Halothermothrix orenii Medium 805 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. Vitamin Solution V7: Composition per liter: Pyridoxine-HCl 50.0mg Nicotinic acid 20.0mg Vitamin B 12 20.0mg Thiamine-HCl·2H 2 O 10.0mg p-Aminobenzoic acid 10.0mg D-Ca-pantothenate 5.0mg Biotin 2.0mg Preparation of Vitamin Solution V7: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Trace Elements Solution SL-12: Composition per liter: FeSO 4 ·7H 2 O 1.1g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.19g MnCl 2 ·2H 2 O 0.05g ZnCl 2 42.0mg NiCl 2 ·6H 2 O 24.0mg Na 2 MoO 4 ·4H 2 O 18.0mg CuCl 2 ·2H 2 O 2.0mg Preparation of Trace Elements Solution SL-12: Add compo- nents. to distilled/deionized water and bring volume to 1.0L. Mix thor- oughly. Filter sterilize. Preparation of Medium: Add components, except sulfide, bicar- bonate, vitamin, and trace elements solutions, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.3. Gently heat while stirring and bring to boiling. Autoclave for 20 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add sul- fide, bicarbonate, vitamin, and trace elements solutions. Mix thorough- ly. Aseptically distribute into sterile screw-cap tubes, bottles, or flasks. Use: For the improved growth during cultivation of pure cultures of Halorhodospira spp. Halorubrum californiense Medium (DSMZ Medium 1194) Composition per liter: NaCl 156.0g MgSO 4 ·7H 2 O 40.6g MgCl 2 ·6H 2 O 26.0g Yesat extract 10.0g KCl 4.0g CaCl 2 ·2H 2 O 0.66g NaBr 0.46g NaHCO 3 0.12g 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. Adjust pH to 7.2. Gently heat while stirring and bring to boiling. Mix thoroughly. Dis- tribute into tubes , bottles, or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Halorubrum californiense. Halothermothrix orenii Medium (DSMZ Medium 761) Composition per liter: NaCl 100.0g Glucose 10.0g KCl 4.0g MgCl 2 ·6H 2 O 2.0g NH 4 Cl 1.0g Na-acetate 1.0g Trypticase™ 0.5g K 2 HPO 4 0.3g KH 2 PO 4 0.3g CaCl 2 ·2H 2 O 0.2g Resazurin 0.001g NaHCO 3 solution 50.0mL Na 2 S·9H 2 O solution 10.0mL Dithionite solution 5.0mL Trace elements solution SL-6 1.0mL pH 7.0 ± 0.2 at 25°C Trace Elements Solution SL-6: Composition per liter: MnCl 2 ·4H 2 O 0.5g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g Na 2 MoO 4 ·2H 2 O 0.03g NiCl 2 ·6H 2 O 0.02g CuCl 2 ·2H 2 O 0.01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Na 2 S·9H 2 O Solution: Composition per 10.0mL: Na 2 S·9H 2 O 0.2g Preparation of Na 2 S·9H 2 O Solution: Add Na 2 S·9H 2 O to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Autoclave for 15 min at 15 psi pressure–121°C. Neutralize to pH 7.0 with sterile HCl. NaHCO 3 Solution: Composition per 100.0mL: NaHCO 3 10.0g Preparation of NaHCO 3 Solution: Add NaHCO 3 to distilled/de- ionized water and bring volume to 100.0mL. Mix thoroughly. Sparge with 80% N 2 + 20% CO 2 . Filter sterilize. Dithionite Solution Composition per 10.0mL: Na-dithionite 2.0mg Preparation of Dithionite Solution: Add Na-dithionite to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Preparation of Medium: Prepare and dispense medium under 80% N 2 + 20% CO 2 gas atmosphere. Add components, except NaHCO 3 solu- tion, dithionite solution, and Na 2 S·9H 2 O solution, to distilled/deionized water and bring volume to 935.0mL. Mix thoroughly. Adjust pH to 7.0. Gently heat and bring to boiling. Cool while sparging with 80% N 2 + 20% CO 2 . Distribute into Hungate tubes under 80% N 2 + 20% CO 2 . Au- toclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically and anaerobically inject NaHCO 3 solution (0.5mL per © 2010 by Taylor and Francis Group, LLC 806 Halothiobacillus Medium 10mL medium), dithionite solution (0.05mL per 10mL medium), and Na 2 S·9H 2 O solution (0.1mL per 10mL medium). Aseptically and an- aerobically distribute into sterile tubes or bottles. Use: For the cultivation of Halothermothrix orenii. Halothiobacillus Medium (DSMZ Medium 864) Composition per 1030.0mL: NaCl 29.0g MgSO 4 ·7H 2 O 1.5g (NH 4 ) 2 SO 4 1.0g KCl 0.7g CaCl 2 ·2H 2 O 0.42g Bromthymol Blue 4.0mg Na-thiosulfate solution 20.0mL Phosphate solution 10.0mL Trace elements solution 1.0mL pH 6.5–7.0 at 25°C Trace Elements Solution: Composition per liter: FeCl 2 ·4H 2 O 1.5g Na 2 -EDTA 0.5g 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: Add FeCl 2 ·4H 2 O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized wa- ter and bring volume to 1.0L. Add remaining components. Mix thor- oughly. Adjust pH to 7.0. Sparge with 80% N 2 + 20% CO 2 . Autoclave for 15 min at 15 psi pressure–121°C. Na-thiosulfate Solution: Composition per 20.0mL: Na 2 S 2 O 3 ·5H 2 O 5.0g Preparation of Na-thiosulfate Solution: Add Na 2 S 2 O 3 ·5H 2 O to distilled/deionized water and bring volume to 20.0mL. Mix thorough- ly. Sparge with 100% N 2 . Filter sterilize. Phosphate Solution: Composition per 10.0mL: K 2 HPO 4 0.5g Preparation of Phosphate Solution: Add K 2 HPO 4 to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Sparge with 100% N 2 . Filter sterilize. Preparation of Medium: Add components, except phosphate solu- tion and Na-thiosulfate solution, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.7. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add 20.0mL sterile Na-thiosulfate solution and 10.0mL sterile phosphate solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Halothiobacillus kellyi. Halovibrio Medium (DSMZ Medium 1176) Composition per liter: NaCl 120.0–240.0g KH 2 PO 4 3.0g NH 4 Cl 0.5g Sodium acetate soltuion 10.0mL HEPES solution 10.0mL Yeast extract solution 2.0mL Magnesium sulfate solution 2.0mL Trace elements solution SL-6 1.0mL pH 7.5 ± 0.2 at 25°C Trace Elements Solution SL-6: Composition per liter: MnCl 2 ·4H 2 O 0.5g H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g Na 2 MoO 4 ·2H 2 O 0.03g NiCl 2 ·6H 2 O 0.02g CuCl 2 ·2H 2 O 0.01g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. HEPES Solution: Composition per 10.0mL: HEPES 2.4g Preparation of HEPES Solution: Add HEPES to distilled/deion- ized water and bring volume to 10.0mL. Mix thoroughly. Sodium Acetate Solution: Composition per 10.0mL: Sodium acetate 1.64g Preparation of Sodium Acetate Solution: Add sodium acetate to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Autoclave for 15 min at 15 psi pressure–121°C. Magnesium Sulfate Solution: Composition per 10.0mL: MgSO 4 ·7H 2 O 2.5g Preparation of Magnesium Sulfate Solution: Add MgSO 4 ·7H 2 O to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. Autoclave for 15 min at 15 psi pressure–121°C. Yeast Extract Solution: Composition per 10.0mL: Yeast extract 1.0g Preparation of Yeast Extract Solution: Add yeast extract to dis- tilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Preparation of Medium: Add components, except yeast extract, magnesium sulfate, and sodium acetate solutions, to distilled/deionized water and bring volume to 970.0mL. Mix thoroughly. Adjust pH to 7.5. Gently heat while stirring and bring to boiling. Cool to room tempera- ture. Dispense into sealed tubes, vials, or bottles. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room temperature. Aseptically add yeast extract, magnesium sulfate, and sodium acetate solutions. Use: For the cultivation of Halovibrio spp. © 2010 by Taylor and Francis Group, LLC Hanahan’s Broth 807 Halovibrio variabilis Medium Composition per liter: NaCl 95.0g MgSO 4 ·7H 2 O 81.0g Yeast extract 7.5g Proteose peptone 2.5g KCl 1.0g Trace elements solution SL-4 10.0mL Vitamin solution 10.0mL pH 7.5 ± 0.2 at 25°C Vitamin Solution: Composition per liter: Pyridoxine HCl 1.0mg Lipoic acid 0.5mg Nicotinic acid 0.5mg p-Aminobenzoic acid 0.5mg Pantothenic acid 0.5mg Riboflavin 0.5mg Thiamine HCl 0.5mg Biotin 0.2mg Folic acid 0.2mg Cyanocobalamin 0.01mg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Filter ster- ilize. Store at 5°C. Trace Elements Solution SL-4: Composition per 900.0mL: EDTA 0.5g FeSO 4 ·7H 2 O 0.2g Trace elements solution SL-6 100.0mL Trace Elements Solution SL-6: Composition per liter: H 3 BO 3 0.3g CoCl 2 ·6H 2 O 0.2g ZnSO 4 ·7H 2 O 0.1g MnCl 2 ·4H 2 O 0.03g CuCl 2 ·2H 2 O 0.01g NiCl 2 ·6H 2 O 0.02g Na 2 MoO 4 ·2H 2 O 0.03g Preparation of Trace Elements Solution SL-6: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Trace Elements Solution SL-4: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except trace elements solution SL-4 and vitamin solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically add 10.0mL of sterile trace elements solution SL-4 and 10.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Halovibrio variabilis. Ham’s F-10 Medium Composition per liter: NaCl 7.4g NaHCO 3 1.2g Glucose 1.1g NaH 2 PO 4 ·H 2 O 0.29g KCl 0.28g L-Arginine·HCl 0.21g L-Glutamine 0.15g MgSO 4 ·7H 2 O 0.15g Sodium pyruvate 0.11g KH 2 PO 4 0.08g CaCl 2 ·2H 2 O 0.04g L-Cystine·2HCl 0.04g L-Histidine·HCl·H 2 O 0.02g L-Lysine·HCl 0.02g L-Asparagine-H 2 O 0.01g L-Aspartic acid 0.01g L-Glutamic acid 0.01g L-Leucine 0.01g L-Proline 0.01g L-Serine 0.01g L-Alanine 8.9mg Glycine 7.5mg D-Phenylalanine 5.0mg L-Methionine 4.5mg Hypoxanthine 4.1mg L-Threonine 3.6mg L-Valine 3.5mg L-Isoleucine 2.6mg L-Tyrosine 1.8mg Vitamin B 12 1.4mg Folic acid 1.3mg Phenol Red 1.2mg Thiamine·HCl 1.0mg FeSO 4 ·7H 2 O 0.8mg Choline chloride 0.7mg D-Calcium pantothenate 0.7mg Thymidine 0.7mg Niacinamide 0.6mg L-Tryptophan 0.6mg Isoinositol 0.5mg Riboflavin 0.4mg Lipoic acid 0.2mg Pyridoxine·HCl 0.2mg ZnSO 4 ·7H 2 O 0.03mg Biotin 0.02mg CuSO 4 ·5H 2 O 3.0μg 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. Filter sterilize. Use: For the growth of Y-1 cell cultures used in the mouse adrenal assay for heat-labile toxin of enterotoxigenic Escherichia coli and Vibrio species. Hanahan’s Broth (SOB Medium) Composition per liter: Casein enzymatic hydrolysate 20.0g Yeast extract 5.0g MgSO 4 2.4g NaCl 0.5g KCl 0.186g pH 7.0 ± 0.2 at 25°C Source: This medium is available as a premixed powder from Sig- maAldrich. © 2010 by Taylor and Francis Group, LLC 808 Hanahan’s HiVeg Broth 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. Use: For the cultivation of recombinant strains of Escherichia coli. Hanahan’s HiVeg Broth Composition per liter: Plant hydrolysate 20.0g Yeast extract 5.0g MgSO 4 2.4g NaCl 0.5g KCl 0.186g pH 7.0± 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 15 psi pressure–121°C. Use: For the cultivation of recombinant strains of Escherichia coli. Harpo’s HTYE Trypticase Peptone Medium Composition per liter: Pancreatic digest of casein 5.0g HEPES (N-[2-Hydroxyethyl]piperazine- N´-2-ethanesulfonic acid) buffer 4.0g Yeast extract 2.0g pH 6.8–7.0 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0–7.2. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the cultivation of Cytophaga arvensicola and Flexibacter columnaris. Harpo’s HTYEM Marine Medium Pancreatic digest of casein 5.0g HEPES 4.0g Yeast extract 2.0g Artificial seawater 1.0L pH 7.5 ± 0.2 at 25°C Artificial Seawater: Composition per liter: NaCl 27.5g MgSO 4 ·7H 2 O 6.78g MgCl 2 ·6H 2 O 5.38g CaCL 2 ·2H 2 O 1.4g KCl 0.72g NaHCO 3 0.2g Preparation of Artificial Seawater: Add components to distilled/ deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to artificial seawater and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.5 with NaOH. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Cytophaga fermentans, Cytophaga later- cula, Cytophaga uliginosa, and Microscilla aggregans. Harrold’s Agar see: M 40 Y Agar Hartley’s Digest Broth Composition per 10.0L: Ox heart 3000.0g 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 Medium: 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 through Whatman #1 filter paper. Cool to room tem- perature. Adjust pH to 7.5. Autoclave for 15 min at 15 psi pressure– 121°C. Use: For the isolation and cultivation of Actinobacillus lignieresii from cattle. Hartley’s Digest HiVeg Broth Composition per liter: Plant hydrolysate 29.0g pH 7.6 ± 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 15 psi pressure–121°C. Use: For the isolation and cultivation of Actinobacillus lignieresii from cattle. Haskins Agar for Tetrahymena Composition per liter: Agar 16.0g Dextrin 8.0g Pancreatic digest of casein 5.0g Yeast extract 5.0g Liver concentrate 0.6g Sodium acetate 0.6g pH 7.2–7.4 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.2–7.4. 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. Overlay agar with a layer of sterile distilled water. Use: For the cultivation of Tetrahymena australis and Tetrahymena vorax. HAY See: Hay Extract Agar © 2010 by Taylor and Francis Group, LLC Hayflick Medium, Modified 809 Hay Extract Agar (HAY) Composition per liter: Hay 50.0g Agar 20.0g pH 6.2 ± 0.2 at 25°C Preparation of Medium: Add finely hashed hay to distilled/deion- ized water and bring volume to 1.0L. Mix thoroughly. Autoclave for 30 min at 15 psi pressure–121°C. Filter through Whatman filter paper. Adjust pH of filtrate to 6.2 with 5% potassium phosphate solution. Add agar. Bring volume to 1.0L wtih distilled/deionized water. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 20 min at 10 psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Alternaria porri, Botryotinia draytonii, Botry- otinia fuckeliana, Botryotinia porri, Botryotinia narcissicola, Botryotinia polyblastis, Botrytis aclada, Botrytis hyacinthi, Botrytis cinerea, Cer- cospora beticola, and Sclerotinia sphaerosperma. Hay Extract Medium See: HE Medium Hay Infusion Agar Composition per liter: Hay, partially decomposed 50.0g Agar 15.0g K 2 HPO 4 2.0g pH 6.2 ± 0.3 at 25°C Preparation of Medium: Add hay to distilled/deionized water and bring volume to 1.0L. Autoclave for 30 min at 15 psi pressure–121°C. Filter through paper and reserve filtrate. Add distilled/deionized water to filtrate and bring volume to 1.0L. Mix thoroughly. Add agar and K 2 HPO 4 . Mix thoroughly. Gently heat and bring to boiling. Adjust pH to 6.2. 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 Alternaria species, Cau- lochytrium protostelioides, Choanephora infundibulifera, Dipsacomy- ces acuminosporus, Eremascus albus, Eremascus fertilis, Eurotium chevalieri, Eurotium halophilicum, Eurotium herbariorum, Mortier- ella bisporalis, Protostelium irregularis, and Saksenaea vasiformis. Hayflick Medium Composition per 107.5mL: Mycoplasma broth base 70.0mL Horse serum 20.0mL Fresh yeast extract solution 10.0mL Penicillin solution 5.0mL Thallous acetate solution 2.5mL pH 7.8 ± 0.2 at 25°C Mycoplasma Broth Base: 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 Preparation of Mycoplasma Broth Base: 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. Auto- clave for 15 min at 15 psi pressure–121°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 live Baker’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90 min at 15 psi pressure–121°C. Allow to stand. Remove supernatant solution. Adjust pH to 6.6–6.8. Filter sterilize. Penicillin Solution: Composition per 5.0mL: Penicillin 20,000U Preparation of Penicillin Solution: Add penicillin to distilled/de- ionized water and bring volume to 5.0mL. Mix thoroughly. Filter ster- ilize. Thallous Acetate Solution: Composition per 10.0mL: Thallous acetate 0.1g Preparation of Thallous Acetate Solution: Add thallous acetate to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. Filter sterilize. Use: For the cultivation of Mycoplasma species. Hayflick Medium, Modified Composition per 1212.0mL: Beef heart, infusion from 500.0g Tryptose 10.0g Noble agar 9.6g NaCl 5.0g Horse serum, normal 200.0mL Fresh yeast extract solution 100.0mL Calf thymus DNA solution 12.0mL 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. Filter sterilize. Calf Thymus DNA Solution: Composition per 20.0mL: Calf thymus DNA 0.04g Preparation of Calf Thymus DNA Solution: Add calf thymus DNA to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Add components, except horse serum, fresh yeast extract solution, and calf thymus DNA solution, to distilled/ deionized water and bring volume to 900.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–50°C. Aseptically add 200.0mL of sterile horse se- rum, 100.0mL of sterile fresh yeast extract solution, and 12.0mL of sterile calf thymus DNA solution. Mix thoroughly. Aseptically distrib- ute into sterile tubes. Use: For the cultivation and maintenance of Mycoplasma mustelae. © 2010 by Taylor and Francis Group, LLC 810 HBT Bilayer Medium HB See: Halophilic Broth 3HB See: Hydroxybutyrate Medium HBT Bilayer Medium (Human Blood Tween™ Bilayer Medium) Composition per 1062.5mL: Agar 13.5g Pancreatic digest of casein 12.0g Casein/meat peptone 10.0g NaCl 5.0g Peptic digest of animal tissue 5.0g Beef extract 3.0g Yeast extract 3.0g Cornstarch 1.0g Human blood, anticoagulated 25.0mL Colistin solution 10.0mL Nalidixic acid solution 10.0mL Amphotericin B solution 10.0mL Polysorbate 80 (Tween™ 80) solution 7.5mL pH 7.3 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Colistin Solution: Composition per liter: Colistin 0.01g Preparation of Colistin: Add colistin to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Filter sterilize. Nalidixic Acid Solution: Composition per liter: Nalidixic acid 0.02g Preparation of Nalidixic Acid Solution: Add nalidixic acid to distilled/deionized water and bring volume to 10.0mL. Mix thorough- ly. Filter sterilize. Amphotericin B Solution: Composition per liter: Amphotericin B 3.0mg Preparation of Amphotericin B Solution: Add Amphotericin B to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. Filter sterilize. Tween™ 80 Solution: Composition per 100.0mL: Tween™ 80 1.0mL Preparation of Tween™ 80 Solution: Add Tween™ 80 to dis- tilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Adjust pH to 7.3. Filter sterilize. Preparation of Medium: Add components, except amphotericin B solution, Tween™ 80, and human blood, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Divide the medium into two 500.0mL fractions. Autoclave both flasks of media for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. To one flask, aseptically add 5.0mL of sterile colistin solution, 5.0mL of sterile nalidixic acid solution, 5.0mL of sterile amphotericin B solution, and 3.75mL of Tween™ 80 solution. Mix thoroughly. Pour into sterile Petri dishes in 7.0mL volumes. Allow agar to harden. To re- maining flask aseptically add 5.0mL of sterile colistin solution, 5.0mL of sterile nalidixic acid solution, 5.0mL of sterile amphotericin B solu- tion, 3.75mL of sterile Tween™ 80 solution, and 25.0mL of sterile hu- man blood. Mix thoroughly. Pour into the same Petri dishes that each contain 7.0mL of the agar medium without blood. The top layer should be approximately 14.0mL per plate. Use: For the selective isolation, cultivation, and differentiation of Gardnerella vaginalis from clinical specimens. HC Agar See: Hemorrhagic coli Agar HC Agar Base Composition per liter: Glucose 20.0g Agar 15.0g Yeast extract 5.0g Na 2 HPO 4 3.5g KH 2 PO 4 3.4g Pancreatic digest of casein 2.5g Peptic digest of animal tissue 2.5g NH 4 Cl 1.4g Na 2 CO 3 1.0g Chloramphenicol 0.1g MgSO 4 ·7H 2 O 0.06g Polysorbate 80 (Tween™ 80) solution 20.0mL 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, except Tween™ 80, to distilled/deionized water and bring volume to 980.0mL. Mix thorough- ly. Gently heat and bring to boiling. Add Tween™ 80. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes. Use: For the cultivation and enumeration of molds in cosmetics and toiletries. HD (1:10 Diluted) (DSMZ Medium 1124) Composition per liter: Casein peptone 0.5g Glucose 0.1g Yeast extract 0.25g pH 5.0 ± 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 5.0. Gently heat while stirring and bring to boiling. Mix thoroughly. Dis- tribute into tubes , bottles, or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation of Edaphobacter modestus. HD Agar, 1:10 Diluted, Modified (DSMZ Medium 1135) Composition per liter: MES (2-[N-morpholino]ethane sulfonic acid) 1.95g Casein peptone 0.5g Yeast extract 0.25g © 2010 by Taylor and Francis Group, LLC Heart Infusion Agar 811 Glucose 0.1g Agar solution 500.0mL pH 5.5 ± 0.2 at 25°C Agar Solution: Composition per 10.0mL: Agar 15.0g Preparation of Agar Solution: Wash agar with double distilled water. Add agar to double distilled/deionized water and bring volume to 500.0mL. Adjsut pH to 5.5. Mix thoroughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Preparation of Medium: Add components, except agar solution, to double distilled/deionized water and bring volume to 500.0mL. Mix thoroughly. Adjust pH to 5.5. Gently heat while stirring and bring to boiling. Mix thoroughly. Autoclave for 15 min at 15 psi–121°C. Cool to 50°C. Aseptically add 500.0mL agar solution. Pour into Petri dishes or aseptically distribute into tubes. Use: For the cultivation of Edaphobacter aggregans. HD- Medium, 1:10 Diluted, Modified (DSMZ Medium 1135) Composition per liter: MES (2-[N-Morpholino]ethane sulfonic acid) 1.95g Casein peptone 0.5g Yeast extract 0.25g Glucose 0.1g pH 5.5 ± 0.2 at 25°C Preparation of Medium: Add components to double distilled/de- ionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 5.5. Gently heat while stirring and bring to boiling. Mix thoroughly. Distribute into tubes , bottles, or flasks. Autoclave for 15 min at 15 psi pressure–121°C . Use: For the cultivation of Edaphobacter aggregans. HE Medium (Hay Extract Medium) Composition per liter: Agar 10.0g Peptone 1.0g Yeast extract 1.0g Hay extract solution 500.0mL pH 6.5 ± 0.2 at 25°C Hay Extract Solution: Composition per liter: Hay, dried 50.0g Preparation of Hay Extract Solution: Add dried barn hay to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gen- tly heat and bring to boiling. Filter through Whatman #40 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 isolation and cultivation of Spirochaeta aurantia. Heart Infusion Agar Composition per liter: Beef heart, infusion from 500.0g Agar 15.0g Tryptose 10.0g NaCl 5.0g pH 7.4 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat 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 a wide variety of fastidious microorganisms. For the cultivation and maintenance of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Serratia rubidaea, Staphylococcus aureus, Tsatumella ptyseos, and Vibrio vulnificus. It can also be used as a base for the preparation of blood agar in determin- ing hemolytic reactions. When using for blood agar, reduce volume to 950.0mL to allow for addition of 50.0mL of defibrinated horse blood. Heart Infusion Agar (HIA) (BAM M159) Composition per liter: Proteose peptone 15.0g Agar 12.0g Yeast extract 5.0g NaCl 5.0g Liver digest 2.5g pH 7.4 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat 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 a wide variety of fastidious microorganisms. For the cultivation and maintenance of Bacillus cere- us, Staphylococcus aureus, Vibrio vulnificus, and Vibrio cholerae. It can also be used as a base for the preparation of blood agar in determin- ing hemolytic reactions. When using for blood agar, reduce volume to 950.0mL to allow for addition of 50.0mL of defibrinated horse blood. Blood is added aseptically after autoclaving. Heart Infusion Agar (HIA) (BAM M60) Composition per liter: Agar 15.0g Tryptose 10.0g NaCl 5.0g Beef heart, infusion from 500.0g 1.0L pH 7.4 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat 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. © 2010 by Taylor and Francis Group, LLC 812 Heart Infusion Agar with Glucose Use: For the isolation and cultivation of a wide variety of fastidious microorganisms. For the cultivation and maintenance of Bacillus cere- us, Staphylococcus aureus, Vibrio vulnificus, and Vibrio cholerae.It can also be used as a base for the preparation of blood agar in determin- ing hemolytic reactions. When using for blood agar, reduce volume to 950.0mL to allow for addition of 50.0mL of defibrinated horse blood. Blood is added aseptically after autoclaving. Heart Infusion Agar with Glucose Composition per liter: Beef heart, infusion from 500.0g Agar 15.0g Tryptose 10.0g NaCl 5.0g Glucose 1.0g pH 7.4 ± 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 Bacillus species and Pseudomonas species. Heart Infusion Agar with 0.1% Glucose Composition per liter: Beef heart, infusion from 500.0g Agar 15.0g Tryptose 10.0g NaCl 5.0g Glucose 1.0g pH 7.4 ± 0.2 at 25°C Source: This medium without glucose is available as a premixed powder from BD Diagnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the isolation and cultivation of Bacillus circulans and Pseudomonas sp. Heart Infusion Agar, HiVeg Composition per liter: Agar 15.0g Plant hydrolysate No. 1 10.0g Plant infusion 10.0g NaCl 5.0g pH 7.4 ± 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 15 psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. Use: For the isolation and cultivation of a wide variety of fastidious microorganisms. Heart Infusion Agar, HiVeg with Blood Composition per liter: Agar 15.0g Plant hydrolysate No. 1 10.0g Plant infusion 10.0g NaCl 5.0g Horse blood, defibrinated 50.0mL pH 7.4 ± 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 950.0mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Auto- clave for 15 min at 15 psi pressure–121°C. Cool to 50°C. Aseptically add 50.0mL defibrinated blood. Mix thoroughly. Pour into sterile Petri dishes or leave in tubes. Use: For the isolation and cultivation of a wide variety of fastidious microorganisms. For determining hemolytic reactions. Heart Infusion Agar with Horse Serum and Fresh Yeast Extract Composition per 930.0mL: Heart infusion agar 720.0mL Horse serum, unheated 200.0mL Fresh yeast extract solution 10.0mL pH 7.4 ± 0.2 at 25°C Heart Infusion Agar: Composition per liter: Beef heart, infusion from 500.0g Agar 15.0g Tryptose 10.0g NaCl 5.0g Preparation of Heart Infusion Agar: Add components to dis- tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gen- tly heat and bring to boiling. Autoclave for 15 min at 15 psi pressure– 121°C. Cool to 45°–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: To 720.0mL of sterile cooled heart infu- sion broth, aseptically add 200.0mL of horse serum and 10.0mL of fresh yeast extract solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Mycoplasma equigenita- lium and Mycoplasma subdolum. Heart Infusion Agar (pH 7.6) with Inactivated Horse Serum (ATCC Medium 493) Composition per liter: Beef heart, infusion from 500.0g Agar 15.0g Tryptose 10.0g © 2010 by Taylor and Francis Group, LLC Heart Infusion Broth 813 NaCl 5.0g Horse serum, inactivated 100.0mL pH 7.6 ± 0.2 at 25°C Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 900.0mL. Mix thorough- ly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse se- rum. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Corynebacterium species. Heart Infusion Agar with Inactivated Horse Serum Composition per liter: Beef heart, infusion from 500.0g Agar 15.0g Tryptose 10.0g NaCl 5.0g Horse serum, inactivated 100.0mL pH 7.4 ± 0.2 at 25°C Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 900.0mL. Mix thorough- ly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse se- rum. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Corynebacterium species. Heart Infusion Agar with Inactivated Horse Serum, Sodium Chloride, and Penicillin Composition per liter: Beef heart, infusion from 500.0g NaCl 35.0g Agar 15.0g Tryptose 10.0g Horse serum, inactivated 100.0mL Penicillin solution 10.0mL pH 7.4 ± 0.2 at 25°C Penicillin Solution: Composition per 10.0mL: Penicillin 1,000,000U Preparation of Penicillin Solution: Add penicillin to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Filter ster- ilize. Preparation of Medium: Add components, except penicillin solu- tion and horse serum, 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 10.0mL of sterile penicillin solution and 100.0mL of sterile horse serum. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Corynebacterium species. Heart Infusion Agar with Sodium Chloride (HIA with NaCl) (BAM M60) Composition per liter: NaCl 20.0g Agar 15.0g Tryptose 10.0g NaCl 20.0g Beef heart, infusion from 500.0g 1.0L pH 7.4 ± 0.2 at 25°C Source: This medium is available as a premixed powder from BD Di- agnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat 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 halophilic Vibrio spp. Heart Infusion Agar with Rabbit Blood Composition per liter: Beef heart, infusion from 500.0g Agar 15.0g Tryptose 10.0g NaCl 5.0g Rabbit blood 50.0mL pH 7.4 ± 0.2 at 25°C Preparation of Medium: Add components, except rabbit blood, to distilled/deionized water and bring volume to 950.0mL. Mix thorough- ly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile rabbit blood. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Neisseria lactamica, Bar- tonella quintana, Bartonella elizabethae, and Bartonella henselae. Heart Infusion Agar with Yeast Extract Composition per liter: Beef heart, infusion from 500.0g Agar 20.0g Tryptose 10.0g NaCl 5.0g Yeast extract 5.0g pH 7.4 ± 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 Moraxella nonliquefa- ciens. Heart Infusion Broth Composition per liter: Beef heart, infusion from 500.0g Tryptose 10.0g NaCl 5.0g pH 7.4 ± 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. Use: For the isolation and cultivation of a wide variety of fastidious microorganisms. © 2010 by Taylor and Francis Group, LLC 814 Heart Infusion Broth Heart Infusion Broth (HI) (BAM M60) Composition per liter: Agar 15.0g Tryptose 10.0g NaCl 5.0g Beef heart, infusion from 500.0g 1.0L pH 7.4 ± 0.2 at 25°C Source: This medium without added NaCl is available as a premixed powder from BD Diagnostic 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. Use: For the isolation and cultivation of a wide variety of fastidious microorganisms. For the cultivation and maintenance of Bacillus cere- us, Staphylococcus aureus and Vibrio vulnificus, and Vibrio cholerae. It can also be used as a base for the preparation of blood agar in deter- mining hemolytic reactions. When using for blood broth, reduce vol- ume to 950.0mL to allow for addition of 50.0mL of defibrinated horse blood. Blood is added aseptically after autoclaving. Heart Infusion Broth with Additives for Staphylococcus Composition per liter: Beef heart, infusion from 500.0g NaCl 30.0 g Tryptose 10.0g Horse serum, inactivated 100.0mL Penicillin solution 10.0mL Fresh yeast extract solution 5.0mL pH 7.4 ± 0.2 at 25°C Penicillin Solution: Composition per 10.0mL: Penicillin 1,000,000U Preparation of Penicillin Solution: Add penicillin to distilled/de- ionized water and bring volume to 10.0mL. Mix thoroughly. Filter ster- ilize. Fresh Yeast Extract Solution: Composition per 100.0mL: Baker’s yeast, live, pressed, starch-free 10.0g 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, fresh yeast extract solution, and penicillin solution—to distilled/deion- ized 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 45°–50°C. Aseptically add sterile horse serum. Mix thorough- ly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Staphylococcus species. Heart Infusion Broth with Additives for Streptobacillus Composition per liter: Beef heart, infusion from 500.0g Tryptose 10.0g Peptone 10.0g NaCl 5.0g Glucose 0.5g Horse serum, inactivated 200.0mL pH 7.5 ± 0.2 at 25°C Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 800.0mL. Mix thorough- ly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Aseptically add sterile horse se- rum. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Streptobacillus monili- formis. Heart Infusion Broth with Glucose Composition per liter: Beef heart, infusion from 500.0g Tryptose 10.0g NaCl 5.0g Glucose 1.0g pH 7.4 ± 0.2 at 25°C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C. Use: For the cultivation and maintenance of Arthrobacter species, Bacillus species, and Pseudomonas species. Heart Infusion Broth with 0.1% Glucose (ATCC Medium 544) Composition per liter: Beef heart, infusion from 500.0g Tryptose 10.0g NaCl 5.0g Glucose 1.0g pH 7.4 ± 0.2 at 25°C Source: Heart infusion broth without glucose is available as a premixed powder from BD Diagnostic 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. Heart Infusion Broth with Glucose and Antibiotics Composition per liter: Beef heart, infusion from 500.0g Tryptose 10.0g NaCl 5.0g Antibiotic inhibitor solution 10.0mL Glucose solution 10.0mL pH 7.4 ± 0.2 at 25°C Antibiotic Inhibitor Solution: Composition per 10.0mL: Streptomycin sulfate 0.1g Tetracycline·HCl 0.025g Preparation of Antibiotic Inhibitor Solution: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thor- oughly. Filter sterilize. Glucose Solution: Composition per 10.0mL: D-Glucose 1.0g © 2010 by Taylor and Francis Group, LLC . flasks of media for 15 min at 15 psi pressure–121°C. Cool to 45°– 50°C. To one flask, aseptically add 5.0mL of sterile colistin solution, 5.0mL of sterile nalidixic acid solution, 5.0mL of sterile. 5.0mL of sterile amphotericin B solu- tion, 3.75mL of sterile Tween™ 80 solution, and 25.0mL of sterile hu- man blood. Mix thoroughly. Pour into the same Petri dishes that each contain 7.0mL of. so- lution. Adjust pH to 6.6–6.8. Preparation of Medium: To 720.0mL of sterile cooled heart infu- sion broth, aseptically add 200.0mL of horse serum and 10.0mL of fresh yeast extract solution. Mix thoroughly.