 20% CO 2 gas mixture. Add components, except sodium formate solution, NaHCO 3 , cysteine solution, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to 25°C while sparging with 80% N 2 + 20% CO 2 . Add solid NaHCO 3 . Mix thoroughly. Adjust pH to 6.8–7.0. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically and anaer- obically add per liter 50.0mL sterile sodium formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. The final pH should be 6.5. Use: For the cultivation of Methanofollis aquaemaris DSM 14661. MB Medium (DSMZ Medium 924) Composition per liter: NaCl 10.0g NaHCO 3 4.0g Yeast extract 2.0g Trypticase™ 2.0g NH 4 Cl 1.0g MgCl 2 ·6H 2 O 1.0g KCl 0.5g CaCl 2 ·2H 2 O 0.4g K 2 HPO 4 0.4g Resazurin 0.5mg Sodium acetate solution 50.0mL Sodium formate solution 50.0mL Na 2 S·9H 2 O solution 10.0mL Cysteine-HCl·H 2 O solution 10.0mL Vitamin solution 10.0mL Trace elements solution 10.0mL Selenite-tungstate solution 1.0mL pH 7.2 ± 0.2 at 25°C Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.25g Preparation of Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Sodium Formate Solution: Composition per 50.0mL: Na-formate 6.8g Preparation of Sodium Formate Solution: Add sodium formate to distilled/deionized water and bring volume to 50.0mL. Mix thor- oughly. Sparge with 100% N 2 . Filter sterilize. Sodium Acetate Solution: Composition per 50.0mL: Na-acetate 1.6g Preparation of Sodium Acetate Solution: Add sodium acetate to distilled/deionized water and bring volume to 50.0mL. Mix thorough- ly. Sparge with 100% N 2 . Filter sterilize. Selenite-Tungstate Solution 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 Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. © 2010 by Taylor and Francis Group, LLC MB Medium 1029 Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.25g 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. Sparge with N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an- aerobically. 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. 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 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Filter sterilize. Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% N 2 + 20% CO 2 gas mixture. Add components, except sodium acetate solution, sodium formate solution, NaHCO 3 , cysteine solution, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 870.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to 25°C while sparging with 80% N 2 + 20% CO 2 . Add solid NaHCO 3 . Mix thoroughly. Adjust pH to 6.8–7.0. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pres- sure–121°C. Aseptically and anaerobically add per liter 50.0mL sterile sodium acetate solution, 50.0mL sterile sodium formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. The final pH should be 7.2. Use: For the cultivation of Methanocalculus taiwanensis DSM 14648. MB Medium (DSMZ Medium 924) Composition per liter: NaCl 5.0g NaHCO 3 4.0g Yeast extract 2.0g Trypticase™ 2.0g NH 4 Cl 1.0g MgCl 2 ·6H 2 O 1.0g KCl 0.5g CaCl 2 ·2H 2 O 0.4g K 2 HPO 4 0.4g Resazurin 0.5mg Sodium acetate solution 50.0mL Sodium formate solution 50.0mL Na 2 S·9H 2 O solution 10.0mL Cysteine-HCl·H 2 O solution 10.0mL Vitamin solution 10.0mL Trace elements solution 10.0mL Selenite-tungstate solution 1.0mL pH 7.2 ± 0.2 at 25°C Cysteine Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.25g Preparation of Cysteine Solution: Add L-cysteine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Sodium Formate Solution: Composition per 50.0mL: Na-formate 6.8g Preparation of Sodium Formate Solution: Add sodium formate to distilled/deionized water and bring volume to 50.0mL. Mix thor- oughly. Sparge with 100% N 2 . Filter sterilize. Sodium Acetate Solution: Composition per 50.0mL: Na-acetate 1.6g Preparation of Sodium Acetate Solution: Add sodium acetate to distilled/deionized water and bring volume to 50.0mL. Mix thorough- ly. Sparge with 100% N 2 . Filter sterilize. Selenite-Tungstate Solution 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 Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.25g 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. Sparge with N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store an- aerobically. © 2010 by Taylor and Francis Group, LLC 1030 M-BCG Yeast and Mold Agar 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. 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 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 80% H 2 + 20% CO 2 . Filter sterilize. Preparation of Medium: Prepare and dispense medium under an oxygen-free 80% N 2 + 20% CO 2 gas mixture. Add components, except sodium acetate solution, sodium formate solution, NaHCO 3 , cysteine solution, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 870.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to 25°C while sparging with 80% N 2 + 20% CO 2 . Add solid NaHCO 3 . Mix thoroughly. Adjust pH to 6.8–7.0. Distribute into tubes or bottles. Autoclave for 15 min at 15 psi pres- sure–121°C. Aseptically and anaerobically add per liter 50.0mL sterile sodium acetate solution, 50.0mL sterile sodium formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. The final pH should be 7.2. Use: For the cultivation of Methanocalculus taiwanensis DSM 14663. M-BCG Yeast and Mold Agar Composition per liter: Glucose 50.0g Agar 15.0g Biopeptone 10.0g Yeast extract 9.0g MgSO 4 ·7H 2 O 2.1g KH 2 PO 4 2.0g Diastase 0.05g Thiamine hydrochloride 0.05g Bromcresol Green 0.026g pH 4.6 ± 0.2 at 25°C Source: This medium is available from HiMedia. 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 detection of fungi in routine analysis of beverages using the membrane filter technique. m-Bismuth Sulfite Broth See: Bismuth Sulfite Broth M-Bismuth Sulfite Broth Composition per liter: Peptic digest of animal tissue 20.0g Bismuth sulfite indicator 16.0g Glucose 10.0g Plant extract 10.0g Na 2 HPO 4 8.0g FeSO 4 0.6g Brilliant Green 0.05g pH 7.7 ± 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 and heat with frequent agitation until boiling. Boil for 1 min. Do not autoclave. Cool to 45°– 50°C. Mix to disperse the precipitate and aseptically distribute into sterile tubes or flasks. Use 2.0–2.2mL of medium for each membrane filter. Use: For the selective isolation of Salmonella typhi and other enteric bacilli and for the detection of Salmonella by the membrane filter method. M-Bismuth Sulfite HiVeg Broth Composition per liter: Plant peptone 20.0g Bismuth sulfite indicator 16.0g Glucose 10.0g Plant extract 10.0g Na 2 HPO 4 8.0g FeSO 4 0.6g Brilliant Green 0.05g pH 7.7 ± 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 and heat with frequent agitation until boiling. Boil for 1 min. Do not autoclave. Cool to 45°– 50°C. Mix to disperse the precipitate and aseptically distribute into sterile tubes or flasks. Use 2.0–2.2mL of medium for each membrane filter. Use: For the selective isolation of Salmonella typhi and other enteric bacilli and for the detection of Salmonella by the membrane filter method. © 2010 by Taylor and Francis Group, LLC M-Brilliant Green HiVeg Broth 1031 MBM Acetate Medium (Mineral Base Medium with Acetate) Composition per liter: Agar 16.0g NaCl 5.0g K 2 HPO 4 1.0g NH 4 H 2 PO 4 1.0g Sodium acetate·3H 2 O 1.0g MgSO 4 ·7H 2 O 0.1g Bromthymol Blue 0.01g pH 6.5 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized wa- ter and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.5. Gently heat and bring to boiling. Distribute into screw-capped tubes in 3.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Allow tubes to cool in a slanted position. Use: For determining the nutritional independence of bacteria. Bacte- ria that are nutritionally independent turn the medium blue. MBM Medium See: Methylene Blue Milk Medium MBM Medium (Modified) (DSMZ Medium 1020) Composition per liter: NaNO 3 0.2g KH 2 PO 4 0.2g NH 4 Cl 0.2g MgCl 2 ·6H 2 O 0.4g KCl 0.2g CaCl 2 ·2H 2 O 0.1g Resazurin 1.0mg Thiosulfate solution 10.0mL Trace elements solution SL-4 2.0mL pH 7.0 ± 0.2 at 25°C Thiosulfate Solution: Composition per 10.0mL: Na 2 S 2 O 3 2.5g 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. 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 Trace Elements Solution SL-6: Composition per liter: MnCl 2 ·4H 2 O 0.5g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g Na 2 MoO 4 ·2H 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. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Preparation of Medium: Add components, except thiosfulfate so- lution and trace elements SL-4 solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Sparge solution with 80% N 2 and 20% CO 2 gas mixture to make it anoxic. Distribute into culture vessels (e.g., 20mL in 120mL serum bottles) under a gas atmo- sphere of 80% N 2 and 20% CO 2 . Autoclave for 15 min at 15 psi pres- sure–121°C. Cool to room temperature. Prior to inoculation, add the trace elements solution and thiosulfate solution to the medium. Adjust pH to 7.0. After inoculation, pressurize culture vessels to 0.5 bar over- pressure with 100% H 2 gas. Use: For the cultivation of Sulfuricurvum kujiense. m-Brilliant Green Broth See: Brilliant Green Broth M-Brilliant Green Broth Composition per liter: Proteose peptone 20.0g Lactose 20.0g Saccharose 20.0g NaCl 10.0g Yeast extract 6.0g Phenol Red 0.16g Brilliant Green 0.025g pH 6.9 ± 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 to1.0L. Mix thoroughly. Distribute into tubes containing inverted Durham tubes, in 10.0mL amounts for testing 1.0mL or less of sample. Gently heat and bring to boiling. Do not au- toclave. Cool the broth rapidly. Medium is sensitive to light. Use: For the detection of coliform microorganisms in foods, dairy products, water, and wastewater, as well as in other materials of sani- tary importance. M-Brilliant Green HiVeg Broth Composition per liter: Plant peptone No. 3 20.0g Lactose 20.0g Saccharose 20.0g NaCl 10.0g Yeast extract 6.0g Phenol Red 0.16g Brilliant Green 0.025g pH 6.9 ± 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 to1.0L. Mix thoroughly. Distribute into tubes containing inverted Durham tubes, in 10.0mL amounts for testing 1.0mL or less of sample. Gently heat and bring to boiling. Do not au- toclave. Cool the broth rapidly. Medium is sensitive to light. © 2010 by Taylor and Francis Group, LLC 1032 M-Broth, HiVeg Use: For the detection of coliform microorganisms in foods, dairy products, water, and wastewater, as well as in other materials of sani- tary importance. M-Broth, HiVeg Composition per liter: Plant hydrolysate 12.5g K 2 HPO 4 5.0g NaCl 5.0g Sodium citrate 5.0g Yeast extract 5.0g D-Mannose 2.0g MgSO 4 0.8g MnCl 2 0.14g FeSO 4 0.04g Polysorbate 80 0.75mL pH 7.0 ± 0.22 at 25°C Source: This medium, without polysorbate 80, is available as a pre- mixed powder from HiMedia. 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 detection of Salmonella in dried foods and feeds. MCA Composition per liter: Carrots 200.0g Agar 15.0g Preparation of Medium: Peel and slice fresh carrots. Place carrots in a blender. Add 500.0mL of distilled/deionized water. Blend for 40 sec at high speed. Filter through four layers of cheesecloth. Squeeze out juice from the residue. Bring volume of filtrate to 1.0L with dis- tilled/deionized water. Add agar. 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 Phytophthora megasperma. McBride Agar, Modified Composition per liter: Agar 15.0g Glycine anhydride 10.0g Tryptose 10.0g NaCl 5.0g Beef extract 3.0g Phenylethanol 2.5g LiCl 0.5g Cycloheximide solution 10.0mL pH 7.3 ± 0.2 at 25°C Cycloheximide Solution: Composition per 10.0mL: Cycloheximide 0.2g Preparation of Cycloheximide Solution: Add cycloheximide to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Filter sterilize. Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for- mation and inhalation. Preparation of Medium: Add components, except cycloheximide solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile cycloheximide solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation of Listeria monocytogenes from dairy products. McBride Listeria Agar Composition per liter: Agar 15.0g Glycine 10.0g Pancreatic digest of casein 5.0g Peptic digest of animal tissue 5.0g NaCl 5.0g Beef extract 3.0g Phenylethyl alcohol 2.5g LiCl 0.5g 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 to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the selective isolation of Listeria monocytogenes from clini- cal and nonclinical specimens containing mixed flora. McBride Listeria HiVeg Agar Base with Blood and Selective Supplement Composition per liter: Agar 15.0g Glycine anhydride 10.0g Plant hydrolysate No. 1 10.0g NaCl 5.0g Plant extract 3.0g Phenyl ethanol 2.5g LiCl 0.5g Sheep blood, defibrinated 50.0mL Selective supplement solution 10.0mL pH 7.3 ± 0.2 at 25°C Source: This medium, without blood or selective supplement solu- tion, is available as a premixed powder from HiMedia. Caution: LiCl is harmful. Avoid bodily contact and inhalation of va- pors. On contact with skin wash with plenty of water immediately. Selective Supplement Solution: Composition per 10.0mL: Cycloheximide 0.2g Preparation of Selective Supplement Solution: Add cyclohex- imide to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for- mation and inhalation. Preparation of Medium: Add components, except blood and se- lective supplement solution, to distilled/deionized water and bring vol- ume to 940.0mL. Mix thoroughly. Gently heat and bring to boiling. © 2010 by Taylor and Francis Group, LLC McClung-Toabe Agar 1033 Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add 50.0mL defibrinated blood and 10.0mL selective supplement solution. Mix thoroughly. Pour into ster- ile Petri dishes or leave in tubes. Use: For the selective isolation of Listeria monocytogenes from clini- cal and nonclinical specimens containing mixed flora. McBride Listeria HiVeg Agar Base, Modified, with Blood and Selective Supplement (Modified McBride Listeria HiVeg Agar Base) Composition per liter: Agar 15.0g Glycine anhydride 10.0g NaCl 5.0g Plant extract 3.0g Phenyl ethanol 2.5g LiCl 0.5g Sheep blood, defibrinated 50.0mL Selective supplement solution 10.0mL pH 7.3 ± 0.2 at 25°C Source: This medium, without blood or selective supplement solu- tion, is available as a premixed powder from HiMedia. Caution: LiCl is harmful. Avoid bodily contact and inhalation of va- pors. On contact with skin wash with plenty of water immediately. Selective Supplement Solution: Composition per 10.0mL: Cycloheximide 0.2g Preparation of Selective Supplement Solution: Add cyclohex- imide to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for- mation and inhalation. Preparation of Medium: Add components, except blood and se- lective supplement solution, to distilled/deionized water and bring vol- ume to 940.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 50°C. Aseptically add 50.0mL defibrinated blood and 10.0mL selective supplement solution. Mix thoroughly. Pour into ster- ile Petri dishes or leave in tubes. Use: For the selective isolation of Listeria monocytogenes from clini- cal and nonclinical specimens containing mixed flora. McCarthy Agar Composition per liter: Cornstarch 10.0g Naladixic acid 0.015g Colistin 0.01g GC agar base 1.0L pH 7.2 ± 0.2 at 25°C GC Agar Base: Composition per liter: Agar 10.0g Pancreatic digest of casein 7.5g Peptic digest of animal tissue 7.5g NaCl 5.0g K 2 HPO 4 4.0g Cornstarch 1.0g KH 2 PO 4 1.0g Preparation of GC Agar Base: Add components to distilled/de- ionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: To 1.0L of GC agar base, add the corn- starch. Gently heat while stirring to dissolve. Add the naladixic acid and colistin. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the isolation and differentiation of Gardnerella vaginalis (Haemophilus vaginalis, Corynebacterium vaginale) from genitouri- nary specimens. Bacteria that can utilize starch appear as colonies sur- rounded by a clear zone. McClung Carbon-Free Broth Composition per liter: NaNO 3 2.0g K 2 HPO 4 0.8g MgSO 4 ·7H 2 O 0.5g FeCl 3 0.01g MnCl 2 ·4H 2 O 8.0mg ZnSO 4 2.0mg pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized wa- ter and bring volume to 1.0L. Mix thoroughly. Gently heat without boiling until salts dissolve. Cool to 25°C. Adjust pH to 7.2. Filter sterilize. Use: For use as a basal medium in determining the carbon assimilation capabilities of microorganisms. McClung-Toabe Agar Composition per liter: Proteose peptone 40.0g Agar 25.0g Na 2 HPO 4 5.0g Glucose 2.0g NaCl 2.0g KH 2 PO 4 1.0g MgSO 4 ·7H 2 O 0.1g Egg yolk emulsion, 50% 100.0mL pH 7.3 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks 11 Whole chicken egg 1 NaCl (0.9% solution) 50.0mL Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution 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. Preparation of Medium: Add components, except egg yolk emul- sion, 50%, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Asepti- © 2010 by Taylor and Francis Group, LLC 1034 McClung-Toabe Agar, Modified cally add 100.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes in 15.0mL volumes. Use: For the isolation and cultivation of Clostridium perfringens in foods. McClung-Toabe Agar, Modified Composition per liter: Proteose peptone No. 2 40.0g Agar 20.0g Na 2 HPO 4 5.0g Glucose 2.0g NaCl 2.0g KH 2 PO 4 1.0g MgSO 4 ·7H 2 O 0.1g Egg yolk emulsion, 50% 100.0mL Hemin solution 1.0mL pH 7.6 ± 0.2 at 25°C Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks 11 Whole chicken egg 1 NaCl (0.9% solution) 50.0mL Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution 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. Hemin Solution: Composition per 100.0mL: Hemin 0.5g NaOH (1N solution) 20.0mL Preparation of Hemin Solution: Add hemin to 20.0mL of 1N NaOH solution. Mix thoroughly. Bring volume to 100.0mL with dis- tilled/deionized water. Preparation of Medium: Add components, except egg yolk emul- sion, 50%, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Asepti- cally add 100.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes. Use: For the cultivation of a wide variety of anaerobic bacteria. For the differentiation of anaerobic bacteria based on lecithinase production and lipase production. Bacteria that produce lecithinase appear as col- onies surrounded by a zone of insoluble precipitate. Bacteria that pro- duce lipase appear as colonies with a pearly iridescent sheen. McClung-Toabe Agar, Modified Composition per liter: Proteose peptone No. 2 40.0g Agar 20.0g Na 2 HPO 4 5.0g Glucose 2.0g NaCl 2.0g KH 2 PO 4 1.0g MgSO 4 ·7H 2 O 0.1g Neomycin 0.1g Egg yolk emulsion, 50% 100.0mL Hemin solution 1.0mL pH 7.6 ± 0.2 at 25°C Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks 11 Whole chicken egg 1 NaCl (0.9% solution) 50.0mL Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution 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. Hemin Solution: Composition per 100.0mL: Hemin 0.5g NaOH (1N solution) 20.0mL Preparation of Hemin Solution: Add hemin to 20.0mL of 1N NaOH solution. Mix thoroughly. Bring volume to 100.0mL with dis- tilled/deionized water. Preparation of Medium: Add components, except egg yolk emul- sion, 50%, to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Asepti- cally add 100.0mL of sterile egg yolk emulsion, 50%. Mix thoroughly. Pour into sterile Petri dishes in 20.0mL volumes. Use: For the cultivation of Clostridium species. For the differentiation of Clostridium species based on lecithinase production and lipase pro- duction. Bacteria that produce lecithinase appear as colonies sur- rounded by a zone of insoluble precipitate. Bacteria that produce lipase appear as colonies with a pearly iridescent sheen. McClung-Toabe Agar, Modified Composition per liter: Proteose peptone No. 2 20.0g Agar 20.0g Yeast extract 5.0g Pancreatic digest of casein 5.0g NaCl 5.0g Sodium thioglycolate 1.0g Egg yolk emulsion, 50% 80.0mL pH 7.6 ± 0.2 at 25°C Egg Yolk Emulsion, 50%: Composition per 100.0mL: Chicken egg yolks 11 Whole chicken egg 1 NaCl (0.9% solution) 50.0mL Preparation of Egg Yolk Emulsion, 50%: Soak eggs with 1:100 dilution 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. Preparation of Medium: Add components, except egg yolk emul- sion, 50%, to distilled/deionized water and bring volume to 920.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Asepti- © 2010 by Taylor and Francis Group, LLC . Cool to 45°–50°C. Aseptically add 20.0mL of sterile Sarci- na lutea suspension, 20.0mL of sterile horse serum, 10.0mL of supplement solution, and 10.0mL of sterile PCAT inhibitor. Mix thor- oughly formate solution, 10.0mL of sterile cysteine solution, and 10.0mL of sterile Na 2 S·9H 2 O solution. Mix thoroughly. The final pH should be 6.5. Use: For the cultivation of Methanofollis aquaemaris. digest of casein 17.0g NaCl 5.0g Papaic digest of soybean meal 3.0g K 2 HPO 4 2.5g Glucose 2.5g pH 7.3 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC 1026 Mating Agar Preparation of Soybean