Burk’s Medium 285 Burke’s Modified Nitrogen-Free Medium with Benzoate Composition per liter: Sodium benzoate 0.72g MgSO 4 ·7H 2 O 0.2g Na 2 HPO 4 0.189g NaHCO 3 0.05g CaSO 4 ·2H 2 O 0.02g KH 2 PO 4 0.011g SrCl 2 ·6H 2 O 0.01g NaCl 0.01g Adenine 0.01g FeSO 4· 7H 2 O 6.0mg Na 2 MoO 3 0.5mg pH 7.8 ± 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 Pseudomonas species and other microor- ganisms which can utilize benzoate as sole carbon source. Burkholderia cepacia Agar Composition per liter: Agar 12.0g Sodium pyruvate 7.0g Peptone 5.0g KH 2 PO 4 4.4g Yeast extract 4.0g Bile salts 1.5g Na 2 HPO 4 1.4g (NH 4 ) 2 SO 4 1.0g MgSO 4 0.2 Phenol Red 0.02g Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O 0.01g Crystal Violet 0.001g Selective supplement solution 10.0mL pH 6.2 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Oxoid Unipath. Selective Supplement Solution: Composition per 10.0mL: Polymyxin B 150,000IU Ticarcillin 100.0mg Gentamicin 5.0mg Preparation of Selective Supplement Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. 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 while stirring and bring to boil- ing. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptially add 10.0mL selective supplement solution. Mix thoroughly. Pour into sterile Petri dishes. Use: For the selective isolation of Burkholderia cepacia from the respiratory secretions of patients with cystic fibrosis and for routine testing of non-sterile inorganic salt solutions containing preservative. Slow growing B. cepacia can be missed on conventional media such as blood or MacConkey agar due to overgrowth caused by other faster growing organisms found in the respiratory tract of CF patients such as mucoid Klebsiella species, Pseudomonas aeruginosa, and Staphylo- coccus species. This may lead to the infection being missed or wrongly diagnosed. Burkholderia pseudomallei Selective Agar (BPSA) Composition per liter: Agar 15.0g Pancreatic Digest of Casein 5.0g Maltose 4.0g Yeast Extract 2.5g Glucose 1.0g Neutral Red 0.1g Gentamicin solution 10.0mL Glycerol 10.0mL Nile Blue solution 1.0mL pH 7.0 ± 0.2 at 25°C Gentamicin Solution: Composition per 10.0mL: Gentamicin 20.0mg Preparation of Gentamicin Solution: Add gentamicin to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Nile Blue Solution: Composition per 10.0mL: Nile Blue 0.2g Preparation of Nile Blue Solution: Add Nile blue to 10.0mL of a 1% solution of dimethyl sulfoxide. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except gentamcin solu- tion, Nile Blue solution, and glycerol, to distilled/deionized water and bring volume to 979.0mL. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°C. Aseptially add 10.0mL sterile gentamicin solution, 1.0mL sterile Nile blue solution, and 10.0mL filter-sterilized glycerol. Mix thoroughly for 5 min on a heated magnetic stirrer at 40°C. Pour into sterile Petri dishes. Use: For the cultivation of Burkholderia pseudomallei from clinical specimens collected from non-sterile sites with improved recovery of the more easily inhibited strains of B. pseudomallei. Burk’s Medium Composition per liter: Sucrose 20.0g MgSO 4 ·7H 2 O 0.2g K 2 HPO 4 0.8g KH 2 PO 4 0.25g CaSO 4 0.13g FeCl 3 1.45mg Na 2 MoO 3 0.253mg Source: This medium is available from HiMedia. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes with inverted Durham tubes. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For thecultivation of nitrogen fixing bacteria, such as Azotrobacter spp., from soil. © 2010 by Taylor and Francis Group, LLC 286 Bushnell-Haas Agar Bushnell-Haas Agar Composition per liter: Agar 15.0g KH 2 PO 4 1.0g K 2 HPO 4 1.0g NH 4 NO 3 1.0g MgSO 4 ·7H 2 O 0.2g FeCl 3 0.05g CaCl 2 ·2H 2 O 0.02g 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. Pour into sterile Petri dishes or leave in tubes. For use in cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0% hy- drocarbon on agar surface or aseptically add sterile hydrocarbon to cooled agar prior to pouring plates. Use: For examining fuels for microbial contamination and for study- ing hydrocarbon utilization by microorganisms. Also for the cultiva- tion of Nocardia species. Bushnell-Haas Broth Composition per liter: KH 2 PO 4 1.0g K 2 HPO 4 1.0g NH 4 NO 3 1.0g MgSO 4 ·7H 2 O 0.2g FeCl 3 0.05g CaCl 2 ·2H 2 O 0.02g pH 7.0 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. For use in cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0% hydrocar- bon on broth surface or add directly to broth. Use: For examining fuels for microbial contamination and for studying the hydrocarbon utilization by microorganisms. Also for the cultivation of Nocardia species. Bushnell-Haas Medium Composition per liter: KH 2 PO 4 1.0g K 2 HPO 4 1.0g NH 4 NO 3 1.0g Cholesterol 0.3g MgSO 4 ·7H 2 O 0.2g FeCl 3 0.05g CaCl 2 ·2H 2 O 0.02g pH 7.0 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. For use in cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0% hydrocar- bon on broth surface or add directly to broth. Use: For the cultivation of Nocardia species. Butanediol Medium Composition per liter: NaH 2 PO 4 ·H 2 O 2.1g 1,4-Butanediol 1.0g NaCl 1.0g NH 4 Cl 1.0g CaCl 2 ·2H 2 O 0.5g MgSO 4 ·7H 2 O 0.5g K 2 HPO 4 0.3g Yeast extract 0.2g Modified Wolfe’s mineral solution 10.0mL pH 7.0 ± 0.2 at 25°C Modified Wolfe’s Mineral Solution: Composition per liter: MgSO 4 ·7H 2 O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO 4 ·H 2 O 0.5g CaCl 2 0.1g CoCl 2 ·6H 2 O 0.1g FeSO 4 ·7H 2 O 0.1g ZnSO 4 ·7H 2 O 0.1g AlK(SO 4 ) 2 ·12H 2 O 0.01g CuSO 4 ·5H 2 O 0.01g H 3 BO 3 0.01g Na 2 MoO 4 ·2H 2 O 0.01g Na 2 SeO 3 0.01g NaWO 4 ·2H 2 O 0.01g NiC1 2 ·6H 2 O 0.01g Preparation of Modified Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH to 6.5 with KOH. Add remaining components one at a time. Add dis- tilled/deionized water to 1.0L. Adjust pH to 6.8. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Pseudomonas putida. Butyrivibrio Species Medium Composition per 1001.0mL: Na 2 CO 3 4.0g Pancreatic digest of casein 2.0g Yeast extract 2.0g K 2 HPO 4 .0.3g Hemin 1.0mg Resazurin 1.0mg Rumen fluid, clarified 150.0mL Minerals solution 75.0mL Carbohydrate solution 20.0mL L-Cysteine·HCl·H 2 O solution 10.0mL Na 2 S·9H 2 O solution 10.0mL Volatile fatty acid mixture 3.1mL pH 6.7 ± 0.2 at 25°C Minerals Solution: Composition per liter: NaCl 12.0g KH 2 PO 4 6.0g (NH 4 ) 2 SO 4 6.0g © 2010 by Taylor and Francis Group, LLC BYE Agar 287 MgSO 4 ·7H 2 O 2.5g CaCl 2 ·2H 2 O 1.6g Preparation of Minerals Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. L-Cysteine·HCl·H 2 O Solution: Composition per 10.0mL: L-Cysteine·HCl·H 2 O 0.25g Preparation of L-Cysteine·HCl·H 2 O Solution: Add L-cys- teine·HCl·H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.25g 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. Mix thoroughly. Sparge with 100% CO 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Carbohydrate Solution: Composition per 20.0mL: Glucose 1.0g Cellobiose 1.0g Glycerol 1.0g Maltose 1.0g Starch, soluble 1.0g Preparation of Carbohydrate Solution: Add components to dis- tilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Sparge under 100% CO 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Volatile Fatty Acid Mixture: Composition per 7.75mL: Acetic acid 4.25mL Propionic acid 1.50mL Butyric acid 1.0mL DL-2-Methyl butyric acid 0.25mL iso-Butyric acid 0.25mL iso-Valeric acid 0.25mL n-Valeric acid 0.25mL Preparation of Volatile Fatty Acid Mixture: Combine compo- nents. Mix thoroughly. Preparation of Medium: Prepare and dispense medium under 100% CO 2 . Add components, except carbohydrate solution, Na 2 CO 3 , L-cysteine·HCl·H 2 O solution, and Na 2 S·9H 2 O solution, to distilled/de- ionized water and bring volume to 960.0mL Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 5 min. Cool to room temperature while sparging with 100% CO 2 . Add Na 2 CO 3 . Continue sparging with 100% CO 2 until pH reaches 6.8. Distribute into rubber- stoppered tubes under 100% CO 2 . Autoclave for 15 min at 15 psi pres- sure–121°C. Aseptically and anaerobically add 20.0mL of sterile car- bohydrate solution, 10.0mL of sterile L-cysteine·HCl·H 2 O solution, and 10.0mL of sterile Na 2 S·9H 2 O solution or, using a syringe, inject the appropriate amount of sterile carbohydrate solution, sterile Na 2 S·9H 2 O solution, and sterile L-cysteine·HCl·H 2 O solution into in- dividual tubes containing medium. Use: For the cultivation of Butyrivibrio species. Butzler Medium See: Campylobacter Selective Medium, Butzler’s Butzler’s Campylobacter Medium See: Campylobacter Selective Medium, Butzler’s BY Agar Medium (ATCC Medium 2038) Composition per liter: Agar 15.0g Yeast extract 5.0g Pancreatic digest of casein 5.0g Beef extract 5.0g NaCl 2.5g K 2 HPO 4 0.1g MgSO 4 ·7H 2 O 0.05g 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. 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 Paracoccus thiocyanatus. BY+ Medium Composition per liter: Glucose 5.0g Peptone 1.0g Yeast extract 1.0g Seawater 1.0L Preparation of Medium: Combine components. Mix thoroughly. Gently heat and bring to boiling. Distribute into flasks or tubes. Auto- clave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Arenariomyces triseptatus, Haliphthoros milfordensis, Haliphthoros philippinensis, Halosphaeria salina, Japonochytrium species, Lignincola laevis, Lindra thalassiae, Schizochytrium aggregatum, Thraustochytrium species, and Torpe- dospora radiata. BYE Agar Composition per liter: Pancreatic digest of casein 16.0g Agar 13.5g Brain heart, solids from infusion 8.0g Peptic digest of animal tissue 5.0g NaCl 5.0g Glucose 2.0g Na 2 HPO 4 2.5g Yeast extract 2.0g Blood, human or animal, sterile 150.0mL pH 7.8–8.0 ± 0.2 at 25°C Preparation of Medium: Add components, except blood, to dis- tilled/deionized water and bring volume to 850.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 150.0mL of sterile blood. Outdated, citrated, or hepa- rinized blood (blood from a blood bank is acceptable). Pour into sterile Petri dishes. © 2010 by Taylor and Francis Group, LLC 288 BYE HiVeg Agar with Blood Use: For the isolation and cultivation of Mycoplasma species and L- forms of bacteria. For the detection of Mycoplasma species in tissue cul- ture and cell lines. BYE HiVeg Agar with Blood Composition per liter: Agar 13.0g Plant infusion 10.0g Plant peptone No. 3 10.0g Plant special infusion 7.5g NaCl 5.0g Na 2 HPO 4 2.5g Glucose 2.0g Yeast extract 2.0g Horse or human blood, sterile 150.0mL pH 7.9 ± 0.2 at 25°C Source: This medium, without blood, is available as a premixed pow- der from HiMedia. Preparation of Medium: Add components, except blood, to dis- tilled/deionized water and bring volume to 850.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 150.0mL of sterile blood. Outdated, citrated, or hepa- rinized blood (blood from a blood bank is acceptable). Pour into sterile Petri dishes. Use: For the isolation and cultivation of Mycoplasma species and L- forms of bacteria. For the detection of Mycoplasma species in tissue cul- ture and cell lines. BYE HiVeg Broth with Blood Composition per liter: Plant infusion 10.0g Plant peptone No. 3 10.0g Plant special infusion 7.5g NaCl 5.0g Na 2 HPO 4 2.5g Glucose 2.0g Yeast extract 2.0g Horse or human blood, sterile 150.0mL pH 7.9 ± 0.2 at 25°C Source: This medium, without blood, is available as a premixed pow- der from HiMedia. Preparation of Medium: Add components, except blood, to dis- tilled/deionized water and bring volume to 850.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 150.0mL of sterile blood. Outdated, citrated, or hepa- rinized blood (blood from a blood bank is acceptable). Use: For the cultivation of Mycoplasma species and L-forms of bacteria. BYEB (Buffered Yeast Extract Broth) Composition per liter: ACES buffer (2-[(2-amino-2-oxoethyl)- amino]-ethane sulfonic acid) 10.0g Yeast extract 10.0g α-Ketoglutarate 1.0g L-Cysteine·HCl·H 2 O 0.4g Fe 4 (P 2 O 7 ) 3 ·9H 2 O 0.25g pH 6.9 ± 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 6.9. Fil- ter sterilize. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Legionella pneumophila. C/10 Agar Composition per liter: Agar 15.0g Pancreatic digest of casein 3.0g CaCl 2 ·2H 2 O 1.36g 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 t o 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 Cytophaga flevensis, Flexibacter filiformis, Myxococcus C/10 Medium Reichenbach Composition per liter: Agar 15.0g Pancreatic digest of casein 3.0g CaCl 2 1.0g pH 7.2 ± 0.2 at 25°C Preparation of Medium: Add components, except agar, to distilled/ deionized water and bring volume to 1.0L. Adjust pH to 7.2. Add agar. Mix thoroughly. Gently heat 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 Flexibacter filiformis. fulvus, and Myxococcus xanthus. C 3G Spiroplasma Medium Composition per liter: Sucrose 100.0g Phenol Red 10.0mg PPLO broth without Crystal Violet 500.0mL Horse serum 150.0mL Fresh yeast extract solution 50.0mL CMRL-1066 medium 5.0mL pH 7.5 ± 0.2 at 25°C Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems. PPLO Broth without Crystal Violet: Composition per 500.0mL: Beef heart, infusion from 11.52g Peptone 2.32g NaCl 1.15g Preparation of PPLO Broth without Crystal Violet: Add components to distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 25.0g © 2010 by Taylor and Francis Group, LLC C 3N Spiroplasma Medium 289 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. Filter sterilize. CMRL-1066 Medium: Composition per liter: NaCl 6.8g NaHCO 3 2.2g D-Glucose 1.0g KCl 0.4g L-Cysteine·HCl·H 2 O 0.26g CaCl 2 , anhydrous 0.2g MgSO 4 ·7H 2 O 0.2g NaH 2 PO 4 ·H 2 O 0.14g L-Glutamine 0.1g Sodium acetate·3H 2 O 0.083g L-Glutamic acid 0.075g L-Arginine·HCl 0.07g L-Lysine·HCl 0.07g L-Leucine 0.06g Glycine 0.05g Ascorbic acid 0.05g L-Proline 0.04g L-Tyrosine 0.04g L-Aspartic acid 0.03g L-Threonine 0.03g L-Alanine 0.025g L-Phenylalanine 0.025g L-Serine 0.025g L-Valine 0.025g L-Cystine 0.02g L-Histidine·HCl·H 2 O 0.02g L-Isoleucine 0.02g Phenol Red 0.02g L-Methionine 0.015g Deoxyadenosine 0.01g Deoxycytidine 0.01g Deoxyguanosine 0.01g Glutathione, reduced 0.01g Thymidine 0.01g Hydroxy- L-proline 0.01g L-Tryptophan 0.01g Nicotinamide adenine dinucleotide 7.0mg Tween™ 80 5.0mg Sodium glucoronate·H 2 O 4.2mg Coenzyme A 2.5mg Cocarboxylase 1.0mg Flavin adenine dinucleotide 1.0mg Nicotinamide adenine dinucleotide phosphate 1.0mg Uridine triphosphate 1.0mg Choline chloride 0.5mg Cholesterol 0.2mg 5-Methyldeoxycytidine 0.1mg Inositol 0.05mg p-Aminobenzoic acid 0.05mg Niacin 0.025mg Niacinamide 0.025mg Pyridoxine 0.025mg Pyridoxal·HCl 0.025mg Biotin 0.01mg D-Calcium pantothenate 0.01mg Folic acid 0.01mg Riboflavin 0.01mg Thiamine·HCl 0.01mg Source: CMRL-1066 medium is available as a premixed powder from BD Diagnostics. Preparation of CMRL-1066 Medium: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 7.2. Filter sterilize. Preparation of Medium: Add components—except horse serum, fresh yeast extract, and CMRL medium—to distilled/deionized water and bring volume to 795.0mL. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 150.0mL of sterile horse se- rum, 50.0mL of sterile fresh yeast extract solution, and 5.0mL of sterile CMRL medium. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Spiroplasma species. C 3N Spiroplasma Medium Composition per 100.0mL: Sucrose 12.0g Phenol Red 10.0mg PPLO broth without Crystal Violet 50.0mL Horse serum 20.0mL Fresh yeast extract solution 5.0mL CMRL-1066 medium 0.5mL pH 7.5 ± 0.2 at 25°C PPLO Broth without Crystal Violet: Composition per 500.0mL: Beef heart, infusion from 11.52g Peptone 2.32g NaCl 1.15g 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 500.0mL. 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. Filter sterilize. CMRL-1066 Medium: Composition per liter: NaCl 6.8g NaHCO 3 2.2g D-Glucose 1.0g KCl 0.4g L-Cysteine·HCl·H 2 O 0.26g CaCl 2 , anhydrous 0.2g MgSO 4 ·7H 2 O 0.2g NaH 2 PO 4 ·H 2 O 0.14g L-Glutamine 0.1g Sodium acetate·3H 2 O 0.083g L-Glutamic acid 0.075g L-Arginine·HCl 0.07g L-Lysine·HCl 0.07g © 2010 by Taylor and Francis Group, LLC 290 CAE Agar Base with Triphenyltetrazolium Chloride L-Leucine 0.06g Glycine 0.05g Ascorbic acid 0.05g L-Proline 0.04g L-Tyrosine 0.04g L-Aspartic acid 0.03g L-Threonine 0.03g L-Alanine 0.025g L-Phenylalanine 0.025g L-Serine 0.025g L-Valine 0.025g L-Cystine 0.02g L-Histidine·HCl·H 2 O 0.02g L-Isoleucine 0.02g Phenol Red 0.02g L-Methionine 0.015g Deoxyadenosine 0.01g Deoxycytidine 0.01g Deoxyguanosine 0.01g Glutathione, reduced 0.01g Thymidine 0.01g Hydroxy- L-proline 0.01g L-Tryptophan 0.01g Nicotinamide adenine dinucleotide 7.0mg Tween™ 80 5.0mg Sodium glucoronate·H 2 O 4.2mg Coenzyme A 2.5mg Cocarboxylase 1.0mg Flavin adenine dinucleotide 1.0mg Nicotinamide adenine dinucleotide phosphate 1.0mg Uridine triphosphate 1.0mg Choline chloride 0.5mg Cholesterol 0.2mg 5-Methyldeoxycytidine 0.1mg Inositol 0.05mg p-Aminobenzoic acid 0.05mg Niacin 0.025mg Niacinamide 0.025mg Pyridoxine 0.025mg Pyridoxal·HCl 0.025mg Biotin 0.01mg D-Calcium pantothenate 0.01mg Folic acid 0.01mg Riboflavin 0.01mg Thiamine·HCl 0.01mg Source: CMRL-1066 medium is available as a premixed powder from BD Diagnostics. Preparation of CMRL-1066 Medium: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad- just pH to 7.2. Filter sterilize. Preparation of Medium: Add components—except horse serum, fresh yeast extract, and CMRL medium—to distilled/deionized water and bring volume to 75.0mL. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 20.0mL of sterile horse se- rum, 5.0mL of sterile yeast extract, and 0.5mL of sterile CMRL medi- um. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Spiroplasma kunkelii. CA See: Carrot Decoction Agar CA YE Broth See: Casamino Acids Yeast Extract Salts Broth, Gorbach Cadmium Fluoride Acriflavin Tellurite Medium See: CFAT Medium CAE Agar Base with Triphenyltetrazolium Chloride (Citrate Azide Enterococcus HiVeg Agar Base) Composition per liter: Agar 15.0g Casein enzymatic hydrolysate 15.0g Sodium citrate 15.0g KH 2 PO 4 5.0g Yeast extract 5.0g Na 2 CO 3 2.0g Polysorbate 80 1.0g NaN 3 0.4g 2,3,5-Triphenyltetrazolium chloride solution 10.0mL pH 7.0 ± 0.2 at 25°C Source: This medium, without triphenyltetrazolium chloride solution, is available as a premixed powder from HiMedia. Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted. 2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride 0.1g Preparation of 2,3,5-Triphenyltetrazolium Chloride Solu- tion: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except 2,3,5-triphe- nyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 10 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation, cultivation, and enumeration of entercocci in water, sewage, and feces by the membrane filter method. For the direct plating of specimens for the detection and enumeration of fecal strep- tococci. CAE HiVeg Agar Base with Triphenyltetrazolium Chloride (Citrate Azide Enterococcus HiVeg Agar Base) Composition per liter: Agar 15.0g Plant hydrolysate 15.0g Sodium citrate 15.0g KH 2 PO 4 5.0g Yeast extract 5.0g Na 2 CO 3 2.0g Polysorbate 80 1.0g NaN 3 0.4g 2,3,5-Triphenyltetrazolium chloride solution 10.0mL pH 7.0 ± 0.2 at 25°C Source: This medium, without triphenyltetrazolium chloride solution, is available as a premixed powder from HiMedia. © 2010 by Taylor and Francis Group, LLC CAL Broth 291 Caution: Sodium azide is toxic. Azides also react with metals and disposal must be highly diluted. 2,3,5-Triphenyltetrazolium Chloride Solution: Composition per 10.0mL: 2,3,5-Triphenyltetrazolium chloride 0.1g Preparation of 2,3,5-Triphenyltetrazolium Chloride Solu- tion: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except 2,3,5-triphe- nyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 10 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add 10.0mL 2,3,5-triphenyltetrazolium chloride solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the isolation, cultivation, and enumeration of entercocci in water, sewage, and feces by the membrane filter method. For the direct plating of specimens for the detection and enumeration of fecal strep- tococci. Caffeic Acid Ferric Citrate Test Medium (CAFC Test Medium) (Caffeic Acid Agar) Composition per liter: Agar 20.0g (NH 4 ) 2 SO 4 5.0g Glucose 5.0g Yeast extract 2.0g K 2 HPO 4 0.8g MgSO 4 ·3H 2 O 0.7g Caffeic acid· 1/2H 2 O 0.18g Chloramphenicol 0.05g Ferric citrate solution 4.0mL pH 6.5 ± 0.2 at 25°C Ferric Citrate Solution: Composition per 20.0mL: Ferric citrate 100.0mg Preparation of Ferric Citrate Solution: Add ferric citrate to 20.0mL of distilled/deionized water. Mix thoroughly. Preparation of Medium: Add components, except chlorampheni- col, to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Heat to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add 0.05g of chloramphenicol. Mix thoroughly. Pour into sterile Petri dishes. Use: For the isolation and presumptive identification of Cryptococcus neoformans. Cryptococcus neoformans appears as dark brown colo- nies. All other Cryptococcus species appear as light brown or nonpig- mented colonies. Caffeine Medium Composition per liter: Agar 15.0g Solution A 400.0mL Solution B 400.0mL Solution C 200.0mL pH 5.0 ± 0.2 at 25°C Solution A: Composition per 400.0mL: Na 2 HPO 4 7.8g KH 2 PO 4 3.0g Caffeine 1.0g NaCl 0.58g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Adjust pH to 5.0. Solution B: Composition per 400.0mL: MgSO 4 ·7H 2 O 0.12g CaCl 2 ·2H 2 O 11.0mg Preparation of Solution B: Add components to distilled/deionized water and bring volume to 400.0mL. Mix thoroughly. Solution C: Composition per 200.0mL: FeCl 3 16.0mg Preparation of Solution C: Add FeCl 3 to distilled/deionized water and bring volume to 200.0mL. Mix thoroughly. Preparation of Medium: To 400.0mL of solution A, add 400.0mL of solution B and 200.0mL of solution C. Adjust pH to 5.0. Add agar. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Pseudomonas species. CAGV Medium See: Casamino Acid Glucose Medium CAL Agar (Cellobiose Arginine Lysine Agar) (Yersinia Isolation Agar) Composition per liter: Agar 20.0g L-Arginine·HCl 6.5g L-Lysine·HCl 6.5g NaCl 5.0g Cellobiose 3.5g Yeast extract 3.0g Sodium deoxycholate 1.5g Neutral Red 0.03g pH 7.3 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Heat to boiling. Do not autoclave. Pour into sterile Petri dishes. Use: For the isolation and characterization of Yersinia enterocolitica from fecal specimens and enumeration of Yersinia enterocolitica from water and other liquid specimens. CAL Broth (Cellobiose Arginine Lysine Broth) Composition per liter: L-Arginine·HCl 6.5g L-Lysine·HCl 6.5g NaCl 5.0g Cellobiose 3.5g Yeast extract 3.0g © 2010 by Taylor and Francis Group, LLC 292 CAL HiVeg Agar Sodium deoxycholate 1.5g Neutral Red 0.03g pH 7.3 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Heat to boiling. Do not autoclave. Distribute into sterile tubes in 6.0–8.0mL volumes. Use: For the isolation and characterization of Yersinia enterocolitica from fecal specimens and enumeration of Yersinia enterocolitica from water and other liquid specimens. CAL HiVeg Agar (Cellobiose Arginine Lysine HiVeg Agar) Composition per liter: Agar 20.0g L-Arginine 6.5g L-Lysine hydrochloride 6.5g NaCl 5.0g Cellobiose 3.5g Yeast extract 3.0g Synthetic detergent No. III 1.5g Neutral Red 0.03g pH 7.1 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Hi- Media. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Heat to boiling. Do not autoclave. Pour into sterile Petri dishes. Use: For the isolation and characterization of Yersinia enterocolitica from fecal specimens and enumeration of Y. enterocolitica from water. CAL HiVeg Broth (Cellobiose Arginine Lysine HiVeg Broth) Composition per liter: L-Arginine 6.5g L-Lysine hydrochloride 6.5g NaCl 5.0g Cellobiose 3.5g Yeast extract 3.0g Synthetic detergent No. III 1.5g Neutral Red 0.03g pH 7.1 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Hi- Media. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Heat to boiling. Do not autoclave. Distribute into sterile tubes. Use: For the isolation and characterization of Yersinia enterocolitica from fecal specimens and enumeration of Yersinia enterocolitica from water and other liquid specimens. Calcium Caseinate Agar Composition per liter: Agar 13.0g Peptic digest of animal tissue 4.0g Calcium caseinate 3.5g Meat extract 2.0g Casein enzymic hydrolysate 2.0g CaCl 2 ·2H 2 O 0.2g Tri-potassium citrate 0.35g Na 2 HPO 4 0.105g KH 2 PO 4 0.035g NaCl 5.0g pH 7.0 ± 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. Distribute into tubes or flasks. Gently heat and bring to boiling. Boil for 10 min. Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly while pouring into Petri dishes. Use: For the detection and enumeration of proteolytic microorganisms in foodstuffs and other materials. Calcium Caseinate Agar with Skim Milk Composition per liter: Agar 13.0g Skim Milk 10.0g Peptic digest of animal tissue 4.0g Calcium caseinate 3.5g Meat extract 2.0g Casein enzymic hydrolysate 2.0g CaCl 2 ·2H 2 O 0.2g Tri-potassium citrate 0.35g Na 2 HPO 4 0.105g KH 2 PO 4 0.035g NaCl 5.0g pH 7.0 ± 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. Distribute into tubes or flasks. Gently heat and bring to boiling. Boil for 10 min. Autoclave for 15 min at 15 psi pressure–121°C. Mix thoroughly while pouring into Petri dishes. Use: For the detection and enumeration of proteolytic microorganisms in foodstuffs and other materials. Caldicellulosiruptor Medium Composition per liter: Pancreatic digest of casein 2.0g K 2 HPO 4 1.5g Cellobiose 1.0g Yeast extract 1.0g NaCl 0.9g NH 4 Cl 0.9g KH 2 PO 4 0.75g L-Cysteine·HCl 0.75g MgCl 2 ·6H 2 O 0.4g FeCl 3 ·6H 2 O 2.5mg Resazurin 0.5mg Trace elements solution SL-10 1.0mL pH 7.2 ± 0.2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg MnCl 2 ·4H 2 O 100.0mg © 2010 by Taylor and Francis Group, LLC Caldisphaera Medium 293 ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/ deionized water and bring volume to 1.0L. Add remaining compo- nents. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components to distilled/deionized water and bring vol- ume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Caldicellulosiruptor saccharolyticus. Caldicellulosiruptor Medium Composition per 1001.0mL: Pancreatic digest of casein 2.0g K 2 HPO 4 1.5g Cellulose 1.0g Cellobiose 1.0g Yeast extract 1.0g NaCl 0.9g NH 4 Cl 0.9g KH 2 PO 4 0.75g MgCl 2 ·6H 2 O 0.4g FeCl 3 ·6H 2 O 2.5mg L-Cysteine·HCl 0.75g Resazurin 0.5mg pH 7.2 ± 0.2 at 25°C Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Preparation of Medium: Prepare and dispense medium under 100% N 2 . Add components to distilled/deionized water and bring vol- ume to 1.0L. Mix thoroughly. Sparge with 100% N 2 . Anaerobically distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Caldicellulosiruptor saccharolyticus. Caldisphaera Medium (DSMZ Medium 991) Composition per liter: Sulfur, powder 10.0g MnCl 2 ·4H 2 O 1.8g (NH 4 ) 2 SO 4 1.3g KH 2 PO 4 0.28g MgSO 4 ·7H 2 O 0.25g CaCl 2 ·2H 2 O 0.07g FeCl 3 ·6H 2 O 0.02g Na 2 B 4 O 7 ·10H 2 O 4.5mg Resazurin 1.0mg ZnSO 4 ·7H 2 O 0.22mg CuCl 2 ·2H 2 O 0.05mg Na 2 MoO 4 ·4H 2 O 0.03mg VOSO 4 ·2H 2 O 0.03mg Na 3 -citrate·2H 2 O 0.03mg CoSO 4 0.01mg Vitamin solution 10.0mL Na 2 S·9H 2 O solution 10.0mL Yeast extract solution 5.0mL pH 4.3 ± 0.2 at 25°C Yeast Extract Solution: Composition per 5.0mL: Yeast extract 0.5g Preparation of Yeast Extract Solution: Add yeast extract to dis- tilled/deionized water and bring volume to 5.0mL. Mix thoroughly. Sparge under 100% N 2 gas for 3 min. Autoclave for 15 min at 15 psi pressure–121°C. Store under N 2 gas. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.5g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. 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: Add components, except vitamin solution, yeast extract solution, sulfur, and Na 2 S·9H 2 O solution, to distilled/deion- ized water and bring volume to 975.0mL. Mix thoroughly. Gently heat and bring to boiling. Boil for 3 min. Cool to room temperature under 80% N 2 + 20% CO 2 . Adjust pH to 3.5 with 10N H 2 SO 4 . Dispense under same gas atmosphere in suitable culture vessels (e.g., 20.0mL of the medium in 120 mL serum bottles). Autoclave for 15 min at 15 psi pres- sure–121°C. Steam sulfur for 3 hr on each of 3 successive days. Asep- © 2010 by Taylor and Francis Group, LLC 294 Calditerrivibrio Medium tically mix the sterilized sulfur with the medium and add vitamins and yeast extract from sterile, anaerobic stock solutions. Prior to inocula- tion change atmosphere to 80% H 2 + 20% CO 2 . Aseptically and anox- ically add Na 2 S·9H 2 O. Adjust pH to 4.0–4.5 if necessary. After inoculation pressurize vials to 1 bar overpressure with 80% H 2 + 20% CO 2 gas mixture. Use: For the cultivation of Caldisphaera spp. Calditerrivibrio Medium (DSMZ Medium 1112) Composition per liter: NaNO 3 0.85g Na-acetate 0.82g NH 4 Cl 0.54g MgCl 2 ·6H 2 O 0.2g CaCl 2 ·2H 2 O 0.15g KH 2 PO 4 0.14g Resazurin 0.5mg NaHCO 3 solution 10.0mL Vitamin solution 10.0mL Na 2 S·9H 2 O solution 10.0mL Trace element solution SL-10 1.0mL Selenite/tungstate solution 1.0mL pH 7.0 ± 0.2 at 25°C NaHCO 3 Solution: Composition per 10.0mL: NaHCO 3 2.5g Preparation of NaHCO 3 Solution: Add components to distilled/ deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 20% CO 2 + 80% H 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Cool to room temperature. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.5g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. 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 . Autoclave for 15 min at 15 psi pressure–121°C. 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. Trace Elements Solution SL-10: Composition per liter: FeCl 2 ·4H 2 O 1.5g CoCl 2 ·6H 2 O 190.0mg MnCl 2 ·4H 2 O 100.0mg ZnCl 2 70.0mg Na 2 MoO 4 ·2H 2 O 36.0mg NiCl 2 ·6H 2 O 24.0mg H 3 BO 3 6.0mg CuCl 2 ·2H 2 O 2.0mg HCl (25% solution) 10.0mL Preparation of Trace Elements Solution SL-10: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized water and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure– 121°C. Preparation of Medium: Add components, except bicarbonate so- lution, sulfide solution, and vitamin solution, to distilled/deionized wa- ter and bring volume to 970.0mL. Mix thoroughly. Gently heat and bring to boiling. Boils for several minutes. Cool to room temperature while sparging with 80% N 2 + 20% CO 2 . Distribute into screw-capped tubes or bottles under an atmosphere of 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Add the bicarbonate solution, sul- fide solution, and vitamin solution. Adjust the final pH to 7.0. Use: For the cultivation of Calditerrivibrio spp. Caldivirga Medium (DSMZ Medium 883) Composition per liter: (NH 4 ) 2 SO 4 1.3g Sulfur, powdered 10.0g Na 2 S·9H 2 O 0.5 g KH 2 PO 4 0.28g MgSO 4 ·7H 2 O 0.25g CaCl 2 ·2H 2 O 0.07g FeCl 3 ·6H 2 O 0.02g Na 2 B 4 O 7 ·10H 2 O 4.5mg MnCl 2 ·4H 2 O 1.8mg Resazurin 0.5mg ZnSO 4 ·7H 2 O 0.22mg CuCl 2 ·2H 2 O 0.05mg Na 2 MoO 4 ·2H 2 O 0.03mg VOSO 4 ·2H 2 O 0.03mg CoSO 4 0.01mg Na 2 S·9H 2 O solution 7.5mL Yeast extract solution 5.0mL Na 3 -citrate·2H 2 O 3.0mL Vitamin solution 1.0mL pH 4.0± 0.2 at 25°C Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.3g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave under 100% N 2 for 15 min at 15 psi pressure–121°C. Cool to room temperature. © 2010 by Taylor and Francis Group, LLC . 10.0mL: Nile Blue 0.2g Preparation of Nile Blue Solution: Add Nile blue to 10.0mL of a 1% solution of dimethyl sulfoxide. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except. 20.0mL of sterile car- bohydrate solution, 10.0mL of sterile L-cysteine·HCl·H 2 O solution, and 10.0mL of sterile Na 2 S·9H 2 O solution or, using a syringe, inject the appropriate amount of sterile. of sterile horse se- rum, 50.0mL of sterile fresh yeast extract solution, and 5.0mL of sterile CMRL medium. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of