Handbook of Microbiological Media, Fourth Edition part 128 pps

Mycoplasma Broth 1265 Basal Medium: Composition per 700.0mL: Sorbitol 50.0g Beef heart, solids from infusion 16.2g Peptone 3.26g NaCl 1.62g Fructose 1.0g Glucose 1.0g Sucrose 1.0g Pancreatic digest of casein 1.0g Preparation of Basal Medium: Add components to distilled/de- ionized water and bring volume to 700.0mL. Mix thoroughly. Adjust pH to 7.5–7.8. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. Preparation of Medium: Filter sterilize horse serum and fresh yeast extract solution. Aseptically add to cooled, sterile basal medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Mycoplasma mycoides, Spiroplasma apis, Spiroplasma citri, and Spiroplasma melliferum. Mycoplasma Broth Composition per 950.0mL: Glucose 1.0g Nicotinamide adenine dinucleotide 0.1g PPLO broth without Crystal Violet 680.0mL Swine serum (56°C, 30 min) 150.0mL Fresh yeast extract solution 100.0mL Phenol Red (0.1% w/v solution) 20.0mL pH 7.8 ± 0.2 at 25°C PPLO Broth without Crystal Violet: Composition per 680.0mL: Beef heart, solids from infusion 11.3g Peptone 2.28g NaCl 1.13g Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems. Preparation of PPLO Broth without Crystal Violet: Add components to distilled/deionized water and bring volume to 680.0mL. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 56°C. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. Preparation of Medium: Mix glucose, nicotinamide adenine dinu- cleotide, swine serum, fresh yeast extract solution, and Phenol Red. Mix thoroughly. Heat to 56°C. Add to cooled, sterile PPLO broth with- out Crystal Violet. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Mycoplasma anseris and Mycoplasma lipofaciens. Mycoplasma Broth (ATCC Medium 555) Composition per 103.0mL: Hartley’s digest broth 30.0mL Pig serum 20.0mL Enzymatic hydrolysate of lactalbumin 10.0mL Hanks’ balanced salt solution, 10X 4.0mL Fresh yeast extract solution 2.0mL Phenol Red (0.25% solution) 1.0mL pH 7.4 ± 0.2 at 25°C Hartley’s Digest Broth: Composition per 10.0L: Ox heart 3,000.0g Pancreatin 50.0g Na 2 CO 3 , anhydrous (0.8% solution) 5.0L HCl, concentrated 80.0mL pH 7.5 ± 0.2 at 25°C Preparation of Hartley’s Digest Broth: Finely mince the ox heart. Add the meat to 5.0L of distilled/deionized water. Gently heat and bring to 80°C. Add the 5.0L of Na 2 CO 3 solution. Cool to 45°C. Add pancreatin and maintain at 45°C for 4 hr while stirring. Add the HCl and steam at 100°C for 30 min. Cool to room temperature. Adjust pH to 8.0 with 1N NaOH. Gently heat and bring to boiling. Continue boiling for 25 min. Filter while hot. Cool to room temperature. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C. Pig Serum: Composition per 100.0mL: Pig serum 100.0mL Preparation of Pig Serum: Adjust pH of pig serum to 4.4 with sterile 1N HCl. Do not let pH go below 4.2. Let serum stand at 4°C for 18-20 hr. Adjust pH to 7.0 with sterile 1N NaOH. Centrifuge at 9000 rpm for 20 min. Discard pellet. Filter supernatant solution through a 0.2μm membrane. Store at −70°C. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. Enzymatic Hydrolysate of Lactalbumin: Composition per 100.0mL: Enzymatic hydrolysate of lactalbumin 5.0g Preparation of Enzymatic Hydrolysate of Lactalbumin: Add enzymatic hydrolysate of lactalbumin to 100.0mL of phosphate buff- ered saline, 1X, pH 7.0. Phosphate Buffered Saline Solution, 1X: Composition per liter: NaCl 8.0g Na 2 HPO 4 ·7H 2 O 2.16g KCl 0.2g KH 2 PO 4 0.2g © 2010 by Taylor and Francis Group, LLC 1266 Mycoplasma Broth Base MgCl 2 ·6H 2 O 0.1g CaCl 2 0.1g Hanks’ Balanced Salt Solution, 10X: Composition per liter: NaCl 80.0g Glucose 10.0g KCl 4.0g CaCl 2 1.4g MgCl 2 ·6H 2 O 1.0g MgSO 4 ·7H 2 O 1.0g Na 2 HPO 4 ·7H 2 O 0.9g KH 2 PO 4 0.6g Preparation of Hanks’ Balanced Salt Solution, 10X: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Combine components in the following order: Hanks’ balanced salt solution, 10X, Phenol Red, Hartley’s di- gest broth, pig serum, enzymatic hydrolysate of lactalbumin, and fresh yeast extract solution. Mix thoroughly. Add 36.0mL of distilled/deion- ized water. Adjust pH to 7.4 with 1N NaOH. Filter sterilize through a 0.2μm membrane. Store at 4°C for up to 3 weeks. Use: For the cultivation of Mycoplasma species. Mycoplasma Broth Base (PPLO Broth Base without Crystal Violet) Composition per liter: Pancreatic digest of casein 7.0g NaCl 5.0g Beef extract 3.0g Yeast extract 3.0g Beef heart, solids from infusion 2.0g pH 7.8 ± 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. Gently heat and bring to boiling. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: Used as a basal medium that should be enriched for the isolation and cultivation of Mycoplasma species. Mycoplasma Broth Base, Frey with Horse Serum Composition per 1100.0mL: Pancreatic digest of casein 7.5g Papaic digest of soybean meal 2.5g KCl 0.4g MgSO 4 0.2g Na 2 PO 4 1.6g KH 2 PO 4 0.1g Horse serum 100.0mL pH 7.7 ± 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 horse serum, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pres- sure–121°C. Cool to 50°C. Add sterile, heat-inactivated horse serum. Mix thoroughly. Aseptically distribute into sterile tubes. Use: For the cultivation of avian mycoplasmas. Mycoplasma Broth Base, Frey with Horse Serum Composition per 1100.0mL: Pancreatic digest 7.5g Yeast extract 5.0g NaCl 5.0g Papaic digest of soybean meal 2.5g Na 2 HPO 4 1.6g KCl 0.4g MgSO 4 ·7H 2 O 0.2g KH 2 PO 4 0.1g Horse serum 100.0mL pH 7.7 ± 0.2 at 25°C Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pres- sure–121°C. Cool to 50°C. Add sterile, heat-inactivated horse serum. Mix thoroughly. Aseptically distribute into sterile tubes. Use: For the cultivation of avian mycoplasmas. Mycoplasma Broth, Supplemented Composition per liter: Pancreatic digest of casein 7.0g NaCl 5.0g Beef extract 3.0g Yeast extract 3.0g Beef heart, solids from infusion 2.0g Horse serum 260.0mL Fresh yeast extract solution 65.0mL pH 7.8 ± 0.2 at 25°C Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. Preparation of Medium: Add components, except horse serum and fresh yeast extract solution, to distilled/deionized 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. Cool to 50°C. To each 75.0mL of cooled, sterile basal medium, add 20.0mL of sterile horse serum and 5.0mL of fresh yeast extract so- lution. Mix thoroughly. Aseptically distribute into sterile tubes. Use: For the isolation and cultivation of Mycoplasma species. Mycoplasma Broth with Supplement G Composition per liter: Bacteriological peptone 10.0g Beef extract 10.0g NaCl 5.0g Special mineral supplement, Oxoid Unipath 0.5g Mycoplasma supplement G 250.0mL pH 7.8 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath. © 2010 by Taylor and Francis Group, LLC Mycoplasma HiVeg Broth Base with Crystal Violet and Tellurite 1267 Mycoplasma Supplement G: Composition per 20.0mL: Thallous acetate 25.0mg Horse serum 20.0mL Yeast extract (25% solution) 10.0mL Penicillin 20,000U Preparation of Mycoplasma Supplement G: Add components to distilled/deionized water and bring volume to 20.0mL. Mix thor- oughly. Filter sterilize. Caution: Thallous acetate is a poison. Preparation of Medium: Add components, except Mycoplasma supplement G, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into flasks in 80.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 20.0mL of sterile Mycoplasma supple- ment G to each 80.0mL of basal medium. Mix thoroughly. Use: For the growth of Mycoplasma species. Mycoplasma Broth with Supplement P Composition per liter: Bacteriological peptone 10.0g Beef extract 10.0g NaCl 5.0g Special mineral supplement, Oxoid Unipath 0.5g Mycoplasma supplement P 250.0mL pH 7.8 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath. Mycoplasma Supplement P: Composition per 20.0mL: Glucose 0.3g Mycoplasma broth base 0.145g Thallous acetate 8.0mg Phenol Red 1.2mg Methylene Blue chloride 0.3mg Penicillin 12,000U Horse serum 6.0mL Yeast extract (25% solution) 3.0mL Preparation of Mycoplasma Supplement P: Add components to distilled/deionized water and bring volume to 20.0mL. Mix thorough- ly. Filter sterilize. Caution: Thallous acetate is a poison. Preparation of Medium: Add components, except Mycoplasma supplement P, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into bot- tles in 1.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 2.0mL of sterile Mycoplas- ma supplement P to each bottle. Use: For the cultivation of Mycoplasma species. Mycoplasma Broth with 10% Swine Serum Composition per liter: Pancreatic digest of casein 5.6g NaCl 4.0g Yeast extract 2.6g Beef extract 2.4g Beef heart, solids from infusion 1.6g Swine serum, heat inactivated 100.0mL Fresh yeast extract solution 100.0mL pH 7.8 ± 0.2 at 25°C Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. Preparation of Medium: Add components, except swine serum and fresh yeast extract solution, to distilled/deionized water and bring volume to 800.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Add sterile swine se- rum and fresh yeast extract solution. Mix thoroughly. Aseptically distrib- ute into sterile tubes. Use: For the cultivation and maintenance of Mycoplasma columbinum and Mycoplasma columborale. Mycoplasma HiVeg Agar Base with Horse Serum and Yeast Extract (PPLO HiVeg Agar Base) Composition per liter: Agar 15.0g Plant peptone 10.0g Plant infusion 6.0g NaCl 5.0g Horse serum 260.0mL Fresh yeast extract solution 65.0mL pH 7.8 ± 0.2 at 25°C Source: This medium, without horse serum and yeast extract solution, is available as a premixed powder from HiMedia. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. Preparation of Medium: Add components, except horse serum and fresh yeast extract solution, to distilled/deionized 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. Cool to 50°C. To each 75.0mL of cooled, sterile basal medium, add 20.0mL of sterile horse serum and 5.0mL of special yeast extract solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the preparation of media for the cultivation of Mycoplasma. Mycoplasma HiVeg Broth Base with Crystal Violet and Tellurite (PPLO HiVeg Broth Base with CV) Composition per liter: Plant peptone 10.0g Plant infusion 6.0g NaCl 5.0g Crystal Violet 0.01g © 2010 by Taylor and Francis Group, LLC 1268 Mycoplasma HiVeg Broth Base without Crystal Violet and with Ascitic Fluid Chapman tellurite solution 2.85mL Ascitic fluid 250.0mL pH 7.8 ± 0.2 at 25°C Source: This medium, without tellurite, is available as a premixed powder from HiMedia. Chapman Tellurite Solution: Composition per 100.0mL: K 2 TeO 3 1.0g Preparation of Chapman Tellurite Solution: Add K 2 TeO 3 to distilled/deionized water and bring volume to 100.0mL. Mix thorough- ly. Filter sterilize. Caution: Potassium tellurite is toxic. Preparation of Medium: Add components, except ascitic fluid and Chapman tellurite solution, to distilled/deionized water and bring vol- ume to 747.15mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to less than 37°C. Aseptically add sterile ascitic fluid and 2.85mL of Chapman tellurite solution. Mix thoroughly. Asep- tically distribute into sterile tubes or flasks. Use: For the isolation of Mycoplasma species from clinical specimens. Mycoplasma HiVeg Broth Base without Crystal Violet and with Ascitic Fluid (PPLO HiVeg Broth Base without CV) Composition per liter: Plant peptone 10.0g Plant infusion 6.0g NaCl 5.0g Ascitic fluid 250.0mL pH 7.8 ± 0.2 at 25°C Source: This medium, without ascitic fluid, is available as a premixed powder from HiMedia. Preparation of Medium: Add components, except ascitic fluid, to distilled/deionized water and bring volume to 750.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to less than 37°C. Aseptically add sterile ascitic fluid. If desired, 0.5g of thallium acetate or 100,000U of penicillin may be added for a more selective medium. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the enrichment of pleuro-pneumonia-like organisms (PPLOs) and Mycoplasma species from clinical specimens. Mycoplasma Horse Serum Broth (ATCC Medium 1959) Mycoplasma broth base 660.0mL Horse serum 200.0mL Fresh yeast extract solution 100.0mL Phenol Red (0.1%) 20.0mL Glucose solution 10.0mL NaOH (1N solution) 6.25mL Arginine solution 5.0mL Mycoplasma Broth Base: Composition per liter: Pancreatic digest of casein 7.0g NaCl 5.0g Beef extract 3.0g Yeast extract 3.0g Beef heart, solids from infusion 2.0g Preparation of Mycoplasma Broth Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. Glucose Solution: Composition per 10.0mL: Glucose 5.0g Preparation of Glucose Solution: Add glucose to 10.0mL of dis- tilled/deionized water. Mix thoroughly. Filter sterilize. Arginine Solution: Composition per 10.0mL: L-Arginine 4.2g Preparation of Arginine Solution: Add arginine to 10.0mL of distilled/deionized water. Mix thoroughly. Filter sterilize. Preparation of Medium: Combine 660.0mL Mycoplasma broth base, 20.0mL Phenol Red, and 6.25mL 1N NaOH. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Asepti- cally add 5.0mL sterile arginine solution, 100.0mL sterile fresh yeast extract solution, 10.0mL sterile glucose solution, and 200.0mL filter sterilized horse serum. Mix thoroughly. Aseptically distribute into ster- ile tubes or flasks. Use: For the preparation of media for the cultivation of Mycoplasma spp. Mycoplasma Liquid Medium Composition per 1004.0mL: Arginine 1.0g Glucose 1.0g L-Cysteine·HCl·H 2 O 1.0g Mycoplasma broth base 850.0mL Horse serum, not inactivated 100.0mL Fresh yeast extract (25% solution) 50.0mL Phenol Red (1.0% solution) 2.0mL DNA calf thymus solution 2.0mL pH 7.8 ± 0.2 at 25°C Mycoplasma Broth Base: Composition per 850.0mL: Pancreatic digest of casein 7.0g NaCl 5.0g Beef extract 3.0g Yeast extract 3.0g Beef heart, solids from infusion 2.0g Preparation of Mycoplasma Broth Base: Add components to distilled/deionized water and bring volume to 850.0mL. Mix thorough- ly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 © 2010 by Taylor and Francis Group, LLC Mycoplasma Medium, Revised 1269 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. DNA Calf Thymus Solution: Composition per 10.0mL: DNA calf thymus 1.0g Preparation of DNA Calf Thymus Solution: Add DNA calf thymus to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Combine components, except Mycoplasma broth base and DNA calf thymus solution, and mix thoroughly. Filter ster- ilize through a 0.2μm membrane. Add sterile solution to 850.0mL of cooled, sterile Mycoplasma broth base. Aseptically add 2.0mL of sterile DNA calf thymus solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Mycoplasma lipophilum and Mycoplasma species. Mycoplasma Medium Composition per liter: Heart infusion broth 25.0g Mucin, bacteriological grade 5.0g Agar, purified (optional) 7.0g Hemoglobin 2.0g Turkey serum, sterile inactivated 100.0mL pH 7.8 ± 0.2 at 25°C Preparation of Medium: Add components, except turkey serum and agar, to 850.0mL distilled water. Adjust pH to 7.8. Heat mixture at 93°– 95°C for 30 min in a water bath. Restore to original volume. Add 0.5% di- atomaceous earth. Mix thoroughly. Filter through Whatman GFA (glass fi- ber paper) in Buchner filter. Clarify using 0.45μm Millipore filter. Add 15% inactivated turkey serum. Sterilize using S3 (0.1μm) Seitz filter. Use positive pressure. For solid medium : Prepare 42.5mL of double-strength broth and 42.5mL of distilled water containing 0.7g of purified agar. Ster- ilize the solutions separately and combine aseptically at 56°C with 15.0mL of sterile inactivated turkey serum for a final volume of 150.0mL. Use: For the cultivation and maintenance of Mycoplasma hyosyn- oviae. Mycoplasma Medium (CIP Medium 89) (DSMZ Medium 1080) Composition per liter: Pancreatic digest of casein 7.0g NaCl 5.0g Beef extract 3.0g Yeast extract 3.0g Beef heart, infusion from (solids) 2.0g Selective supplement solution 210.0mL Yeast extract solution 100.0mL Phenol Red solution 20.0mL Yeast Extract Solution: Composition per 100.0mL: Yeast extract 25.0g Preparation of Yeast Extract Solution: Add yeast extract to dis- tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Phenol Red Solution: Composition per 100.0mL: Phenol Red 1.0g Preparation of Phenol Red Solution: Add Phenol Red to dis- tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C. Selective Supplement Solution: Composition per 210.0mL: Ampicillin 1.0g Horse serum 200.0mL Arginine solution 10.0mL Arginine Solution: Composition per 100.0mL: L-Arginine 50.0g Preparation of Arginine Solution: Add L-arginine to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.0. Preparation of Selective Supplement Solution: Add ampicillin to 10.0mL arginine solution. Add horse serum. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except yeast extract, Phenol Red, and selective supplement solutions, to distilled/deionized water and bring volume to 670.0mL. Mix thoroughly. Adjust pH to 7.2. Distribute into tubes or flasks. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add yeast extract, Phenol Red, and selective supplement solutions. Mix thoroughly. Aseptically distribute into tubes or flasks. Use: For the cultivation of Mycoplasma spp. Mycoplasma Medium, Revised Composition per 1030.0mL: Noble agar 10.0g Distilled water 360.0mL Heart infusion broth 300.0mL Pig serum, heat inactivated 200.0mL Enzymatic hydrolysate of lactalbumin 100.0mL Hanks’ balanced salt solution, 10X 40.0mL Fresh yeast extract solution 20.0mL Phenol Red (0.25% solution) 10.0mL Heart Infusion Broth: Composition per liter: Beef heart, infusion from 500.0g Tryptose 10.0g NaCl 5.0g Preparation of Heart Infusion Broth: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gen- tly heat and bring to boiling. Enzymatic Hydrolysate of Lactalbumin: Composition per 100.0mL: Enzymatic hydrolysate of lactalbumin 5.0g Phosphate buffered saline, 1X, pH7.0 100.0mL Preparation of Enzymatic Hydrolysate of Lactalbumin: Add enzymatic hydrolysate of lactalbumin to 100.0mL of phosphate buff- ered saline, 1X, pH 7.0. © 2010 by Taylor and Francis Group, LLC 1270 Mycoplasma pneumoniae Isolation Medium Phosphate Buffered Saline Solution, 1X: Composition per liter: NaCl 8.0g Na 2 HPO 4 ·7H 2 O 2.16 KCl 0.2g KH 2 PO 4 0.2g MgCl 2 ·6H 2 O 0.1g CaCl 2 0.1g Preparation of Phosphate Buffered Saline Solution, 1X: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Hanks’ Balanced Salt Solution, 10X: Composition per liter: Na 2 Cl 80.0g Glucose 10.0g KCl 4.0g CaCl 2 1.4g MgCl 2 ·6H 2 O 1.0g MgSO 4 ·7H 2 O 1.0g Na 2 HPO 4 ·7H 2 O 0.9g KH 2 PO 4 0.6g Preparation of Hanks’ Balanced Salt Solution, 10X: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g Preparation of Fresh Yeast Extract Solution: Add the live Bak- er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so- lution. Adjust pH to 6.6–6.8. Preparation of Medium: Add agar and heart infusion broth to dis- tilled/deionized water and bring volume to 660.0mL. Mix thoroughly. Ad- just pH to 7.4. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add pig serum, enzymatic hydrolysate of lactalbumin, Hanks’ balanced salt solution, 10X, fresh yeast extract solution, and Phenol Red solution. Mix thoroughly. Aseptically distribute into sterile tubes. Use: For the cultivation and maintenance of Mycoplasma dispar, Mycoplasma flocculare, and Mycoplasma hyopneumoniae. Mycoplasma pneumoniae Isolation Medium Composition per 1200.0mL: Beef heart for infusion 50.0g Peptone 10.0g NaCl 5.0g Water 900.0mL Yeast extract solution 100.0mL α-Gamma horse serum, unheated 200.0mL pH 7.6–7.8 at 25°C Yeast Extract Solution: Composition per 10.0mL: Yeast, active, dry, Baker’s 250.0g Preparation of Yeast Extract Solution: Add yeast to 1.0L of dis- tilled/deionized water. Mix thoroughly. Gently heat and bring to boil- ing. Filter through Whatman #2 filter paper. Adjust the pH of the filtrate to 8.0 with NaOH. Distribute into tubes in 10.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C. Store at −20°C. Preparation of Medium: Add components, except yeast extract so- lution and α-gamma horse serum, 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 yeast extract solution and α-gamma horse serum. Mix thoroughly. Aseptically distribute into sterile tubes. Use: For the isolation and cultivation of Mycoplasma pneumoniae. Mycoplasmal Agar Composition per liter: Papaic digest of soy meal 20.0g Agarose 10.0g NaCl 5.0g Phenol Red (2% solution) 1.0mL pH 7.3 ± 0.2 at 25°C Preparation of Medium: Add components, except agarose, to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 7.3 with 1N NaOH. Add agarose. 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 and cultivation of human mycoplasmas and ure- aplasmas. Mycorrhiza Medium Composition per liter: Agar 15.0g Glucose 4.0g Ammonium tartrate 1.0g Malt extract 1.0g KH 2 PO 4 0.2g MgSO 4 ·7H 2 O 0.1g CaCl 2 ·2H 2 O 26.0mg NaCl 20.0mg Inositol 10.0mg ZnSO 4 ·7H 2 O 0.88mg MnSO 4 ·4H 2 O 0.81mg FeCl 3 ·6H 2 O 0.8mg Nicotinamide 100.0μg p-Aminobenzoic acid 100.0μg Pantothenic acid 100.0μg Pyridoxine 100.0μg Thiamine 100.0μg Biotin 25.0μg Riboflavin 25.0μg Preparation of Medium: Add components to distilled/deionized wa- ter and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling. Distribute into tubes or flasks. Autoclave for 10 min at 15 psi pressure– 121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Thelephora terrestris. Mycosel™ Agar (Cycloheximide Chloramphenicol Agar) Composition per liter: Agar 15.5g Papaic digest of soybean meal 10.0g Glucose 10.0g © 2010 by Taylor and Francis Group, LLC MYX Agar 1271 Cycloheximide 0.4g Chloramphenicol 0.05g pH 6.9 ± 0.2 at 25°C Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for- mation and inhalation. 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. Autoclave for 15 min at 14 psi pressure– 118°C. Avoid overheating. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the selective isolation of pathogenic fungi from specimens with other fungi and bacteria. MYCT Medium (DSMZ Medium 972) Composition per liter: KH 2 PO 4 13.6g Cyclomaltoheptaose (ß-cyclodextrin) 7.0g (NH 4 ) 2 SO 4 4.0g Yeast extract 3.0g Casein hydrolysate 3.0g Tween™ 80 1.0g MgCl 2 0.2g Sodium citrate 0.25g FeSO 4 ·7H 2 O 25.0mg MnSO 4 ·4H 2 O 25.0mg pH 6.5 ± 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 Lactobacillus sp. Mykorrhiza Agar Composition per liter: Agar 15.0g Malt extract 8.0g Glucose 7.0g Casein hydrolysate 1.0g Yeast extract 1.0g Asparagine 0.5g KH 2 PO 4 0.5g MgSO 4 ·7H 2 O 0.5g 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 Klebsiella pneumoniae. MYP Agar See: Mannitol Yolk Polymyxin Agar MYP Agar Base, HiVeg with Egg Yolk and Polymyxin B (Phenol Red Egg Yolk Polymyxin Agar Base, HiVeg) Composition per liter: Agar 15.0g D-Mannitol 10.0g Plant peptone 10.0g NaCl 10.0g Plant extract No. 1 1.0g Phenol Red 0.025g Egg yolk emulsion, 20% 10.0mL Polymyxin B solution 1.0mL pH 7.1 ± 0.2 at 25°C Source: This medium, without egg yolk and polymyxin B, is avail- able as a premixed powder from HiMedia. Egg Yolk Emulsion, 20%: Composition per 100.0mL: Chicken egg yolks 11 Whole chicken egg 1 NaCl (0.9% solution) 80.0mL Preparation of Egg Yolk Emulsion, 20%: 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. Measure 20.0mL of egg yolk emulsion and add to 80.0mL of 0.9% NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C. Polymyxin B Solution: Composition per 1.0mL: Polymyxin B 1.0mg Preparation of Polymyxin B Solution: Add polymyxin B to dis- tilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Fil- ter sterilize. Preparation of Medium: Add components—except egg yolk emul- sion, 20%, and polymyxin B solution—to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Gently heat and bring to boil- ing. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL of sterile egg yolk emulsion, 20%, and 1.0mL of sterile polymyxin B solution. Mix thoroughly. Pour into sterile Petri dishes. Mysorens Medium Composition per liter: Peptone 10.0g Meat extract 10.0g Yeast extract 5.0g NaCl 3.0g 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 Arthrobacter mysorens. MYX Agar (DSMZ Medium 729) Composition per liter: Na 2 -glutamate 5.0g Yeast extract 1.0g MgSO 4 ·7H 2 O 1.0g Glucose solution 10.0mL pH 7.2 ± 0.2 at 25°C Glucose Solution: Composition per10.0mL: Glucose 2.0g Preparation of Glucose Solution: Add glucose to distilled/deion- ized water and bring volume to 10.0mL. Mix thoroughly. Filter steril- ize. © 2010 by Taylor and Francis Group, LLC 1272 Myxobacteria Medium Preparation of Medium: Add components, except glucose solution, to distilled/deionized water and bring volume to 990.0mL. Mix thor- oughly. Adjust pH to 7.2. Aseptically add 10.0mL glucose solution. Mix thoroughly. Aseptically distribute into sterile tubes or bottles. Use: For the cultivation of Taxeobacter spp. Myxobacteria Medium Composition per liter: Agar 15.0g Skim milk powder 5.0g Yeast extract 0.5g Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Do not adjust pH. Gently heat and bring to boiling. Distribute into tubes or flasks. Auto- clave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Archangium primige- nium, Chondrococcus macrosporus, and Myxococcus coralloides. Myxococcus flavescens Medium Composition per liter: Agar 15.0g Soluble starch 5.0g Casitone 2.5g Galactose 1.0g Raffinose 1.0g Sucrose 1.0g Yeast extract 1.0g MgSO 4 ·7H 2 O 0.5g K 2 HPO 4 0.25g pH 6.0 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.0. 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 Myxococcus flavescens. Myxococcus Medium Composition per liter: Agar 12.0g Pancreatic digest of casein 1.0g Meat extract 1.0g Glucose solution 50.0mL pH 7.2 ± 0.2 at 25°C Glucose Solution: Composition per 50.0mL: Glucose 1.0g Preparation of Glucose Solution: Add glucose to distilled/deion- ized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Preparation of Medium: Add components, except glucose solu- tion, to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Adjust pH to 7.2. 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. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes or bottles. Allow tubes or bottles to cool in a slanted position. Use: For the cultivation of Myxococcus species. Myxococcus xanthus Medium Composition per liter: Agar 20.0g Pancratic digest of casein 10.0g MgSO 4 ·7H 2 O 0.5g K 2 HPO 4 0.148g KH 2 PO 4 0.017g pH 7.6 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Myxococcus xanthus. N plus C Medium Composition per liter: Pancreatic digest of casein 10.0g Glucose 10.0g Citric acid·H 2 O 4.04g KH 2 PO 4 2.0g Yeast extract 1.5g CaCl 2 ·2H 2 O 0.6g MgSO 4 ·7H 2 O 0.6g FeCl 2 ·4H 2 O 0.06g ZnSO 4 ·7H 2 O 0.034g Hemin solution 1.0mL pH 4.6 ± 0.2 at 25°C Hemin Solution: Composition per 100.0mL: NaOH 1.0g Hemin 250.0mg Preparation of Hemin Solution: Add components to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Auto- clave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except hemin solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 4.6. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Prior to inoculation, add 0.1mL of hemin solution per 100.0mL of medium. Use: For the cultivation and maintenance of Physarum polycephalum. N DeVogel Medium (Vogel N Medium) Composition per liter: Sucrose 15.0g KH 2 PO 4 5.0g Trisodium citrate·2H 2 O 3.0g NH 4 NO 3 2.0g MgSO 4 ·7H 2 O 0.2g CaCl 2 ·H 2 O solution 20.0mL Biotin solution 5.0mL Trace elements solution 5.0mL CaCl 2 ·H 2 O Solution: Composition per 20.0mL: CaCl 2 ·H 2 O 0.1g © 2010 by Taylor and Francis Group, LLC NAM Medium 1273 Preparation of CaCl 2 ·H 2 O Solution: Add CaCl 2 ·H 2 O to dis- tilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Biotin Solution: Composition per 100.0mL: Biotin 5.0mg Preparation of Biotin Solution: Add biotin to 50% ethanol and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Store at 5°C. Trace Elements Solution: Composition per 100.0mL: Citric acid·H 2 O 5.0g ZnSO 4 ·7H 2 O 5.0g Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 1.0g CuSO 4 ·5H 2 O 0.25g H 3 BO 3 , anhydrous 0.05g MnSO 4 ·H 2 O 0.05g Na 2 MoO 4 ·2H 2 O 0.05g Preparation of Trace Elements Solutions: Add components successively to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly after addition of each component. Filter sterilize. Add 2–3.0mL of chloroform as a preservative. Store at 25°C. Preparation of Medium: Add components, except biotin solution and trace elements solution, to distilled/deionized water and bring vol- ume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pres- sure–121°C. Aseptically add 5.0mL of sterile biotin solution and 5.0mL of sterile trace elements solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Neurospora crassa. NAG Medium (DSMZ Medium 366) Composition per liter: Agar 15.0g Glucose 10.0g Peptone 5.0g Meat extract 3.0g 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. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Do not overheat. Pour into sterile Petri dishes or leave in tubes. Use: For the isolation and cultivation of Xanthomonas fragariae. Nakayama Glucose Agar Composition per 1001.0mL: Yeast extract 15.0g Agar 10.0g Glucose 10.0g Peptone 10.0g Solution A 10.0mL Solution B 10.0mL Solution C 1.0mL pH 6.8 ± 0.2 at 25°C Solution A: Composition per 100.0mL: K 2 HPO 4 0.5g KH 2 PO 4 0.5g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution B: Composition per 100.0mL: MgSO 4 ·7H 2 O 3.0g MnSO 4 ·5H 2 O 0.1g NaCl 0.1g CuSO 4 ·5H 2 O 0.01g Preparation of Solution B: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Solution C: Composition per 100.0mL: Trisodium citrate 2.0g FeSO 4 ·7H 2 O 0.1g Preparation of Solution C: Add components to distilled/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 solution A, so- lution B, and solution C, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Asepti- cally add 10.0mL of sterile solution A, 10.0mL of sterile solution B, and 1.0mL of sterile solution C. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Bacillus laevolacticus. NAM Medium Composition per liter: Pancreatic digest of casein 15.0g Agar 15.0g Papaic digest of soybean meal 5.0g NaCl 5.0g Sheep blood, defibrinated 50.0mL Hemin solution 10.0mL N-Acetyl muramic acid (NAM) solution 1.0mL pH 7.3 ± 0.2 at 25°C Hemin Solution: Composition per 100.0mL: Hemin .0.050g NaOH (1N solution) 1.0mL Preparation of Hemin Solution: Add hemin to NaOH solution. Mix thoroughly. Adjust volume to 100.0mL with distilled/deionized wa- ter. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. N-Acetyl Muramic Acid (NAM) Solution: Composition per 10.0mL: N-Acetyl muramic acid 100.0mg Preparation of N-Acetyl Muramic Acid (NAM) Solution: Add N-acetyl muramic acid to distilled/deionized water and bring vol- ume to 10.0mL. Filter sterilize. Preparation of Medium: Add components, except sheep blood, hemin solution, and N-acetyl muramic acid (NAM) solution, to dis- tilled/deionized water and bring volume to 49.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Aseptically add 50.0mL of sterile sheep blood, 10.0mL of sterile hemin solution, and 1.0mL of sterile N-acetyl muramic acid (NAM) solution. © 2010 by Taylor and Francis Group, LLC 1274 NANAT Agar Mix thoroughly. Aseptically and anaerobically distribute into sterile tubes under a gas phase of 80% N 2 + 10% CO 2 + 10% H 2 . Use: For the cultivation of Bacteroides forsythus. NAMn See: Nutrient Agar with Manganese NANAT Agar (Nalidixic Acid Novobiocin Actidione Tellurite Agar) Composition per liter: Pancreatic digest of casein 17.0g Agar 15.0g NaCl 5.0g Tween™ 80 5.0g Papaic digest of soybean meal 3.0g K 2 HPO 4 2.5g Glucose 2.5g Yeast extract 1.0g Tellurite solution 10.0mL Antibiotic solution 10.0mL pH 7.2 ± 0.2 at 25°C Tellurite Solution: Composition per 100.0mL: K 2 TeO 3 0.05g Preparation of Tellurite Solution: Add K 2 TeO 3 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Antibiotic Solution: Composition per 10.0mL: Actidione (cycloheximide) 0.04g Polymyxin B (optional) 0.03g Novobiocin 0.025g Nalidixic acid 0.02g Preparation of Antibiotic Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Caution: Potassium tellurite is toxic. Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for- mation and inhalation. Preparation of Medium: Add components, except tellurite solu- tion and antibiotic solution, to distilled/deionized water and bring vol- ume to 980.0mL. 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 tellurite solution and antibiotic solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation and cultivation of Rhodococcus (Corynebacte- rium) equi from animal feces, especially from horses and swine. The addition of polymyxin B inhibits the growth of Pseudomonas aerugi- nosa which may interfere with the isolation of Rhodococcus equi. Nannocystis Agar Composition per liter: Agar 15.0g CaCl 2 ·2H 2 O 1.0g pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes. After the agar has solidified, overlay the surface with 0.5mL of a suspension of dead (autoclaved) Escherichia coli cells. Use: For the cultivation and maintenance of Nannocystis species. Naphthalene Medium Composition per liter: NH 4 NO 3 2.5g Na 2 HPO 4 ·2H 2 O 1.0g Naphthalene 0.64g MgSO 4 ·7H 2 O 0.5g Fe(SO 4 ) 3 ·5H 2 O 0.01g Co(NO 3 ) 2 ·6H 2 O 5.0mg CaCl 2 ·2H 2 O 1.0mg KH 2 PO 4 0.5mg MnSO 4 ·2H 2 O 0.1mg (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.1mg 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 Pseudomonas alcaligenes. Naphthalene Mineral Salts Medium See: Medium for Hydrocarbon-Degrading Bacteria Naphthalene Sulfonic Acid Medium Composition per 1004.0mL: Na 2 HPO 4 ·2H 2 O 3.5g KH 2 PO 4 1.0g NH 4 Cl 0.31g MgCl 2 ·6H 2 O 0.1g Ca(NO 3 ) 2 ·4H 2 O 0.05g Solution A 100.0mL Solution B 3.0mL Trace elements solution SL-4 1.0mL pH 7.0 ± 0.2 at 25°C Solution A: Composition per liter: Glucose 3.0g Glycerol 3.0g Sodium succinate 3.0g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Solution B: Composition per liter: Naphthalene sulfonic acid 2.3g Preparation of Solution B: Add naphthalene sulfonic acid to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. 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 Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. © 2010 by Taylor and Francis Group, LLC . Hydrolysate of Lactalbumin: Composition per 100.0mL: Enzymatic hydrolysate of lactalbumin 5.0g Preparation of Enzymatic Hydrolysate of Lactalbumin: Add enzymatic hydrolysate of lactalbumin to 100.0mL of. hydrolysate of lactalbumin 5.0g Phosphate buffered saline, 1X, pH7.0 100.0mL Preparation of Enzymatic Hydrolysate of Lactalbumin: Add enzymatic hydrolysate of lactalbumin to 100.0mL of phosphate. Prepare 42.5mL of double-strength broth and 42.5mL of distilled water containing 0.7g of purified agar. Ster- ilize the solutions separately and combine aseptically at 56°C with 15.0mL of sterile