Regan-Lowe Semisolid Transport Medium 1485 Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into screw- capped tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the transport and isolation of bacteria from dental plaque, especially Streptococcus mutans, Streptococcus sanguis, and Lactoba- cillus species. Reduced Transport Fluid Composition per liter: (NH 4 ) 2 SO 4 9.0g NaCl 9.0g K 2 HPO 4 4.5g KH 2 PO 4 4.5g Na 2 CO 3 4.0g EDTA (ethylenediamine tetraacetic acid) 3.8g Dithiothreitol 2.0g MgSO 4 ·7H 2 O 1.8g pH 8.0 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Asep- tically distribute into sterile tubes with rubber stoppers. Use: For the transport and isolation of bacteria from dental plaque, especially Streptococcus mutans and Streptococcus sanguis. Also used for the cultivation of a variety of Gram-positive bacteria from the oral cavity, especially streptococci, actinomycetes, lactobacilli, clostridia, Bacteroides species, Fusobacterium species, and Veillonella species. Reduced Transport Fluid Composition per liter: Stock mineral salt solution No. 1 75.0mL Stock mineral salt solution No. 2 75.0mL Dithiothreitol (1% solution) 20.0mL Ethylenediamine tetraacetic acid (1M solution) 10.0mL Na 2 CO 3 (8% solution) 5.0mL Resazurin (0.1% solution) 1.0mL pH 8.0 ± 0.2 at 25°C Stock Mineral Salt Solution No. 1: Composition per 100.0mL: K 2 HPO 4 0.6g Preparation of Stock Mineral Salt Solution No. 1: Add K 2 HPO 4 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Stock Mineral Salt Solution No. 2: Composition per 100.0mL: NaCl 1.2g (NH 4 ) 2 SO 4 1.2g K 2 HPO 4 0.6g MgSO 4 ·7H 2 O 0.25g Preparation of Stock Mineral Salt Solution No. 2: Add com- ponents to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Asep- tically distribute into sterile tubes with rubber stoppers. Use: For the transport and isolation of bacteria from dental plaque, especially Streptococcus mutans and Streptococcus sanguis. Also used for the cultivation of a variety of Gram-positive bacteria from the oral cavity, especially streptococci, actinomycetes, lactobacilli, clostrida, Bacteroides, Fusobacteria, and Veillonela. Regan-Lowe Charcoal Agar (Regan-Lowe Medium) Composition per liter: Agar 12.0g Beef extract 10.0g Pancreatic digest of gelatin 10.0g Soluble starch 10.0g NaCl 5.0g Charcoal 4.0g Niacin 0.01g Horse blood, defibrinated 100.0mL Cephalexin solution 10.0mL pH 7.4 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Cephalexin Solution: Composition per 10.0mL: Cephalexin 0.04g Preparation of Cephalexin Solution: Add cephalexin to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except horse blood and cephalexin solution, to distilled/deionized water and bring volume to 890.0mL. 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 horse blood and sterile cephalexin solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Swirl me- dium while dispensing to keep charcoal in suspension. Use: For the selective isolation and cultivation of Bordetella pertussis and Bordetella parapertussis from clinical specimens. Regan-Lowe Semisolid Transport Medium Composition per liter: Agar 6.0g Beef extract 5.0g Pancreatic digest of gelatin 5.0g Soluble starch 5.0g NaCl 2.5g Charcoal 2.0g Niacin 0.01g Horse blood, defibrinated 100.0mL Cephalexin solution 10.0mL pH 7.4 ± 0.2 at 25°C Cephalexin Solution: Composition per 10.0mL: Cephalexin 0.04g Preparation of Cephalexin Solution: Add cephalexin to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except horse blood and cephalexin solution, to distilled/deionized water and bring volume to 890.0mL. 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 ster- ile horse blood and sterile cephalexin solution. Mix thoroughly. Aseptical- © 2010 by Taylor and Francis Group, LLC 1486 Reinforced AE Medium ly distribute into small, sterile, screw-capped tubes. Fill tubes half-full. Swirl medium while dispensing to keep charcoal in suspension. Use: For the transport of Bordetella pertussis and Bordetella paraper- tussis isolated from clinical specimens. Reinforced AE Medium (RAE Medium) (LMG Medium 239) Composition per liter: Base medium 500.0mL Growth medium 500.0mL pH 5.0 ± 0.2 at 25°C Base Medium: Composition per liter: Agar 10.0g Preparation of Base Medium: Add agar to distilled/deionized wa- ter and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour as a base layer into sterile Petri dishes. Growth Medium: Composition per liter: Glucose 40.0g Agar 20.0g Yeast extract 10.0g Peptone 10.0g Na 2 HPO 4 ·2H 2 O 3.38g Citric acid·2H 2 O 1.5g Ethanol 20.0mL Acetic acid 10.0mL Preparation of Growth Medium: Add components, except etha- nol and acetic acid, to distilled/deionized water and bring volume to 970.0mL. 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 20.0mL filter sterilized ethanol and 10.0mL filter sterilized acetic acid. Mix thoroughly. Preparation of Growth Medium: This medium is used as a dou- ble layer. Pour as a layer of base medium into sterile Petri dishes. Al- low to solidify. Pour a thin layer of growth medium over the solid base medium. Allow to solidify. Use: For the isolation and cultivation of Gluconacetobacter spp. and Acetobacter pomorum. Reinforced Clostridial Agar Composition per liter: Agar 13.5g Beef extract 10.0g Pancreatic digest of casein 10.0g NaCl 5.0g Glucose 5.0g Yeast extract 3.0g Sodium acetate 3.0g Soluble starch 1.0g L-Cysteine·HCl·H 2 O 0.5g pH 6.8 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems and 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 10 psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and enumeration of Clostridium species, Bifi- dobacterium species, other anaerobes (e.g., lactobacilli), and faculta- tive organisms from clinical specimens and foods. Reinforced Clostridial Agar with Tween™ (LMG Medium 146) Composition per liter: Agar 13.5g Beef extract 10.0g Pancreatic digest of casein 10.0g NaCl 5.0g Glucose 5.0g Yeast extract 3.0g Sodium acetate 3.0g Tween™ 80 1.0g Soluble starch 1.0g L-Cysteine·HCl·H 2 O 0.5g pH 6.8 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 10 psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Bifidobacterium meryci- cum. Reinforced Clostridial HiVeg Agar Composition per liter: Agar 13.5g Plant extract 10.0g Plant hydrolysate 10.0g Glucose 5.0g NaCl 5.0g Yeast extract 3.0g Sodium acetate 3.0g Starch, soluble 1.0g L-Cysteine·HCl 0.5g pH 6.8 ± 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. Autoclave for 15 min at 10 psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and enumeration of clostridia and other anaer- obes. Reinforced Clostridial HiVeg Broth Composition per liter: Plant extract 10.0g Plant hydrolysate 10.0g Glucose 5.0g NaCl 5.0g Sodium acetate 3.0g Yeast extract 3.0g © 2010 by Taylor and Francis Group, LLC Reinforced Clostridial Medium with Uric Acid 1487 Starch, soluble 1.0g Agar 0.5g L-Cysteine·HCl 0.5g pH 6.8 ± 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. Autoclave for 15 min at 10 psi pressure–115°C. Use: For the cultivation and enumeration of clostridia and other anaer- obes. Reinforced Clostridial Medium Composition per liter: Tryptose 10.0g Beef extract 10.0g Glucose 5.0g NaCl 5.0g Yeast extract 3.0g Sodium acetate 3.0g Soluble starch 1.0g L-Cysteine·HCl·H 2 O 0.5g Agar 0.5g pH 6.8 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems and 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 10 psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes. Use: For the nonselective cultivation and enumeration of Clostridium species, other anaerobes such as lactobacilli, and facultative organisms from clinical specimens and foods. Reinforced Clostridial Medium with Casamino Acids Composition per liter: Casamino acids 15.0g Agar 13.5g Beef extract 10.0g Pancreatic digest of casein 10.0g NaCl 5.0g Glucose 5.0g Yeast extract 3.0g Sodium acetate 3.0g Soluble starch 1.0g L-Cysteine·HCl·H 2 O 0.5g pH 6.8 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Clostridium aminophilum. Reinforced Clostridial Medium with Glycerol Composition per liter: Agar 13.5g Beef extract 10.0g Pancreatic digest of casein 10.0g NaCl 5.0g Glucose 5.0g Glycerol 5.0g Yeast extract 3.0g Sodium acetate 3.0g Soluble starch 1.0g L-Cysteine·HCl·H 2 O 0.5g pH 6.8 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 10 psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Anaerovibrio glycerini. Reinforced Clostridial Medium, Modified (ATCC Medium 2107) Composition per liter: Tryptose 10.0g Beef extract 10.0g Glucose 5.0g NaCl 5.0g Yeast extract 3.0g Sodium acetate 3.0g Soluble starch 1.0g L-Cysteine·HCl·H 2 O 0.5g pH 6.8 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 10 psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Clostridium saccharobutylicum, Clostrid- ium frigidicarnis, and Mitsuokella jalaludinii. Reinforced Clostridial Medium with Sodium Lactate Composition per liter: Tryptose 10.0g Beef extract 10.0g Glucose 5.0g NaCl 5.0g Yeast extract 3.0g Sodium acetate 3.0g Soluble starch 1.0g L-Cysteine·HCl·H 2 O 0.5g Agar 0.5g Sodium lactate (60% solution) 15.0mL pH 6.8 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 10 psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes. Use: For the nonselective cultivation and enumeration of Clostridium species, other anaerobes such as lactobacilli, and facultative organisms from clinical specimens and foods. Reinforced Clostridial Medium with Uric Acid Composition per liter: Agar 13.5g Beef extract 10.0g Pancreatic digest of casein 10.0g NaCl 5.0g © 2010 by Taylor and Francis Group, LLC 1488 Renibacterium KDM2 Medium Glucose 5.0g Uric acid 3.0g Yeast extract 3.0g Sodium acetate 3.0g Soluble starch 1.0g L-Cysteine·HCl·H 2 O 0.5g pH 6.8 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Clostridium acidurici. Renibacterium KDM2 Medium Composition per liter: Agar 15.0g Peptone 10.0g L-Cysteine·HCl·H 2 O 1.0g Yeast extract 0.5g Fetal calf serum 200.0mL pH 6.5 ± 0.2 at 25°C Preparation of Medium: Add components, except fetal calf serum and agar, to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Adjust pH to 6.5 with NaOH. Add agar. 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 fetal calf serum. Mix thor- oughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Renibacterium salmoni- narum. Reuters Sorbic Acid Agar Base Composition per liter: D-Glucose 20.0g Agar 16.0g Casein enzymic hydrolysate 10.0g Meat extract 10.0g Yeast extract 5.0g Sodium acetate 5.0g Sodium citrate 3.0g Tween 80 1.0g MgSO 4 ·7H 2 O 0.2g MnSO 4 ·2H 2 O 0.05g Selective supplement solution 10.0mL pH 5.0 ± 0.2 at 25°C Source: This medium is available from HiMedia. Selective Supplement Solution: Composition per 10.0mL: Sorbic acid 0.4g Preparation of Selective Supplement Solution: Add sorbic acid to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except selective sup- plement solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Mix to dis- solve components completely. Cool to 50°C. Aseptically add selective supplement solution. Mix thoroughly. Sterilize for 30 min at 0 psi pres- sure–100°C. Pour into Petri dishes or aseptically distribute into sterile tubes. Use: For the isolation and differentiation of lactobacilli from food- stuffs, feces, etc. RF Medium Composition per liter: Yeast extract 0.05g Peptone 0.05g (NH 4 ) 2 SO 4 0.05g L-Cysteine·HCl·H 2 O 0.05g Salt solution 50.0mL Rumen fluid, clarified 30.0mL Resazurin (1% solution) 0.1mL pH 7.4 ± 0.2 at 25°C Salt Solution: Composition per liter: NaHCO 3 10.0g NaCl 2.0g K 2 HPO 4 1.0g KH 2 PO 4 1.0g CaCl 2 , anhydrous 0.2g MgSO 4 0.2g Preparation of Salts Solution: Add CaCl 2 and MgSO 4 to 300.0mL of distilled/deionized water. Mix thoroughly until dissolved. Bring vol- ume to 800.0mL with distilled/deionized water. Add remaining com- ponents while stirring. Bring volume to 1.0L. Mix thoroughly. Store at 4°C. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.2–6.3 with 4N HCl. Gently heat and bring to boiling under 100% N 2 . Anaer- obically distribute into tubes in 7.0mL volumes. Cap with rubber stop- pers. Place tubes in a press. Autoclave for 20 min at 15 psi pressure– 121°C with fast exhaust. The pH of the medium should be 7.4 after au- toclaving. Use: For the cultivation and maintenance of Treponema bryantii. RFC Agar See: Rumen Fluid Cellobiose Agar RGCA Medium (Rumen Fluid Glucose Cellobiose Agar) Composition per 300.3mL: Rumen fluid 120.0mL Solution IV 65.0mL Mineral solution I 45.0mL Mineral solution II 45.0mL Na 2 CO 3 solution 20.0mL L-Cysteine·HCl·H 2 O solution 5.0mL Solution III 0.3mL pH 6.6 ± 0.2 at 25°C Mineral Solution I: Composition per 100.0mL: K 2 HPO 4 0.3g Preparation of Mineral Solution I: Add K 2 HPO 4 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Mineral Solution II: Composition per 100.0mL: (NH 4 ) 2 SO 4 0.6g NaCl 0.6g © 2010 by Taylor and Francis Group, LLC Rhizobium BIII Defined Agar 1489 KH 2 PO 4 0.3g MgSO 4 0.06g CaCl 2 0.06g Preparation of Mineral Solution II: Add components to dis- tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Solution III: Composition per 10.0mL: Resazurin 0.01g Preparation of Solution III: Add resazurin to 10.0mL of distilled/ deionized water. Mix thoroughly. Solution IV: Composition per 65.0mL: Agar 4.5g Glucose 0.6g Cellobiose 0.6g Preparation of Solution IV: Add components to distilled/deion- ized water and bring volume to 65.0mL. Mix thoroughly. L-Cysteine·HCl·H 2 O Solution: Composition per 100.0mL: L-Cysteine·HCl·H 2 O 3.0g 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 100.0mL. Mix thoroughly. Filter sterilize. Na 2 CO 3 Solution: Composition per 100.0mL: Na 2 CO 3 6.0g 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. Filter sterilize. Rumen Fluid: Composition per 120.0mL: Rumen fluid 120.0mL Preparation of Rumen Fluid: Filter rumen contents, obtained from a cow on an alfalfa-hay concentrate ration, through two layers of cheesecloth to remove larger particles. Store under CO 2 in quart milk bottles in the refrigerator. Much of the particulate matter settles out. Use the supernatant fluid. Preparation of Medium: Combine 45.0mL of mineral solution I, 45.0mL of mineral solution II, 0.3mL of solution III, and 65.0mL of so- lution IV in a 500.0mL flask. Gently heat and bring to boiling. Add 120.0mL of rumen fluid. Gently heat and bring to boiling under 100% CO 2 . Cap with a rubber stopper and wire the stopper secure. Autoclave for 20 min at 15 psi pressure–121°C. Cool to 45°–50°C. Remove stop- per and gas with 100% CO 2 to eliminate O 2 . Aseptically add 5.0mL of sterile L-cysteine·HCl·H 2 O solution and 20.0mL of sterile Na 2 CO 3 so- lution. Mix thoroughly. Aseptically and anaerobically distribute into tubes under 100% CO 2 in 6.0mL volumes. Cap with rubber stoppers. Use: For the cultivation and maintenance of Ruminococcus albus, Ruminococcus flavifaciens, and Succinimonas amylolytica. Rhamnose Salts Medium Composition per liter: Rhamnose 10.0g Yeast extract 3.0g K 2 HPO 4 2.9g KH 2 PO 4 2.1g NH 4 ·Cl 2.0g MgSO 4 ·7H 2 O 0.4g NaCl 30.0mg CaCl 2 3.0mg FeSO 4 ·7H 2 O 1.0mg pH 7.0 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Rhodococcus chlorophenolicus. Rhizobium Agar (LMG 201) Composition per liter: Agar 20.0g Mannitol 10.0g Yeast extract 1.0g Sodium glutamate 0.5g KH 2 PO 4 0.5g MgSO 4 ·7H 2 O 0.1g CaCl 2 ·2H 2 O 40.0mg FeCl 3 4.0mg pH 6.8 ± 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 6.8. Gently heat and bring to boiling. Distribute into tubes or flasks. Auto- clave 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 Rhizobium fredii, Rhizo- bium galegae, Rhizobium leguminosarum, Rhizobium loti, Rhizobium meliloti, and Rhizobium tropici. Rhizobium BIII Defined Agar Composition per liter: Agar 13.0g Mannitol 10.0g Sodium glutamate 1.1g K 2 HPO 4 0.23g MgSO 4 ·7H 2 O 0.1g Trace elements stock 1.0mL Vitamin stock 1.0mL pH 7.0 ± 0.2 at 25°C Trace Elements Stock: Composition per liter: Nitrilotriacetic acid 7.0g CaCl 2 ·2H 2 O 6.62g H 3 BO 3 0.145g FeSO 4 ·7H 2 O 0.125g Na 2 MoO 4 0.125g ZnSO 4 ·7H 2 O 0.108g CoSO 4 ·7H 2 O 0.07g CuSO 4 ·5H 2 O 5.0mg MnCl 2 ·4H 2 O 4.3mg Preparation of Trace Elements Stock: Add components to 500.0mL of distilled/deionized water in the order: CaCl 2 ·2H 2 O, H 3 BO 3 , FeSO 4 ·7H 2 O, CoSO 4 ·7H 2 O, CuSO 4 ·5H 2 O, MnCl 2 ·4H 2 O, ZnSO 4 ·7H 2 O, and Na 2 MoO 4 . Adjust pH to 5.0. Add nitrilotriacetic acid. Bring volume to 1.0L with distilled/deionized water. © 2010 by Taylor and Francis Group, LLC 1490 Rhizobium BIII Defined Broth Vitamin Stock: Composition per liter: Inositol 0.12g p-Aminobenzoic acid 0.02g Biotin 0.02g Calcium pantothenate 0.02g Nicotinic acid 0.02g Pyridoxine·HCl 0.02g Riboflavin 0.02g Thiamine·HCl 0.02g Sodium phosphate buffer (50.0mM solution, pH 7.0) 1.0L Preparation of Vitamin Stock: Combine components. Mix thor- oughly. Filter sterilize. Store at 4°C in the dark. Preparation of Medium: Add components, except vitamin stock, to distilled/deionized water and bring volume to 999.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pres- sure–121°C. Cool to 45°–50°C. Aseptically add 1.0mL of sterile vitamin stock. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation and cultivation of Rhizobium species from root nodules. Rhizobium BIII Defined Broth Composition per liter: Mannitol 10.0g Sodium glutamate 1.1g K 2 HPO 4 0.23g MgSO 4 ·7H 2 O 0.1g Trace elements stock 1.0mL Vitamin stock 1.0mL pH 7.0 ± 0.2 at 25°C Trace Elements Stock: Composition per liter: Nitrilotriacetic acid 7.0g CaCl 2 ·2H 2 O 6.62g H 3 BO 3 0.145g FeSO 4 ·7H 2 O 0.125g Na 2 MoO 4 0.125g ZnSO 4 ·7H 2 O 0.108g CoSO 4 ·7H 2 O 0.07g CuSO 4 ·5H 2 O 5.0mg MnCl 2 ·4H 2 O 4.3mg Preparation of Trace Elements Stock: Add components, except nitrilotriacetic acid, to 500.0mL of distilled/deionized water in the or- der listed. Adjust pH to 5.0. Add nitrilotriacetic acid. Bring volume to 1.0L with distilled/deionized water. Vitamin Stock: Composition per liter: Inositol 0.12g p-Aminobenzoic acid 0.02g Biotin 0.02g Calcium pantothenate 0.02g Nicotinic acid 0.02g Pyridoxine·HCl 0.02g Riboflavin 0.02g Thiamine·HCl 0.02g Sodium phosphate buffer (50.0mM solution, pH 7.0) 1.0L Preparation of Vitamin Stock: Combine components. Mix thor- oughly. Filter sterilize. Store at 4°C in the dark. Preparation of Medium: Add components, except vitamin stock, to distilled/deionized water and bring volume to 999.0mL. Mix thor- oughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Aseptically add 1.0mL of sterile vitamin stock. Mix thoroughly. Asep- tically distribute into sterile tubes or flasks. Use: For the isolation and cultivation of Rhizobium species. Rhizobium japonicum Agar Composition per liter: Agar 15.0g Mannitol 10.0g Yeast extract 1.0g Soil extract 200.0mL Soil Extract: Composition per liter: African Violet soil 77.0g Na 2 CO 3 0.2g Preparation of Soil Extract: Add components to 1.0L of tap water. Autoclave for 15 min at 15 psi pressure–121°C. Filter through What- man filter paper. Bring volume to 1.0L with tap water. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Bradyrhizobium japoni- cum. Rhizobium Medium 1 Composition per liter: Agar 15.0g Yeast extract 10.0g K 2 HPO 4 0.5g MgSO 4 ·7H 2 O 0.2g NaCl 0.2g FeCl 3 ·6H 2 O 0.002g pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components, except agar, to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 7.2. 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 of members of the Rhizobiaceae. Rhizobium Medium 2 Composition per liter: Agar 15.0g Glycerol 4.6g CaSO 4 1.3g K 2 HPO 4 1.0g L-Arabinose 1.0g Yeast extract 1.0g KNO 3 0.7g MgSO 4 ·7H 2 O 0.36g FeCl 3 ·6H 2 O 4.0mg pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components, except agar, to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 7.2. Add agar. Gently heat and bring to boiling. Distribute © 2010 by Taylor and Francis Group, LLC Rhizomonas Medium 1491 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 members of the Rhizobiaceae. Rhizobium X Medium Composition per liter: Agar 15.0g Mannitol 10.0g Yeast extract 1.0g Soil extract 200.0mL pH 7.2 ± 0.2 at 25°C Soil Extract: Composition per 200.0mL: African Violet soil 77.0g Na 2 CO 3 0.2g Preparation of Soil Extract: Add components to tap water and bring volume to 200.0mL. Autoclave for 60 min at 15 psi pressure– 121°C. Filter through Whatman #1 filter paper. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Bradyrhizobium japoni- cum, Rhizobium species, and Sinorhizobium xinjiangensis. Rhizobium X Medium with Thiram Composition per liter: Agar 15.0g Mannitol 10.0g Yeast extract 1.0g Soil extract 200.0mL Thiram solution 10.0mL pH 7.2 ± 0.2 at 25°C Thiram Solution: Composition per 10.0mL: Thiram 1.0mg Ethanol, absolute 10.0mL Preparation of Thiram Solution: Add thiram to 10.0mL of abso- lute ethanol. Mix thoroughly. Filter sterilize. Soil Extract: Composition per 200.0mL: African Violet soil 77.0g Na 2 CO 3 0.2g Preparation of Soil Extract: Add components to tap water and bring volume to 200.0mL. Preparation of Medium: Add components, except thiram 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 50°C. Aseptically add 10.0mL of sterile thi- ram solution. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Bradyrhizobium japoni- cum, Rhizobium species, and Sinorhizobium xinjiangensis. Rhizoctonia Isolation Medium Composition per liter: Agar 20.0g K 2 HPO 4 1.0g KCl 0.5g MgSO 4 ·7H 2 O 0.5g NaNO 2 0.2g FeSO 4 ·7H 2 O 0.01g Dexon ® solution 10.0mL Antibiotic solution 10.0mL Gallic acid solution 10.0mL Antibiotic Solution: Composition per 10.0mL: Chloramphenicol 0.05g Streptomycin 0.05g Preparation of Antibiotic Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Dexon ® Solution: Composition per 10.0mL: Dexon ® (Chemagro ® ) wettable powder 0.09g Preparation of Dexon ® Solution: Add Dexon ® to distilled/deion- ized water and bring volume to 10.0mL. Mix thoroughly. Filter steril- ize. Gallic Acid Solution: Composition per 10.0mL: Gallic acid 0.4g Preparation of Gallic Acid Solution: Add gallic acid to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components—except Dexon ® solu- tion, antibiotic solution, and gallic acid solution—to distilled/deion- ized water and bring volume to 970.0mL. 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 Dexon ® solution, sterile an- tibiotic solution, and sterile gallic acid solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation and cultivation of Rhizoctonia species. Rhizomonas Medium Composition per liter: Noble agar 11.0g Pancreatic digest of casein 5.0g Glucose 2.5g K 2 HPO 4 1.0g MgSO 4 ·7H 2 O 0.5g KNO 3 0.5g Ca(NO 3 ) 2 ·4H 2 O 0.06g pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.2. 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 Rhizomonas suberifa- ciens. © 2010 by Taylor and Francis Group, LLC 1492 Rhizomonas suberifaciens Medium Rhizomonas suberifaciens Medium Composition per liter: Pancreatic digest of casein 5.0g K 2 HPO 4 ·3H 2 O 1.3g Noble agar 1.1g KNO 3 0.5g MgSO 4 ·7H 2 O 0.5g Ca(NO 3 ) 2 ·4H 2 O 60.0mg pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 7.2. 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 Rhizomonas suberifaciens. Rhodobacter adriaticus Medium Composition per 1001.0mL: NaCl 25.0g NaHCO 3 3.0g K 2 HPO 4 .1.0g NH 4 Cl 1.0g MgCl 2 ·6H 2 O 0.5g Sodium ascorbate 0.5g CaCl 2 ·2H 2 O 0.1g Trace elements solution SLA 1.0mL Vitamin solution 1.0mL pH 7.0 ± 0.2 at 25°C Trace Elements Solution SLA: Composition per liter: CuCl 2 ·2H 2 O 10.0g FeCl 2 ·4H 2 O 1.8g H 3 BO 3 0.5g CoCl 2 ·6H 2 O 0.25g ZnCl 2 0.1g MnCl 2 ·4H 2 O 70.0mg Na 2 MoO 4 ·2H 2 O 30.0mg Na 2 SeO 3 ·5H 2 O 10.0mg NiCl 2 ·6H 2 O 10.0mg Preparation of Trace Elements Solution SLA: Add compo- nents to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Bring pH to 2.0–3.0. Vitamin Solution: Composition per liter: Nicotinamide 35.0mg Thiamine·HCl 30.0mg p-Aminobenzoic acid 20.0mg Pyridoxal·HCl 10.0mg Calcium DL-pantothenate 10.0mg Biotin 10.0mg Vitamin B 12 5.0mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter ster- ilize. Preparation of Medium: Add components, except vitamin solu- tion, to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 1.0mL of sterile vitamin solution. Mix thoroughly. Aseptically dis- tribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Rhodobacter adraiticus. Rhodobacter changlensis Medium (DSMZ Medium 1197) Composition per 1001.0mL: Yeast extract 0.4g Sodium pyruvate 3.0g NH 4 Cl 0.6g MgCl 2 ·6H 2 O 0.5g KH 2 PO 4 0.5g NaCl 0.4g NH 4 Cl 0.6g CaC l 2 ·2H 2 O 0.05g Trace elements solution SL-7 1.0mL Vitamin solution 1.0mL pH 7.2 ± 0.2 at 25°C Trace Elements Solution SL-7: Composition per liter: CoCl 2 ·6H 2 O 200.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg H 3 BO 3 60.0mg Na 2 MoO 4 ·2H 2 O 40.0mg CuCl 2 ·2H 2 O 20.0mg NiCl 2 ·6H 2 O 20.0mg HCl (25%) 1.0mL Preparation of Trace Elements Solution SL-7: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Vitamin Solution: Composition per 100.0mL: Vitamin B 12 20.0mg Preparation of Vitamin Solution: Add vitamin B 12 to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except vitamin solu- tion, to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Adjust pH to 7.2. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Asep- tically add vitamin solution. Mix thoroughly. Aseptically distribute into culture vessels. Use: For the cultivation of Rhodobacter changlensis. Rhodobacter Medium (LMG Medium 80) Composition per liter: Yeast extract 1.0g Disodium succinate 1.0g KH 2 PO 4 0.5g MgSO 4 ·7H2O 0.4g NaCl 0.4g NH 4 Cl 0.4g CaCl 2 ·2H 2 O 50.0mg Ferric citrate solution 5.0mL Trace elements solution 1.0mL Ethanol 0.5mL pH 5.8 ± 0.2 at 25°C © 2010 by Taylor and Francis Group, LLC Rhodobacter veldkampii Medium 1493 Ferric Citrate Solution : Composition per 100.0mL: Ferric citrate 0.1g Preparation of Ferric Citrate Solution: Add ferric citrate to dis- tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Trace Elements Solution: Composition per liter: 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 30.0mg MnCl 2 ·4H 2 O 30.0mg NiCl 2 ·6H 2 O 20.0mg CuCl 2 ·2H 2 O 10.0mg Preparation of Trace Elements Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute 40 mL me- dium into 50 mL screw-capped bottles. Flush each bottle for 1 to 2 min with nitrogen gas and then close immediately with rubber septa and screw caps. Autoclave for 15 min at 15 psi pressure–121°C. Sterile sy- ringes are used to inoculate and remove the samples. Incubate in light using a tungsten lamp. Use: For the cultivation of Rhodobacter capsulatus, Rhodobacter spha- eroides, and Rhodospirillum rubrum. Rhodobacter veldkampii Medium (DSMZ Medium 867) Composition per 2780.0mL: Solution 1 1540.0mL Solution 3 1000.0mL Solution 4 120.0mL Solution 5 120.0mL pH 4.0 ± 0.1 at 25°C Solution 1: Composition per 2500.0mL: CaCl 2 2.0g Preparation of Solution 1: Add CaCl 2 to distilled/deionized water and bring volume to 2.5L. Mix thoroughly. Solution 3: Composition per liter: NaHCO 3 4.5g Solution 2 100.0mL Preparation of Solution 3: Add NaHCO 3 to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Sparge with gas- eous CO 2 for at least 30 min. Add 100.0mL solution 2. Immediately fil- ter sterilize using CO 2 pressure to push the liquid through (no suction). Solution 2: Composition per 100.0mL: Sodium ascorbate 2.4g KH 2 PO 4 1.0g KCl 1.0g NH 4 Cl 0.8g MgCl 2 ·6H 2 O 0.8g Heavy metal solution 50.0mL Vitamin solution 15.0mL Vitamin B 12 solution 3.0mL Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Heavy Metal Solution: Composition per liter: EDTA 1.50g FeSO 4 ·7H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g MnCl 2 ·7H 2 O 0.02g Modified Hoagland trace elements solution 6.0mL Preparation of Heavy Metal Solution: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Modified Hoagland Trace Elements Solution: Composition per 3.6L: H 3 BO 3 11.0g MnCl 2 ·4H 2 O 7.0g ZnCl 2 1.0g CuCl 2 1.0g NiCl 2 1.0g CoCl 2 1.0g AlCl 3 1.0g KI 1.0g KBr 0.5g LiCl 0.5g SnCl 2 ·2H 2 O 0.5g BaCl 2 0.5g Na 2 MoO 4 0.5g Na 2 SeO 3 0.5g NaVO 3 ·H 2 O 0.1g Preparation of Modified Hoagland Trace Elements Solu- tion: Add components sequentially to distilled/deionized water and bring final volume to 3.6L. Mix thoroughly after adding each compo- nent until dissolved. Adjust pH to just below 7.0. Adjust the final pH to 3–4. The flaky yellow precipitate which is formed after mixing transforms after standing for one or a few days into a very fine white precipitate. Mix thoroughly before use. Vitamin B 12 Solution: Composition per 3.0mL: Vitamin B 12 (cyanocobalamine) 2.0mg Preparation of Vitamin B 12 Solution: Add vitamin B 12 to dis- tilled/deionized water and bring volume to 3.0mL. Mix thoroughly. Vitamin Solution: Composition per 100.0mL: Pyridoxamine·2HCl 5.0mg Nicotinic acid 2.0mg Thiamine 1.0mg Pantothenic acid 0.5mg Biotin 0.2mg p-Aminobenzoic acid 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 100% CO 2 for 30 min. Solution 4: Composition per 200.0mL: Na 2 S·9H 2 O 3.0g Preparation of Solution 4: Add Na 2 S·9H 2 O to distilled/deionized water in a flask with a magnetic stirrer and bring volume to 200.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pres- sure–121°C. Cool to room temperature. Partially neutralize the steril- © 2010 by Taylor and Francis Group, LLC 1494 Rhodobacter veldkampii Medium ized solution by adding, on a magnetic stirrer, drop by drop, 2.0mL sterile 2M H 2 SO 4 . Solution 5: Composition per 200.0mL: Na-acetate 6.0g Preparation of Solution 5: Add Na-acetate to distilled/deionized water and bring volume to 200.0mL. Mix thoroughly. Sparge with 100% N 2 for 5 min. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. Preparation of Medium: Distribute solution 1 in 77.0mL amounts into 20 127mL screw-capped Boston round bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 50.0mL sterile solution 3 to each of the 20 127mL bottles contain- ing 77.0mL of sterile solution 1 so that the solution completely fills the bottle. Mix thoroughly. Remove 6.0mL of the medium from the com- pletely filled bottles. Add 6.0mL of neutralized solution 4 so that the bottles are again completely filled. Mix thoroughly. Remove 6.0mL of the medium from the completely filled bottles. Add 6.0mL of solution 5 so that the bottles are again completely filled. Mix thoroughly. Adjust pH to 4.0. Allow the bottles to stand overnight to develop a hazy, white precipitate before inoculating. Mix the solution thoroughly before use. To inoculate, remove 6.0mL of completed medium and replace it with an equal volume of inoculum. Grow cultures under tungsten light. Use: For the cultivation of Rhodobacter veldkampii. Rhodobacter veldkampii Medium Composition per 127.0mL: Solution 1 76.2mL Solution 2 + Solution 3 44.8mL Solution 4 6.0mL Solution 1: Composition per 2.5L: CaCl 2 2.0g Preparation of Solution 1: Add CaCl 2 to distilled/deionized water and bring volume to 2.5L. Distribute in 80.0mL volumes into 127.0mL screw-capped bottles. Autoclave for 15 min at 15 psi pressure–121°C. Solution 2: Composition per 100.0mL: Sodium ascorbate 2.4g Sodium acetate 1.0g KC1 1.0g KH 2 PO 4 1.0g MgCl 2 ·6H 2 O 0.8g NH 4 Cl 0.8g Heavy metal solution 50.0mL Vitamin solution 15.0mL Vitamin B 12 solution 3.0mL Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Heavy Metal Solution: Composition per liter: Ethylenediamine tetraacetate (EDTA) 1.5g FeSO 4 ·7H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g MnCl 2 ·4H 2 O 0.02g Modified Hoagland trace elements solution 6.0mL Preparation of Heavy Metal Solution: Dissolve EDTA in 800.0mL of distilled/deionized water. Add remaining components. Bring volume to 1.0L with distilled/deionized water. Mix thoroughly. Modified Hoagland Trace Elements Solution: Composition per 3.6L: H 3 BO 3 11.0g MnCl 2 ·4H 2 O 7.0g AlCl 3 1.0g CoCl 2 1.0g CuCl 2 1.0g KI 1.0g NiCl 2 1.0g ZnCl 2 1.0g BaCl 2 0.5g KBr 0.5g LiCl 0.5g Na 2 MoO 4 0.5g SeCl 4 0.5g SnCl 2 ·2H 2 O 0.5g NaVO 3 ·H 2 O 0.1g Preparation of Modified Hoagland Trace Elements Solu- tion: Prepare each component as a separate solution. Dissolve each salt in approximately 100.0mL of distilled/deionized water. Adjust the pH of each solution to below 7.0. Combine all the salt solutions and bring the volume to 3.6L with distilled/deionized water. Adjust the pH to 3–4. A yellow precipitate may form after mixing. After a few days, it will turn into a fine white precipitate. Mix the solution thoroughly be- fore using. Vitamin Solution: Composition per 100.0mL: Pyridoxamine·2HCl 5.0mg Nicotinic acid 2.0mg Thiamine 1.0mg Pantothenic acid 0.5mg Biotin 0.2mg p-Aminobenzoic acid 0.1mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL. Mix thoroughly. Vitamin B 12 Solution: Composition per 100.0mL: Vitamin B 12 (cyanocobalamin) 2.0mg Preparation of Vitamin B 12 Solution: Add vitamin B 12 to dis- tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Solution 3: Composition per 900.0mL: NaHCO 3 4.5g Preparation of Solution 3: Add NaHCO 3 to distilled/deionized water and bring volume to 900.0mL. Mix thoroughly. Bubble 100% CO 2 through the solution for 30 min. After CO 2 saturation of solution 3, add solution 2 and immediately filter the mixture through a Seitz fil- ter (or a Millipore) using positive CO 2 pressure to push the liquid through. Solution 4: Composition per 200.0mL: Na 2 S·9H 2 O 3.0g Preparation of Solution 4: Add Na 2 S·9H 2 O to distilled/deionized water and bring volume to 200.0mL. Add a magnetic stir bar to the flask. Autoclave for 15 min at 15 psi pressure–121°C. On a magnetic stirrer, © 2010 by Taylor and Francis Group, LLC . refrigerator. Much of the particulate matter settles out. Use the supernatant fluid. Preparation of Medium: Combine 45.0mL of mineral solution I, 45.0mL of mineral solution II, 0.3mL of solution III,. the transport and isolation of bacteria from dental plaque, especially Streptococcus mutans and Streptococcus sanguis. Also used for the cultivation of a variety of Gram-positive bacteria from. thoroughly. Preparation of Growth Medium: This medium is used as a dou- ble layer. Pour as a layer of base medium into sterile Petri dishes. Al- low to solidify. Pour a thin layer of growth medium over