Handbook of Microbiological Media, Fourth Edition part 172 ppt

3.0mg Preparation of Selenite-Tungstate Solution: Add components to distilled/deionized water and bring volume to 1.0L.. 3.0mg Preparation of Selenite-Tungstate Solution: Add components

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Thauera aromatica AR-1 Medium 1705

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 Deinococcus species.

Thauera aromatica AR-1 Medium

(DSMZ Medium 855)

Solution A 870.0mL

Solution C 100.0mL

Solution D 10.0mL

Solution E (Vitamin solution) 10.0mL

Solution B (Trace elements solution SL-10) 1.0mL

Selenite-tungstate solution 1.0mL

pH 7.2 ± 0.2 at 25°C

Solution A:

Na2SO4 3.0g

NaCl 1.0g

KNO3 0.6g

KCl 0.5g

MgCl2·6H2O 0.4g

NH4Cl 0.3g

KH2PO4 0.2g

CaCl2·2H2O 0.15g

Resazurin 1.0mg

Preparation of Solution A: Add components to distilled/deionized

water and bring volume to 870.0mL Mix thoroughly

Solution B (Trace Elements Solution SL-10):

Compositionper liter:

FeCl2·4H2O 1.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

Na2MoO4·2H2O 36.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

HCl (25% solution) 10.0mL

Preparation of Solution B (Trace Elements Solution SL-10):

Add FeCl2·4H2O to 10.0mL of HCl solution Mix thoroughly Add

dis-tilled/deionized water and bring volume to 1.0L Add remaining

com-ponents Mix thoroughly Sparge with 100% N2 Autoclave for 15 min

at 15 psi pressure–121°C

Solution C:

NaHCO3 5.0g

Preparation of Solution C: Add NaHCO3 to distilled/deionized

water and bring volume to 100.0mL Mix thoroughly Filter sterilize

Flush with 80% N2 + 20% CO2 to remove dissolved oxygen

Solution D:

Na-benzoate 0.7g

Preparation of Solution D: Add Na-benzoate to

distilled/deion-ized water and bring volume to 10.0mL Mix thoroughly Sparge with

100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Solution E (Vitamin Solution):

Pyridoxine-HCl 10.0mg Thiamine-HCl·2H2O 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 B12 0.10mg

Solution E (Vitamin Solution): Add components to distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Selenite-Tungstate Solution Compositionper liter:

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Preparation of Selenite-Tungstate Solution: Add components

to distilled/deionized water and bring volume to 1.0L Mix thoroughly Sparge with 100% N2 Filter sterilize

Preparation of Medium: Gently heat solution A and bring to boil-ing Boil solution A for a few minutes Cool to room temperature Gas with 80% N2 + 20% CO2 gas mixture to reach a pH below 6 Autoclave for 15 min at 15 psi pressure–121°C Cool to room temperature Se-quentially add 1.0mL solution B, 100.0mL solution C, 10.0mL solu-tion D, 10.0mL solusolu-tion E, and 1.0mL sterile selenite-tungstate solution Distribute anaerobically under 80% N2 + 20% CO2 into ap-propriate vessels Addition of 10–20mg sodium dithionite per liter from a 5% (w/v) solution, freshly prepared under N2 and filter-steril-ized, may stimulate growth

Use: For the anaerobic cultivation of Thauera aromatica.

Thauera aromatica AR-1 Medium

Solution A 870.0mL Solution C 10.0mL Solution D (Vitamin solution) 10.0mL Solution B (Trace elements solution SL-10) 1.0mL Selenite-tungstate solution 1.0mL

pH 7.2 ± 0.2 at 25°C

Solution A:

NaCl 1.0g

Na2SO4 3.0g MgCl2·6H2O 0.4g

KH2PO4 0.2g

NH4Cl 0.3g KCl 0.5g CaCl2·2H2O 0.15g Resazurin 1.0mg

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 870.0mL Mix thoroughly

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1706 Thauera aromatica Medium

Solution B (Trace Elements Solution SL-10):

FeCl2·4H2O 1.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

Preparation of Solution B (Trace Elements Solution SL-10):

Add FeCl2·4H2O to 10.0mL of HCl solution Mix thoroughly Add

dis-tilled/deionized water and bring volume to 1.0L Add remaining

com-ponents Mix thoroughly Sparge with 100% N2 Autoclave for 15 min

Solution C:

Na-benzoate 0.7g

Preparation of Solution C: Add Na-benzoate to

distilled/deion-ized water and bring volume to 10.0mL Mix thoroughly Sparge with

Solution D (Vitamin Solution):

Pyridoxine-HCl 10.0mg

Thiamine-HCl·2H2O 5.0mg

Riboflavin 5.0mg

Nicotinic acid 5.0mg

Lipoic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.10mg

Solution D (Vitamin Solution): Add components to

distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Sparge with

Selenite-Tungstate Solution

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

to distilled/deionized water and bring volume to 1.0L Mix thoroughly

Sparge with 100% N2 Filter sterilize

Preparation of Medium: Adjust pH of solution A to 7.0 Autoclave

for 15 min at 15 psi pressure–121°C Cool to room temperature

Se-quentially add 1.0mL solution B, 10.0mL solution C, 10.0mL solution

D, and 1.0mL sterile selenite-tungstate solution Adjust pH to 7.2

Aseptically distribute into tubes or flasks

Use: For the aerobic cultivation of Thauera aromatica.

Thauera aromatica Medium

Composition 1015.0mL:

Solution A 500.0mL

Solution B 500.0mL

Trace elements solution SL-10 10.0mL Vitamin solution 5.0mL

pH 7.5 ± 0.2 at 25°C

Solution A:

K2HPO4 5.92g

KH2PO4 0.816g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 500.0mL Mix thoroughly Adjust pH to 7.5 Autoclave for 15 min at 15 psi pressure–121°C Cool to room temper-ature

Solution B:

KNO3 2.0g Sodium benzoate 0.72g

NH4Cl 0.267g MgSO4·7H2O 0.197g CaCl2·2H2O 0.025g

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 500.0mL Mix thoroughly Adjust pH to 7.5 Autoclave for 15 min at 15 psi pressure–121°C Cool to room temper-ature

Trace Elements Solution SL-10:

FeCl2·4H2O 1.5g CoCl2·6H2O 190.0mg MnCl2·4H2O 100.0mg ZnCl2 70.0mg

Na2MoO4·2H2O 36.0mg NiCl2·6H2O 24.0mg

H3BO3 6.0mg CuCl2·2H2O 2.0mg HCl (25% solution) 10.0mL

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

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% N2 Autoclave for 15 min at 15 psi pressure– 121°C Cool to room temperature

Vitamin Solution:

Pyridoxine·HCl 10.0mg

Lipoic acid 5.0mg Nicotinic acid 5.0mg Riboflavin 5.0mg Thiamine·HCl 5.0mg Calcium DL-pantothenate 5.0mg Biotin 2.0mg Folic acid 2.0mg Vitamin B12 0.1mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L Mix thoroughly Filter ster-ilize

Preparation of Medium: Aseptically combine 500.0mL of sterile solution A, 500.0mL of sterile solution B, 10.0 mL of sterile trace ele-ments solution SL-10, and 5.0 mL of sterile vitamin solution Mix thor-oughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of Thauera aromatica

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Thayer-Martin HiVeg Medium Base with Hemoglobin and Vitox Supplement 1707

Thauera mechernichi Medium

Na2HPO4 4.20g

Na-acetate 2.93g

KH2PO4 1.5g

NH4Cl 0.3g

MgSO4·7H2O 0.1g

Trace elements solution 2.0mL

pH 7.1 ± 0.2 at 25°C

Trace Elements Solution:

Na2-EDTA 50.0g

CaCl2·2H2O 5.5g

MnCl2·4H2O 5.06g

FeSO4·7H2O 5.0g

ZnSO4·7H2O 2.0g

CoCl2·6H2O 1.61g

CuSO4·5H2O 1.57g

(NH4)6Mo7O24·4H2O 1.1g

Preparation of Trace Elements Solution: Add components to

distilled/deionized water and bring volume to 1.0L Mix thoroughly

Adjust pH to 6.0

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 Thauera mechernichensis.

Thayer-Martin Agar, Modified

(MTM II) (Modified Thayer-Martin Agar)

Agar 12.0g

Hemoglobin 10.0g

Pancreatic digest of casein 7.5g

Selected meat peptone 7.5g

NaCl 5.0g

K2HPO4 4.0g

Cornstarch 1.0g

KH2PO4 1.0g

CNVT inhibitor 10.0mL

Supplement solution 10.0mL

pH 7.2 ± 0.2 at 25°C

CNVT Inhibitor:

Colistin sulfate 7.5mg

Trimethoprim lactate 5.0mg

Vancomycin 3.0mg

Nystatin 12,500U

Preparation of CNVT Inhibitor: Add components to

distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Filter

ster-ilize

Supplement Solution:

Glucose 100.0g

L-Cysteine·HCl 25.9g

L-Glutamine 10.0g

L-Cystine 1.1g Adenine 1.0g Nicotinamide adenine dinucleotide 0.25g Vitamin B12 0.1g Thiamine pyrophosphate 0.1g Guanine·HCl 0.03g Fe(NO3)3·6H2O 0.02g

p-Aminobenzoic acid 0.013g

Thiamine·HCl 3.0mg

Source: The supplement solution IsoVitaleX® enrichment is available from BD Diagnostic Systems This enrichment may be replaced by supplement VX from BD Diagnostic Systems

Preparation of Supplement Solution: Add components to dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except CNVT inhibitor and supplement solution, to distilled/deionized water and bring volume

to 990.0mL Mix thoroughly Gently heat and bring to boiling Distrib-ute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add 10.0mL of sterile CNVT inhibitor and 10.0mL of sterile supplement solution Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the isolation of Neisseria species from specimens containing

mixed flora of bacteria and fungi

Thayer-Martin HiVeg Medium Base with Hemoglobin and Vitox Supplement

Plant special peptone 23.0g Agar 13.0g NaCl 5.0g Starch 1.0g Hemoglobin solution 250.0mL Vitox supplement 10.0mL

pH 7.0 ± 0.2 at 25°C

Source: This medium, without hemoglobin or Vitox supplement, is available as a premixed powder from HiMedia

Hemoglobin Solution:

Hemoglobin 5.0g

Preparation of Hemoglobin Solution: Add hemoglobin to dis-tilled/deionized water and bring volume to 250.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Vitox Supplement:

Glucose 2.0g

L-Glutamine 0.2g

L-Cystine 0.022g Adenine sulfate 0.01g Nicotinamide adenine dinucleotide 5.0mg Cocarboxylase 2.0mg Guanine·HCl 0.6mg Fe(NO3)3·6H2O 0.4mg

Vitamin B12 0.2mg Thiamine·HCl 0.06mg

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1708 Thayer-Martin Medium

Preparation of Vitox Supplement: Add components to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Filter

sterilize

Preparation of Medium: Add components, except hemoblobin and

Vitox supplement, to distilled/deionized water and bring volume to

740.0mL Mix thoroughly Gently heat until boiling Autoclave for 15

min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add

250.0mL of sterile hemoglobin solution and 10.0mL of sterile Vitox

supplement Mix thoroughly Pour into sterile Petri dishes or distribute

into sterile tubes

Use: For the isolation and cultivation of fastidious microorganisms,

especially Neisseria species For the selective isolation of gonococci

from pathological specimens

Thayer-Martin Medium

GC agar base 740.0mL

Hemoglobin solution 250.0mL

Vitox supplement 10.0mL

pH 7.3 ± 0.2 at 25°C

GC Agar Base:

Special peptone 15.0g

Agar 10.0g

NaCl 5.0g

K2HPO4 4.0g

Cornstarch 1.0g

KH2PO4 1.0g

pH 7.2 ± 0.2 at 25°C

base and the hemoglobin to distilled/deionized water and bring volume

to 740.0mL Mix thoroughly Gently heat until boiling Autoclave for

15 min at 15 psi pressure–121°C Cool to 45°–50°C

Hemoglobin 5.0g

Preparation of Hemoglobin Solution: Add hemoglobin to

dis-tilled/deionized water and bring volume to 250.0mL Mix thoroughly

Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Glucose 2.0g

L-Glutamine 0.2g

L-Cystine 0.022g

Adenine sulfate 0.01g

Nicotinamide adenine dinucleotide 5.0mg

Cocarboxylase 2.0mg

Guanine·HCl 0.6mg

Fe(NO3)3·6H2O 0.4mg

Vitamin B12 0.2mg

Thiamine·HCl 0.06mg

sterilize

Preparation of Medium: To 740.0mL of cooled sterile GC agar

base, aseptically add 250.0mL of sterile hemoglobin solution and

10.0mL of sterile Vitox supplement Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

especially Neisseria species.

Thayer-Martin Medium

Hemoglobin 10.0g

GC medium base 980.0mL CNVT inhibitor 10.0mL Supplement B 10.0mL

pH 7.3 ± 0.2 at 25°C

Source: This medium is available as a prepared medium in tubes from

BD Diagnostic Systems

GC Medium Base:

Proteose peptone No 3 15.0g Agar 10.0g NaCl 5.0g

K2HPO4 4.0g Cornstarch 1.0g

KH2PO4 1.0g

pH 7.2 ± 0.2 at 25°C

Preparation of GC Medium Base: Add components of GC

medi-um base and the hemoglobin to distilled/deionized water and bring vol-ume to 1.0L Mix thoroughly Gently heat until boiling Autoclave for

15 min at 15 psi pressure–121°C Cool to 45°–50°C

CNVT Inhibitor:

Colistin sulfate 7.5mg Trimethoprim lactate 5.0mg Vancomycin 3.0mg Nystatin 12,500U

Preparation of CNVT Inhibitor: Add components to distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Filter ster-ilize

Preparation of Medium: To 980.0mL of cooled sterile GC medium base, aseptically add 10.0mL of sterile CNVT inhibitor and 10.0mL of sterile supplement B Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

especially Neisseria species.

Thayer-Martin Medium, Modified (Modified Thayer-Martin Agar)

GC agar base 720.0mL Hemoglobin solution 250.0mL

GC supplement 30.0mL

pH 7.3 ± 0.2 at 25°C

GC Agar Base:

Special peptone 15.0g Agar 10.0g NaCl 5.0g

K2HPO4 4.0g

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Thayer-Martin Medium, Selective 1709

Cornstarch 1.0g

KH2PO4 1.0g

pH 7.2 ± 0.2 at 25°C

base to distilled/deionized water and bring volume to 720.0mL Mix

thoroughly Gently heat until boiling Autoclave for 15 min at 15 psi

pressure–121°C Cool to 45°–50°C

Hemoglobin 5.0g

GC Supplement:

Yeast autolysate 10.0g

Glucose 1.5g

NaHCO3 0.15g

Colistin sulfate 7.5mg

Trimethoprim lactate 5.0mg

Vancomycin 3.0mg

Nystatin 12,500U

Preparation of GC Supplement: Add components to

distilled/de-ionized water and bring volume to 30.0mL Mix thoroughly Filter

ster-ilize

Preparation of Medium: To 720.0mL of cooled sterile GC agar

base, aseptically add 250.0mL of sterile hemoglobin solution and

30.0mL of sterile GC supplement Mix thoroughly Pour into sterile

Pe-tri dishes or disPe-tribute into sterile tubes

Use: For the selective isolation and cultivation of fastidious

microor-ganisms, especially Neisseria species.

Thayer-Martin Medium, Modified

(Modified Thayer-Martin Agar)

GC agar base 730.0mL

Hemoglobin solution 250.0mL

Vitox supplement 10.0mL

VCNT antibiotic solution 10.0mL

pH 7.3 ± 0.2 at 25°C

GC Agar Base:

Special peptone 15.0g

Agar 10.0g

NaCl 5.0g

K2HPO4 4.0g

Cornstarch 1.0g

KH2PO4 1.0g

pH 7.2 ± 0.2 at 25°C

base in to distilled/deionized water and bring volume to 730.0mL Mix

thoroughly Gently heat until boiling Autoclave for 15 min at 15 psi

pressure–121°C Cool to 45°–50°C

Hemoglobin 5.0g

Preparation of Hemoglobin Solution: Add hemoglobin to dis-tilled/deionized water and bring volume to 250.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Glucose 2.0g L-Cysteine·HCl 0.518g L-Glutamine 0.2g L-Cystine 0.022g Adenine sulfate 0.01g Nicotinamide adenine dinucleotide 5.0mg Cocarboxylase 2.0mg Guanine·HCl 0.6mg Fe(NO3)3·6H2O 0.4mg

Vitamin B12 0.2mg Thiamine·HCl 0.06mg

Preparation of Vitox Supplement: Add components to distilled/ deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

VCNT Antibiotic Solution:

Colistin methane sulfonate 7.5mg Trimethoprim lactate 5.0mg Vancomycin 3.0mg Nystatin 12,500U

Preparation of VCNT Antibiotic Solution: Add components to distilled/deionized water and bring volume to 10.0mL Mix

thorough-ly Filter sterilize

Preparation of Medium: To 730.0mL of cooled, sterile GC agar base, aseptically add 250.0mL of sterile hemoglobin solution, 10.0mL

of sterile Vitox supplement, and 10.0mL of VCNT antibiotic solution Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Thayer-Martin Medium, Selective

GC agar base 730.0mL Hemoglobin solution 250.0mL Vitox supplement 10.0mL VCN antibiotic solution 10.0mL

pH 7.3 ± 0.2 at 25°C

GC Agar Base:

Special peptone 15.0g Agar 10.0g NaCl 5.0g

K2HPO4 4.0g Cornstarch 1.0g

KH2PO4 1.0g

pH 7.2 ± 0.2 at 25°C

base to distilled/deionized water and bring volume to 730.0mL Mix thoroughly Gently heat until boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

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1710 Thayer-Martin Selective Agar

Hemoglobin 5.0g

Glucose 2.0g

L-Glutamine 0.2g

L-Cystine 0.022g

Adenine sulfate 0.01g

Nicotinamide adenine dinucleotide 5.0mg

Cocarboxylase 2.0mg

Guanine·HCl 0.6mg

Fe(NO3)3·6H2O 0.4mg

Vitamin B12 0.2mg

Thiamine·HCl 0.06mg

sterilize

VCN Antibiotic Solution:

Colistin methane sulfonate 7.5mg

Vancomycin 3.0mg

Nystatin 12,500U

Preparation of VCN Antibiotic Solution: Add components to

distilled/deionized water and bring volume to 10.0mL Mix

thorough-ly Filter sterilize

Preparation of Medium: To 730.0mL of cooled, sterile GC agar

base, aseptically add 250.0mL of sterile hemoglobin solution, 10.0mL

of sterile Vitox supplement, and 10.0mL of VCN antibiotic solution

Mix thoroughly Pour into sterile Petri dishes or distribute into sterile

tubes

Thayer-Martin Selective Agar

Agar 12.0g

Hemoglobin 10.0g

Pancreatic digest of casein 7.5g

Selected meat peptone 7.5g

NaCl 5.0g

K2HPO4 4.0g

Cornstarch 1.0g

KH2PO4 1.0g

Supplement solution 10.0mL

VCN inhibitor 10.0mL

pH 7.2 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD

Di-agnostic Systems

Supplement Solution:

Glucose 100.0g

L-Glutamine 10.0g L-Cystine 1.1g Adenine 1.0g Nicotinamide adenine dinucleotide 0.25g Vitamin B12 0.1g Thiamine pyrophosphate 0.1g Guanine·HCl 0.03g Fe(NO3)3·6H2O 0.02g

Thiamine·HCl 3.0mg

Source: The supplement solution IsoVitaleX® enrichment is available from BD Diagnostic Systems This enrichment may be replaced by supplement VX from BD Diagnostic Systems

Preparation of Supplement Solution: Add components to dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly Filter sterilize

VCN Inhibitor:

Colistin 7.5mg Vancomycin 3.0mg Nystatin 12,500U

Preparation of VCN Inhibitor: Add components to distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Filter ster-ilize

Preparation of Medium: Add components, except supplement solu-tion and VCN inhibitor, to distilled/deionized water and bring volume 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 VCN inhibitor and sterile supplement solution Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the selective isolation of Neisseria gonorrhoeae and

Neis-seria meningitidis from specimens containing mixed flora of bacteria

and fungi

Thermincola Medium

Composition per liter:

NH4Cl 1.0g MgCl2·6H2O 0.33g

KH2PO4 0.5g KCl 0.33g Na-acetate 0.2g CaCl2·2H2O 0.1g Resazurin 0.5mg Vitamin solution 20.0mL Bicarbonate solution 10.0mL Carbonate solution 10.0mL Sulfide solution 10.0mL Yeast extract solution 10.0mL Wolfe's mineral elixir 1.0mL

pH 8.0 ± 0.2 at 25°C

Sulfide Solution :

Na2S·9H2O 1.0g

Preparation of Sulfide Solution: Add Na2S·9H2O to distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C Cool to room temperature

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Thermoacetogenium phaeum Medium 1711

Bicarbonate Solution :

NaHCO3 0.5g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Sparge

with gas mixture of 80% N2 + 20% CO2 Filter sterilize

Carbonate Solution :

Na2CO3 0.5g

Preparation of Carbonate Solution: Add Na2CO3 to

distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Sparge

with gas mixture of 80% N2 + 20% CO2 Filter sterilize

Yeast Extract Solution:

Yeast extract 0.2g

Preparation of Yeast Extract Solution: Add yeast extract to

Sparge with 100% N2 gas Autoclave for 15 min at 15 psi pressure–

121°C

Wolfe’s Mineral Elixir:

MgSO4·7H2O 30.0g

NaCl 10.0g

MnSO4·2H2O 5.0g

(NH4)2NiSO4·6H2O 2.8g

CoCl2·6H2O 1.8g

ZnSO4·7H2O 1.8g

FeSO4·7H2O 1.0g

CaCl2·2H2O 1.0g

KAl(SO4)2·12H2O 0.18g

CuSO4·5H2O 0.1g

H3BO3 0.1g

Na2MoO4·2H2O 0.1g

Na2SeO4 0.1g

Na2WO4·2H2O 0.1g

Preparation of Wolfe’s Mineral Elixir: Adjust pH of 1.0L of

dis-tilled/deionized water to 1.0 with dilute H2SO4 Add components one

at a time Mix thoroughly to dissolve

Riboflavin 5.0mg

Lipoic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly Sparge

with 80% H2 + 20% CO2 Filter sterilize

Preparation of Medium: Add components, except vitamin

solu-tion, to distilled/deionized water and bring volume to 950.0mL Mix

thoroughly Gently heat and bring to boiling Boil for 1 min Cool to

room temperature while sparging with 100% N2 gas Dispense into

cul-ture vessels under an atmostphere of 100% CO (carbon monoxide)

Culture vessels should be filled to approximately 20% Autoclave for

15 min at 15 psi pressure–121°C Cool to room temperature

Aspetical-ly add carbonate, bicarbonate, vitamin, and sulfide solutions Mix

thor-oughly Adjust the pH to 8.0 with sterile, anoxic 1N HCl Prior to

inoculation aseptically add yeast extract solution

Use: For the cultivation of Thermincola carboxydiphila

Thermoacetogenium phaeum Medium

KHCO3 3.5g

NH4Cl 1.0g NaCl 0.6g

KH2PO4 0.3g MgCl2·6H2O 0.1g CaCl2·2H2O 0.08g Resazurin 0.5mg Sodium pyruvate solution 50.0mL Vitamin solution 10.0mL

Na2S·9H2O solution 10.0mL Cysteine solution 10.0mL Trace elements solution 1.0mL Selenite-tungstate solution 1.0mL

pH 7.0–7.1 at 25°C

Sodium Pyruvate Solution:

Sodium pyruvate 5.0g

Preparation of Sodium Pyruvate Solution: Add sodium pyru-vate to distilled/deionized water and bring volume to 50.0mL Mix thoroughly Sparge with 100% N2 Filter sterilize

Selenite-Tungstate Solution Compositionper liter:

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

to distilled/deionized water and bring volume to 1.0L Mix thoroughly Sparge with 100% N2 Filter sterilize

Na 2 S·9H 2 O Solution:

Na2S·9H2O 0.3g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Cysteine Solution:

L-Cysteine·HCl·H2O 0.3g

Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL Mix

thorough-ly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure– 121°C

Trace Elements Solution SL-10:

FeCl2·4H2O 1.5g

Na2-EDTA 0.5g CoCl2·6H2O 190.0mg MnCl2·4H2O 100.0mg

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1712 Thermoacidurans Agar

ZnCl2 70.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

Preparation of Trace Elements Solution SL-10: Add FeCl2·4H2O

to 10.0mL of HCl solution Mix thoroughly Add distilled/deionized

water and bring volume to 1.0L Add remaining components Mix

thor-oughly Adjust pH to 6.5 Sparge with 100% N2 Autoclave for 15 min

Riboflavin 5.0mg

Lipoic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

deionized water and bring volume to 1.0L Mix thoroughly Sparge

with 80% H2 + 20% CO2 Filter sterilize

Preparation of Medium: Prepare and dispense medium under 80%

N2 + 20% CO2 gas atmosphere Add components, except KHCO3,

sodi-um pyruvate solution, cysteine solution, and Na2S·9H2O solution, to

distilled/deionized water and bring volume to 930.0mL Mix thoroughly

Gently heat and bring to boiling Boil for 10 min Cool to room

temper-ature while sparging with 80% N2 + 20% CO2 Add 3.5g KHCO3 Mix

thoroughly while sparging with 80% N2 + 20% CO2 gas atmosphere

Autoclave for 15 min at 15 psi pressure–121°C Aseptically and

anaero-bically add 50.0mL sodium pyruvate solution, 10.0mL cysteine

solu-tion, and 10.0mL Na2S·9H2O solution Mix thoroughly Final pH is 7.0–

7.1 Aseptically and anaerobically distribute into sterile tubes or bottles

Use: For the cultivation of Thermacetogenium phaeum.

Thermoacidurans Agar

Agar 20.0g

Yeast extract 5.0g

Proteose peptone 5.0g

Glucose 5.0g

K2HPO4 4.0g

pH 5.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD

Di-agnostic Systems

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 Bacillus thermoacidurans

from food products

Thermoacidurans HiVeg Agar

Agar 20.0g Glucose 5.0g Plant peptone No 3 5.0g Yeast extract 5.0g

K2HPO4 4.0g

pH 5.0 ± 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 12 psi pressure–118°C Do not overheat Pour into sterile Petri dishes or leave in tubes

Use: For the isolation and cultivation of Bacillus thermoacidurans

from food products

Thermoactinomyces dichotomicus Medium

Maize, split 50.0g Agar 20.0g Starch 10.0g NaCl 5.0g Peptone 5.0g CaCl2 0.5g

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add split maize (crushed corn) to 1.0L of boiling water Steam for 30 min Filter through Whatman #1 filter pa-per Add remaining components to maize filtrate Gently heat and bring

to boiling Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation of Thermoactinomyces dichotomicus

Thermoactinomyces Medium

Polypeptone™ 30.0g Agar 15.0g Glycerol 2.0g

L-Asparagine 1.0g

K2HPO4 1.0g Vitamin B solution 10.0mL Trace salts solution 1.0mL

pH 7.2 ± 0.2 at 25°C

Trace Salts Solution:

FeSO4·7H2O 0.1g MnCl2·4H2O 0.1g ZnSO4·7H2O 0.1g

Preparation of Trace Salts Solution: Add components to dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly

Vitamin B Solution:

Composition per 200.0mL:

Thiamine-HCl 10.0mg Riboflavin 10.0mg Nicotinate 10.0mg Pyridoxine-HCl 10.0mg

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Thermoanaerobacter ethanolicus Medium 1713

Inositol 10.0mg

Calcium pantothenate 10.0mg

p-Aminobenzoate 10.0mg

D-Biotin 5.0mg

Preparation of Vitamin B Solution: Add components to distilled/

sterilize

Preparation of Medium: Add components, except vitamin B

solu-tion, to distilled/deionized water and bring volume to 900.0mL Mix

thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to

room temperature Aseptically add 100.0mL vitamin B solution

Asep-tically distribute into sterile tubes or flasks

Use: For the cultivation of Thermoactinomyces peptonophilus.

Agar 20.0g

Malt extract 10.0g

Yeast extract 4.0g

Glucose 4.0g

pH 7.3 ± 0.2 at 25°C

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 Thermoactinomyces

sac-chari.

Composition per liter:

Polypeptone 30.0g

Agar 15.0g

Glycerol 2.0g

L-Asparagine 1.0g

KH2PO4 1.0g

Vitamin solution 10.0mL

Trace elements solution 1.0mL

pH 7.2 ± 0.2 at 25°C

Riboflavin 10.0mg

Inositol 10.0mg

Biotin 5.0mg

sterilize

FeSO4·7H2O 0.1g

MnCl2·4H2O 0.1g

ZnSO4·7H2O 0.1g

Preparation of Trace Elements Solution: Add components to dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly

Preparation of Medium: Add components, except vitamin solu-tion, to distilled/deionized water and bring volume to 990.0mL Mix thoroughly Adjust the pH to 7.2 Autoclave for 15 min at 15 psi pres-sure–121°C Cool to room temperature Aspetically add vitamin solu-tion Mix thoroughly Aseptically dispense into culture vessels

Use: For the cultivation of Thermoactinomyces spp.

Thermoactinopolyspora Medium

Maltose 20.0g Agar 15.0g Papaic digest of soybean meal 15.0g Yeast extract 2.0g

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to tap water and bring volume to 1.0L Mix thoroughly Gently heat and bring to boiling Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation and maintenance of Thermoactinomyces and

Thermoactinopolyspora species.

Thermoanaerobacter ethanolicus Medium

Glucose 8.0g

Na2HPO4·12H2O 4.2g Yeast extract 2.0g

KH2PO4 1.5g

NH4Cl 0.5g MgCl2·6H2O 0.18g Reducing solution 40.0mL Wolfe’s modified mineral solution 5.0mL Resazurin (0.1% solution) 1.0mL Vitamin solution 0.5mL

Caution: This medium contains Na2S, and H2S production will occur, especially upon prolonged boiling H2S is hazardous and preparation of this medium should be done in a chemical fume hood

Reducing Solution:

Composition per 200.0mL:

Cysteine·HCl·H2O 2.5g

Na2S·9H2O 2.5g

NaOH (0.2N solution) 200.0mL

Preparation of Reducing Solution: Gently heat the NaOH solu-tion and bring to boiling Gas with 95% N2 + 5% H2 Cool to room tem-perature Add the cysteine·HCl·H2O and Na2S·9H2O Anaerobically distribute into tubes Cap with rubber stoppers Autoclave for 15 min

Pyridoxine·HCl 0.1g

Calcium pantothenate 0.05g Nicotinic acid 0.05g Thioctic acid 0.05g Biotin 0.02g Folic acid 0.02g Riboflavin 5.0mg

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1714 Thermoanaerobacter subterraneus Medium

Thiamine·HCl 5.0mg

Vitamin B12 1.0mg

deionized water and bring volume to 500.0mL Mix thoroughly Store

solution in the dark at −10°C

Wolfe’s Modified Mineral Solution:

MgSO4·7H2O 3.0g

Nitrilotriacetic acid 1.5g

NaCl 1.0g

MnSO4·H2O 0.5g

CaCl2 (anhydrous) 0.1g

Co(NO3)2·6H2O 0.1g

FeSO4·7H2O 0.1g

ZnSO4·7H2O 0.1g

AlK(SO4)2 (anhydrous) 0.01g

CuSO4·5H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Na2SeO3 (anhydrous) 1.0mg

nitrilotriacetic acid to 500.0mL of distilled/deionized water Dissolve

by adjusting pH to 6.5 with KOH Add remaining components Add

distilled/deionized water to 1.0L

Preparation of Medium: Add components, except reducing

solu-tion, to distilled/deionized water and bring volume to 1.0L Gently heat

and bring to boiling under 95% N2 + 5% H2 Continue boiling until

or changes from blue to pink Add the reducing solution The pink

col-or will disappear, indicating that the solution has been reduced

Distribute into tubes or flasks under 95% N2 + 5% H2 using anerobic

techniques Cap tubes with rubber stoppers Autoclave for 15 min at 15

psi pressure–121°C

Use: For the cultivation and maintenance of thermophilic anaerobes

such as Thermoanaerobacter species and some Clostridium species.

Thermoanaerobacter subterraneus Medium

Yeast extract 2.0g

MgCl2·6H2O 1.0g

NH4Cl 1.0g

NaCl 0.6g

Cysteine-HCl·H2O 0.5g

K2HPO4 0.3g

KH2PO4 0.3g

KCl 0.2g

CaCl2·2H2O 0.1g

Resazurin 0.5mg

D-Glucose solution 30.0mL

NaHCO3 solution 20.0mL

Trace mineral solution 10.0mL

Na2S2O3 solution 10.0mL

Na2S·9H2O solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Na 2 S·9H 2 O Solution :

Na2S·9H2O 0.45g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to

Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C Cool

to room temperature

NaHCO3 4.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to distilled/de-ionized water and bring volume to 20.0mL Mix thoroughly Sparge with 80% N2 + 20% CO2 Filter sterilize

Glucose Solution:

D-Glucose 4.0g

Preparation of Glucose Solution: Add D-glucose to distilled/de-ionized water and bring volume to 30.0mL Mix thoroughly Sparge with 100% N2 Filter sterilize

Na 2 S 2 O 3 Solution :

Na2S2O3·5H2O 2.5g

Preparation of Na 2 S 2 O 3 Solution : Add Na2S2O3·5H2O to distilled/ deionized water and bring volume to 10.0mL Mix thoroughly Auto-clave under 100% N2 for 15 min at 15 psi pressure–121°C Cool to room temperature

MgSO4·7H2O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO4·2H2O 0.5g CoSO4·7H2O 0.18g ZnSO4·7H2O 0.18g CaCl2·2H2O 0.1g FeSO4·7H2O 0.1g NiCl2·6H2O 0.025g KAl(SO4)2·12H2O 0.02g

H3BO3 0.01g

Na2MoO4·4H2O 0.01g CuSO4·5H2O 0.01g

Na2SeO3·5H2O 0.3mg

Preparation of Trace Elements Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water Dissolve by adjusting pH

to 6.5 with KOH Add remaining components Add distilled/deionized water to 1.0L Mix thoroughly

Preparation of Medium: Prepare and dispense medium under 80%

N2 + 20% CO2 gas atmosphere Add components, except NaHCO3 solu-tion, glucose solusolu-tion, Na2S·9H2O solution, and Na2S2O3 solution, to distilled/deionized water and bring volume to 930.0mL Mix thoroughly Sparge with 80% N2 + 20% CO2 Distribute into sterile tubes or bottles Autoclave for 15 min at 15 psi pressure–121°C Aseptically and anaero-bically per 1.0L of medium add 20.0mL NaHCO3 solution, 30.0mL glu-cose solution, 10.0mL Na2S·9H2O solution, and 10.0mL Na2S2O3 solution Mix thoroughly The final pH should be 7.0

Use: For the cultivation of Thermoanaerobacter subterraneus.

Thermoanaerobacter sulfurophilus Medium

Composition per 1055.0mL:

Sulfur, powdered 10.0g

NH4Cl 0.33g

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