Handbook of Microbiological Media, Fourth Edition part 130 pot

0.05g Folic acid...4.0μg Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L.. 0.01mg Biotin ...1.0μg Preparation of Medium: Add compon

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Neurospora Minimal Medium 1285

Vitamin Solution:

Compositionper liter:

Ribonucleic acid, alkali hydrolyzed 0.5g

Inositol 0.4g

Choline 0.2g

Nicotinamide 0.2g

Pantothenic acid 0.2g

Thiamine 0.1g

p-Aminobenzoic acid 0.05g

Pyridoxine 0.05g

Riboflavin 0.05g

Folic acid 4.0μg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly Filter

ster-ilize

Preparation of Medium: Add components, except vitamin

solu-tion, to distilled/deionized water and bring volume to 990.0mL Mix

thoroughly Gently heat and bring to boiling Autoclave for 15 min at

15 psi pressure–121°C Cool to 45°–50°C Aseptically add 10.0mL of

sterile vitamin solution Mix thoroughly Pour into sterile Petri dishes

or distribute into sterile tubes

Use: For the cultivation of Neurospora species on complete medium.

Neurospora Medium

Sucrose 15.0g

Ammonium tartrate 5.0g

KH2PO4 1.0g

NH4NO3 1.0g

MgSO4·7H2O 0.5g

CaCl2 0.1g

NaCl 0.1g

ZnCl2 2.0mg

FeCl3 0.2mg

Cu Cl2 0.1mg

MnCl2 0.02mg

Na2MoO4·2H2O 0.02mg

H3BO3 0.01mg

Biotin 1.0μg

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 Neurospora species on minimal medium.

Neurospora Minimal Medium

Sucrose 20.0g

Ammonium tartrate 5.0g

KH2PO4 1.0g

NaNO3 1.0g

MgSO4·7H2O 0.5g

CaCl2·2H2O 0.1g

NaCl 0.1g

ZnSO4·7H2O 5.5mg

FeSO4·7H2O 0.54mg

CuSO4·5H2O 0.39mg

MnSO4·4H2O 0.063mg

H3BO3 0.057mg

Na2MoO4·2H2O 0.05mg Biotin 5.0μg

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 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 Neurospora species and the detection of

Neurospora mutants.

Neurospora Minimal Medium

Sucrose 20.0g Agar 20.0g

KH2PO4 5.0g Trisodium citrate·2H2O 2.5g

NH4NO3 2.0g MgSO4·7H2O 0.2g CaCl2·2H2O 0.1g Sucrose-agar solution 200.0mL Biotin solution 1.0mL Trace elements solution 0.1mL

pH 5.8 ± 0.2 at 25°C

Sucrose-Agar Solution:

Composition per 200.0mL:

Sucrose 20.0g Agar 20.0g

Preparation of Sucrose-Agar Solution: Add components to dis-tilled/deionized water and bring volume to 200.0mL Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pres-sure–121°C Cool to 45°–50°C

Biotin Solution:

Biotin 50.0μg

Preparation of Biotin Solution: Add biotin to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Trace Elements Solution:

Composition per 100.0mL

Citric acid·H2O 5.0g ZnSO4·7H2O 5.0g Fe(NH4)2(SO4)2·6H2O 1.0g CuSO4·5H2O 0.25g MnSO4·H2O 0.05g

H3BO3 0.05g

Na2MoO4·2H2O 0.05g

Preparation of Trace Elements Solution: Add components one

at a time to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Filter sterilize Add 1.0mL of chloroform as a preser-vative Store at room temperature

Preparation of Medium: Add trisodium citrate·2H2O, KH2PO4,

NH4NO3, MgSO4·7H2O, and CaCl2·2H2O to distilled/deionized water and bring volume to 800.0mL Make sure that one component is dis-solved completely before adding the next one This is conveniently done in a Fernbach flask on a shaker Mix thoroughly Filter sterilize Warm solution to 45°–50°C Add 200.0mL of sterile sucrose-agar so-lution, 1.0mL of sterile biotin soso-lution, and 0.1mL of sterile trace ele-ments solution Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

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1286 Nevskia Medium

Use: For the cultivation and maintenance of Cochliobolus sativus,

Fusarium solani, Neurospora crassa, and Neurospora sitophila

Neutral Red Broth

See: LICNR Broth Nevskia Medium

(DSMZ Medium 828) Compositionper liter:

Na-lactate 0.56g

MgSO4·7H2O 0.05g

CaCl2·2H2O 0.014g

Potassium phosphate buffer, 1M, pH 7 27.5mL

Trace elements solution SL-9 0.5mL

Seven vitamin solution 0.5mL

pH 7.1 ± 0.2 at 25°C

Trace Elements Solution SL-9:

Nitrilotriacetic acid 12.8g

FeCl2·4H2O 3.5g

CoCl2·6H2O 190.0mg

MnCl2·4H2O 100.0mg

ZnCl2 70.0mg

NiCl2·6H2O 24.0mg

H3BO3 6.0mg

CuCl2·2H2O 2.0mg

HCl (25% solution) 10.0mL

Preparation of Trace Elements Solution SL-9: 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 6.0 Sparge with 80% N2 + 20% CO2 Autoclave

for 15 min at 15 psi pressure–121°C

Seven Vitamin Solution:

Pyridoxine hydrochloride 300.0mg

Thiamine-HCl·2H2O 200.0mg

Nicotinic acid 200.0mg

Vitamin B12 100.0mg

Calcium pantothenate 100.0mg

p-Aminobenzoic acid 80.0mg

D(+)-Biotin 20.0mg

Preparation of Seven Vitamin Solution: Add components to

dis-tilled/deionized water and bring volume to 1.0L Sparge with 100% N2

Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except seven vitamin

solution, to distilled/deionized water and bring volume to 999.5mL

Mix thoroughly Adjust pH to 7.1 Autoclave for 15 min at 15 psi

pres-sure–121°C Cool to room temperature Aseptically add 0.5mL seven

vitamin solution Mix thoroughly Aseptically distribute into sterile

tubes or flasks

Use: For the cultivation of Nevskia ramosa.

New York City Medium Compositionper liter:

NYC basal medium 640.0mL

Horse blood cells 200.0mL

Horse plasma, citrated 120.0mL

Yeast dialysate 25.0mL

Glucose solution 10.0mL Antibiotic VCNT solution 5.0mL

pH 7.4 ± 0.2 at 25°C

NYC Basal Medium:

Compositionper 640.0mL:

Solution 1 400.0mL Solution 3 200.0mL Solution 2 40.0mL

Preparation of NYC Basal Medium: Combine solution 1, solu-tion 2, and solusolu-tion 3 Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Solution 1:

Agar 20.0g

Preparation of Solution 1: Add agar to distilled/deionized water and bring volume to 400.0mL Mix thoroughly Melt agar in autoclave for 10 min at 0 psi pressure–100°C Cool to 45°–50°C

Solution 2:

Cornstarch 1.0g

Preparation of Solution 2: Add cornstarch to distilled/deionized water and bring volume to 40.0mL Mix thoroughly Warm to 45°– 50°C

Solution 3:

Proteose peptone No 3 15.0g NaCl 5.0g

K2HPO4 4.0g

KH2PO4 1.0g

Preparation of Solution 3: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring

to boiling Cool to 45°–50°C

Horse Blood Cells:

Horse blood cells, sedimented 6.0mL

Preparation of Horse Blood Cells: Cow blood may be used in-stead of horse blood but do not use sheep blood Use cells freshly packed by sedimentation Do not pack by centrifugation Aseptically add 6.0mL of sedimented blood cells to 200.0mL of sterile distilled/de-ionized water Mix thoroughly

Horse Plasma, Citrated:

Composition per 6.0L:

Horse blood 5400.0mL Citrate solution 600.0mL

Preparation of Horse Plasma, Citrated: Place 600.0mL of ster-ile citrate solution into a receiving bottle Draw horse blood to the 6.0L mark Allow cells to sediment out Aseptically remove plasma

Citrate Solution:

Sodium citrate 150.0g NaCl 81.13g

Preparation of Citrate Solution: Add components to distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Filter sterilize

Glucose Solution:

D-Glucose 5.0g

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Niacin Assay HiVeg Medium 1287

Preparation of Glucose Solution: Add D-glucose to

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

for 10 min at 10 psi pressure–115°C Cool to 45°–50°C

Yeast Dialysate:

Baker’s yeast, fresh 908.0g

Preparation of Yeast Dialysate: Add fresh baker’s yeast to

2500.0mL of distilled/deionized water Mix thoroughly Autoclave for

10 min at 15 psi pressure–121°C Cool to 25°C Put into dialysis

tub-ing Dialyze against 2.0L of distilled/deionized water for 48 hr at 4°C

Antibiotic VCNT Solution:

Colistin 7.5mg

Trimethorprim lactate 3.0mg

Vancomycin·HCl 2.0mg

Nystatin 12.5U

Preparation of Antibiotic Solution: Add components to distilled/

deionized water and bring volume to 5.0mL Mix thoroughly Filter

sterilize

Preparation of Medium: Have all solutions prepared and at 45°–

50°C Aseptically combine components Mix thoroughly Pour into

sterile Petri dishes

Use: For the isolation and cultivation of pathogenic Neisseria species.

Used as a transport medium for urogenital and other clinical specimens

For the isolation and presumptive identification of Mycoplasmatales,

including large-colony species (Mycoplasma pneumoniae) and

T–myco-plasmas from urogenital specimens

New York City Medium, Modified

NYC basal medium 840.0mL

α-Gamma horse serum (Flow Labs) 120.0mL

Yeast dialysate 25.0mL

Glucose solution 10.0mL

Antibiotic LCNT solution 5.0mL

pH 7.4 ± 0.2 at 25°C

NYC Basal Medium:

Horse blood 5400.0mL

Solution 1 600.0mL

Solution 3 200.0mL

Solution 2 40.0mL

Preparation of NYC Basal Medium: Combine solution 1,

solu-tion 2, and solusolu-tion 3 Mix thoroughly Autoclave for 15 min at 15 psi

pressure–121°C Cool to 45°–50°C

Solution 1:

Agar 20.0g

Preparation of Solution 1: Add agar to distilled/deionized water

and bring volume to 600.0mL Mix thoroughly Melt agar in autoclave

for 10 min at 0 psi pressure–100°C Cool to 45°–50°C

Solution 2:

Cornstarch 1.0g

Preparation of Solution 2: Add cornstarch to distilled/deionized

water and bring volume to 40.0mL Mix thoroughly Warm to 45°–

50°C

Solution 3:

Proteose peptone No 3 15.0g NaCl 5.0g

K2HPO4 4.0g

KH2PO4 1.0g

Preparation of Solution 3: Add components to distilled/deionized water and bring volume to 200.0mL Mix thoroughly Gently heat and bring to boiling Cool to 45°–50°C

Glucose Solution:

D-Glucose 5.0g

Preparation of Glucose Solution: Add glucose to distilled/deion-ized water and bring volume to 10.0mL Mix thoroughly Autoclave for

10 min at 10 psi pressure–115°C Cool to 45°–50°C

Yeast Dialysate:

Baker’s yeast, fresh 908.0g

Preparation of Yeast Dialysate: Add fresh Baker’s yeast to 2500.0mL of distilled/deionized water Mix thoroughly Autoclave for

10 min at 15 psi pressure–121°C Cool to 25°C Put into dialysis tub-ing Dialyze against 2.0L of distilled/deionized water for 48 hr at 4°C

Antibiotic LCNT Solution:

Colistin 7.5mg Lincomycin·HCl 4.0mg Trimethorprim lactate 3.0mg Nystatin 12.5U

Preparation of Antibiotic LCNT Solution: Add the components

to distilled/deionized water and bring volume to 5.0mL Mix

thorough-ly Filter sterilize the solution

Preparation of Medium: Have all solutions prepared and at 45°– 50°C Aseptically combine components Mix thoroughly Pour into sterile Petri dishes

Use: For the isolation and cultivation of pathogenic Neisseria species.

Used as a transport medium for urogenital and other clinical specimens For the isolation and presumptive identification of Mycoplasmatales,

including large-colony species (Mycoplasma pneumoniae) and

T–myco-plasmas from urogenital specimens

Niacin Assay HiVeg Medium Compositionper liter:

Glucose 40.0g Sodium acetate 20.0g Plant acid hydrolysate, vitamin free 12.0g

KH2PO4 1.0g

K2HPO4 1.0g

L-Cystine 0.4g MgSO4 0.4g

DL-Tryptophan 0.2g Adenine sulfate 0.02g FeSO4 0.02g Guanine hydrochloride 0.02g MnSO4 0.02g Uracil 0.02g NaCl 0.02g Pyridoxine hydrochloride 0.4mg Riboflavin (Vitamin B2) 0.4mg

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1288 Niacin Assay Medium

Thiamine hydrochloride 0.2mg

p-Aminobenzoic acid (PABA) 0.1mg

Biotin 0.08mg

pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from

Hi-Media

water and bring volume to 1.0L Mix thoroughly Gently heat and bring

to boiling Continue boiling for 2–3 min Distribute into tubes in 5.0mL

volumes Add standard solution or test solutions to each tube Adjust

the volume of each tube to 10.0mL with distilled/deionized water

Au-toclave for 10 min at 15 psi pressure–121°C

Use: For the microbiological assay of nicotinic acid or nicotinamide

(niacin) using Lactobacillus plantarum as the test organism.

Niacin Assay Medium Composition per liter:

Glucose 40.0g

Sodium acetate 20.0g

Vitamin assay casamino acids 12.0g

K2HPO4 1.0g

KH2PO4 1.0g

L-Cystine 0.4g

MgSO4·7H2O 0.4g

DL-Tryptophan 0.2g

Adenine sulfate 0.02g

FeSO4·5H2O 0.02g

Guanine·HCl 0.02g

MnSO4·H2O 0.02g

NaCl 0.02g

Uracil 0.02g

Pyridoxine·HCl 0.4mg

Riboflavin 0.4mg

Thiamine·HCl 0.2mg

Biotin 0.8μg

pH 6.7 ± 0.2 at 25°C

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

Di-agnostic Systems

to boiling Continue boiling for 2–3 min Distribute into tubes in 5.0mL

volumes Add standard solution or test solutions to each tube Adjust

the volume of each tube to 10.0mL with distilled/deionized water

Use: For the microbiological assay of nicotinic acid or nicotinamide

(niacin) using Lactobacillus plantarum as the test organism.

Nickels and Leesment Agar, Modified

Part 1 750.0mL

Part 2 100.0mL

Part 3 100.0mL

Part 4 50.0mL

pH 6.65 ± 0.2 at 25°C

Part 1:

Pancreatic digest of casein 20.0g Lactose 10.0g Yeast extract 5.0g NaCl 4.0g Gelatin 2.5g Sodium citrate 2.0g

Preparation of Part 1: Add components to distilled/deionized wa-ter and bring volume to 750.0mL Mix thoroughly Adjust pH to 6.65

Part 2:

Nonfat dry milk 50.0g

Preparation of Part 2: Add nonfat dry milk to distilled/deionized

water and bring volume to 500.0mL Inoculate with Lactobacillus

bul-garicus Incubate at 20°C for 24 hr Centrifuge at 5000 rpm for 10 min

to separate the curd Collect the supernatant solution Autoclave for 15 min at 15 psi pressure–121°C Store at 4°C

Part 3:

Calcium citrate 13.3g Carboxymethylcellulose 0.8g

Preparation of Part 3: Combine the calcium citrate and carboxy-methylcellulose in a mortar and grind until a fine powder Add powder

to 100.0mL of hot distilled/deionized water Mix thoroughly Filter through cheesecloth

Part 4:

Calcium lactate 8.0g

Preparation of Part 4: Add calcium lactate to distilled/deionized water and bring volume to 50.0mL Mix thoroughly Gently heat until dissolved

Preparation of Medium: Prepare each of the four parts separately Autoclave each part for 15 min at 15 psi pressure–121°C Aseptically combine part 1, part 2, part 3, and part 4 Mix thoroughly Pour into sterile Petri dishes Swirl flask while dispensing medium

Use: For the isolation and cultivation of acid-producing microorgan-isms from foods

Nickerson Medium

See: BiGGY Agar

Nicotinic Acid Medium (DSMZ Medium 152) Compositionper liter:

Yeast extract 10.0g Nicotinic acid 5.0g Cysteine-HCl·H2O 0.5g NaHCO3 0.4g NaCl 80.0mg

KH2PO4 40.0mg

K2HPO4 40.0mg CaCl2·2H2O 8.0mg MgSO4·7H2O 8.0mg Distilled water 1000.0mL

pH 8.2 ± 0.2 at 25°C

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Nitrate Assimilation Medium, Auxanographic Method for Yeast Identification 1289

Preparation of Medium: Add nicotinic acid to distilled/deionized

water and bring volume to 1.0L Gently heat and bring to boiling Boil

with mixing until nicotinic acid is fully dissolved Cool to 25°C Add

remaining components Readjust volume to 1.0L with

distilled/deion-ized water Mix thoroughly Adjust pH to 8.2 Distribute into tubes or

flasks under 100% nitrogen atmosphere Autoclave for 15 min at 15 psi

pressure–121°C

Use: For the cultivation and maintenance of Eubacterium barkeri.

Niger Seed Agar

See: Bird Seed Agar

Niger Seed Salts Agar with Yeast Extract

Niger seeds 50.0g

Agar 20.0g

Glucose 1.0g

Yeast extract 1.0g

KH2PO4 1.0g

MgSO4·7H2O 0.5g

Preparation of Medium: Add Niger seeds to 250.0mL of distilled/

deionized water in a blender container Soak the seeds at 60°–70°C for

1.5 hr Blend Filter through Whatman #1 filter paper Reserve filtrate

Combine filtrate with remaining components Add distilled/deionized

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 Arthroderma otae and

Arthroderma vanbreuseghemii

NIH Agar Composition per liter:

Pancreatic digest of casein 15.0g

Agar 15.0g

Glucose 5.5g

Yeast extract 5.0g

NaCl 2.5g

L-Cystine 0.05g

pH 7.1 ± 0.2 at 25°C

Di-agnostic Systems

water and bring volume to 1.0L Mix thoroughly Gently heat while

stirring 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 microorganisms isolated

from sterility testing of biological products Also used as a solid

medium for sterility testing

NIH Thioglycolate Broth Composition per liter:

Glucose 5.5g

Yeast extract 5.0g

NaCl 2.5g

L-Cystine 0.5g Sodium thioglycolate 0.5g

pH 7.1 ± 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 while stirring and bring to boiling Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

Use: For sterility testing of biological products that are turbid or oth-erwise cannot be cultivated in fluid thioglycolate broth because of its viscosity

Nine K Medium (9K Medium) Compositionper liter:

FeSO4·7H2O 50.0g (NH4)2SO4 3.0g Ca(NO3)2 1.0g

K2HPO4 0.5g MgSO4·7H2O 0.5g KCl 0.1g

H2SO4, 10N 1.0mL

pH 3.0 ± 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 3.0 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C

Use: For the cultivation of Thiobacillus ferrooxidans.

Nitrate Agar Composition per liter:

Agar 12.0g Peptone 5.0g Beef extract 3.0g KNO3 1.0g

pH 6.8 ± 0.2 at 25°C

to boiling Distribute into tubes Autoclave for 15 min at 15 psi pres-sure–121°C Allow tubes to cool in a slanted position

Use: For the differentiation of aerobic and facultative Gram-negative microorganisms based on their ability to reduce nitrate Test for nitrates with sulfanilic acid and α-naphthylamine reagents Bacteria that reduce nitrate to nitrite turn the reagents red or pink

Nitrate Assimilation Medium, Auxanographic Method for Yeast Identification Compositionper liter:

Noble agar 20.0g Glucose 10.0g

KH2PO4 1.0g MgSO4·7H2O 0.5g NaCl 0.1g CaCl2·2H2O 0.1g

DL-Methionine 0.02g

DL-Tryptophan 0.02g

L-Histidine·HCl 0.01g

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1290 Nitrate Broth

Inositol 2.0mg

H3BO3 0.5mg

ZnSO4·7H2O 0.4mg

MnSO4·4H2O 0.4mg

Thiamine·HCl 0.4mg

Pyridoxine 0.4mg

Niacin 0.4mg

Riboflavin 0.2mg

FeCl3 0.2mg

Na2MoO4·4H2O 0.2mg

KI 0.1mg

CuSO4·5H2O 0.04mg

Folic Acid 2.0μg

Biotin 2.0μg

pH 4.5 ± 0.2 at 25°C

to boiling Distribute into screw-capped tubes in 20.0mL volumes

Use: For nitrate assimilation tests by the auxanographic method

Nitrate Broth (International Streptomyces Project Medium 8)

(ISP Medium 8) (ATCC Medium 872) Composition per liter:

Peptone 5.0g

Beef extract 3.0g

KNO3 1.0g

pH 7.0 ± 0.2 at 25°C

Di-agnostic Systems

Use: For the differentiation of aerobic and facultative Gram-negative

microorganisms based on their ability to reduce nitrate Test for

nitrates with sulfanilic acid and α-naphthylamine reagents Bacteria

that reduce nitrate to nitrite turn the reagents red or pink

Nitrate Broth Composition per liter:

Pancreatic digest of gelatin 20.0g

KNO3 2.0g

pH 7.2 ± 0.2 at 25°C

Di-agnostic Systems

microorganisms based on their ability to reduce nitrate Test for

Nitrate Broth, Campylobacter

Beef heart, solids from infusion 500.0g Tryptose 10.0g NaCl 5.0g KNO3 2.0g

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0 Dis-tribute 4.0mL volumes into test tubes that contain inverted Durham tubes Autoclave for 15 min at 15 psi pressure–121°C

Use: For the differentiation of Campylobacter species based on their

ability to reduce nitrate

Nitrate Broth, Enriched Compositionper liter:

Pancreatic digest of casein 13.0g NaCl 5.0g Yeast extract 5.0g Heart muscle, solids from infusion 2.0g KNO3 2.0g

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 Distribute into test tubes that contain an inverted Durham tube Autoclave for 15 min at 15 psi pressure–121°C

Use: For the differentiation of aerobic and facultative Gram-negative microorganisms based on their ability to reduce nitrate to nitrite or form N2 gas Test for nitrates with sulfanilic acid and α-naphthylamine reagents Bacteria that reduce nitrate to nitrite turn the reagents red or pink

Nitrate HiVeg Agar Compositionper liter:

Agar 12.0g Plant peptone 5.0g Plant extract 3.0g KNO3 1.0g

pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-Media

to boiling Distribute into tubes Autoclave for 15 min at 15 psi pres-sure–121°C Allow tubes to cool in a slanted position

Use: For the detection of nitrate reduction by bacteria

Nitrate HiVeg Broth Compositionper liter:

Plant peptone 5.0g Plant extract 3.0g KNO3 1.0g

pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Hi-Media

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Nitrate Mineral Salts Medium with Methanol 1291

microorganisms based on their ability to reduce nitrate Test for ntrates

with sulfanilic acid and α-naphthylamine reagents Bacteria that

re-duce nitrate to nitrite turn the reagents red or pink

Nitrate Liquid Medium Compositionper liter:

Solution A 500.0mL

Solution B 250.0mL

Solution C 250.0mL

Solution A:

Mannitol 10.0g

KNO3 0.6g

Na2HPO4·12H2O 0.45g

Na2SO4 0.03g

Preparation of Solution A: Add components to distilled/deionized

water and bring volume to 500.0mL Mix thoroughly Autoclave for 15

min at 15 psi pressure–121°C Cool to 25°C

Solution B:

MgSO4·7H2O 0.12g

CaCl2·6H2O 0.1g

FeCl3·6H2O 0.01g

Preparation of Solution B: Add components to distilled/deionized

water and bring volume to 250.0mL Mix thoroughly Autoclave for 15

min at 15 psi pressure–121°C Cool to 25°C

Solution C:

Thiamine·HCl 0.1mg

Biotin 0.5μg

Preparation of Solution C: Add components to distilled/deionized

water and bring volume to 250.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Aseptically combine 500.0mL of cooled,

sterile solution A, 250.0mL of cooled, sterile solution B, and 250.0mL

of sterile solution C Mix thoroughly Aseptically distribute into sterile

tubes or flasks

Use: For the isolation and cultivation of Rhizobium species.

Nitrate Methanol Medium

NaNO3 5.0g

K2HPO4 2.0g

NaCl 1.0g

MgSO4·7H2O 0.02g

Na2MoO4·H2O 1.0mg

Riboflavin 0.2mg

Pyridoxine·HCl 0.2mg

Thiamine·HCl 0.1mg

Biotin 0.01mg Methanol 10.0mL

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 990.0mL Mix

thorough-ly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C Filter sterilize methanol Aseptically add sterile methanol to cooled sterile medium Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Methylobacterium

rhodi-num.

Nitrate Mineral Salts Medium (NMS Medium)

Noble agar 12.5g MgSO4·7H2O 1.0g KNO3 1.0g

KH2PO4 0.272g CaCl2·6H2O 0.2g Ferric ammonium EDTA 4.0mg Trace elements solution 0.5mL

pH 6.8 ± 0.2 at 25°C

Composition per liter:

Disodium EDTA 0.5g FeSO4·7H2O 0.2g

H3BO3 0.03g CoCl2·6H2O 0.02g ZnSO4·7H2O 0.01g MnCl2·4H2O 3.0mg

Na2MoO4·2H2O 3.0mg NiCl2·6H2O 2.0mg CaCl2·2H2O 1.0mg

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

to boiling Adjust pH to 6.8 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 Methylobacterium spe-cies, Methylococcus capsulatus, Methylomonas agile, and

Methylomo-nas methanica.

Nitrate Mineral Salts Medium with Methanol

(NMS Medium with Methanol) Compositionper liter:

Noble agar 12.5g MgSO4·7H2O 1.0g KNO3 1.0g

KH2PO4 0.272g CaCl2·6H2O 0.2g Ferric ammonium EDTA 4.0mg

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1292 Nitrate Reduction Broth

Trace elements solution 0.5mL

Methanol 1.0mL

pH 6.8 ± 0.2 at 25°C

Disodium EDTA 0.5g

FeSO4·7H2O 0.2g

H3BO3 0.03g

CoCl2·6H2O 0.02g

ZnSO4·7H2O 0.01g

MnCl2·4H2O 3.0mg

Na2MoO4·2H2O 3.0mg

NiCl2·6H2O 2.0mg

CaCl2·2H2O 1.0mg

Preparation of Trace Elements Solution: Add components to

dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components, except methanol, to

distilled/deionized water and bring volume to 999.0mL Mix

thorough-ly Gently heat and bring to boiling Adjust pH to 6.8 Distribute into

tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Cool

to 45°–50°C Filter sterilize methanol Aseptically add sterile methanol

to cooled sterile medium Mix thoroughly Pour into sterile Petri dishes

or leave in tubes

Use: For the cultivation and maintenance of Methylobacterium

fuji-sawaense, Methylobacterium species, and Methylomonas clara.

Nitrate Reduction Broth Compositionper liter:

Pancreatic digest of gelatin 5.0g

Beef extract 3.0g

KNO3 1.0g

pH 6.9 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Distribute into test

tubes that contain an inverted Durham tube Autoclave for 15 min at 15

psi pressure–121°C

Use: For the differentiation of members of the Pseudomonadaceae

based on their ability to reduce nitrate to nitrite or form N2 gas Test for

NaCl 5.0g

Yeast extract 5.0g

Heart muscle, solids from infusion 2.0g

KNO3 or NaNO3 2.0g

pH 7.4 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Distribute into test

tubes that contain an inverted Durham tube Autoclave for 15 min at 15

psi pressure–121°C

Use: For the differentiation of a variety of Gram-negative bacteria

based on their ability to reduce nitrate to nitrite or form N2 gas Test for

Pancreatic digest of casein 13.0g NaCl 5.0g Yeast extract 5.0g Heart muscle, solids from infusion 2.0g KNO3 or NaNO3 2.0g

pH 7.4 ± 0.2 at 25°C

Use: For the differentiation of a variety of nonfermenting Gram-neg-ative bacteria based on their ability to reduce nitrate to nitrite or form

N2 gas Test for nitrates with sulfanilic acid and α-naphthylamine re-agents Bacteria that reduce nitrate to nitrite turn the reagents red or pink

Nitrate Reduction Broth, Clark Compositionper liter:

Peptone 20.0g KNO3 or NaNO3 2.0g

Use: For the differentiation of a variety of Gram-negative bacteria based on their ability to reduce nitrate to nitrite or form N2 gas Test for nitrates with sulfanilic acid and α-naphthylamine reagents Bacteria that reduce nitrate to nitrite turn the reagents red or pink

Nitrate Reduction Broth for

Pseudomonas and Related Genera

Peptone 5.0g NaCl 5.0g Yeast extract 2.0g Beef extract 1.0g NaNO3 0.1g

pH 7.4 ± 0.2 at 25°C

Use: For the differentiation of members of the Pseudomonadaceae based on their ability to reduce nitrate to nitrite or form N2 gas Test for nitrates with sulfanilic acid and α-naphthylamine reagents Bacteria that reduce nitrate to nitrite turn the reagents red or pink

Nitratiruptor and Nitratifactor Medium

(DSMZ Medium 1024) Composition per liter:

Sulfur, elemental 3.0g

Na2S2O3·5H2O 1.0g NaNO3 1.0g Bicarbonate solution 10.0mL

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Nitriliruptor alkaliphilus Medium 1293

Vitamin solution 10.0mL

DMJ synthetic seawater .1.0L

pH 7.0 ± 0.2 at 25°C

Bicarbonate Solution:

NaHCO3 1.0g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Filter

sterilize

DMJ Synthetic Seawater:

Composition perliter:

NaCl 30.0g

MgCl2·6H2O 4.18g

MgSO4·7H2O 3.4g

KCl 0.33g

NH4Cl 0.25g

K2HPO4 0.14g

CaCl2·2H2O 0.14g

Fe(NH4)2(SO4)2·6H2O 0.01g

NiCl2·6H2O 0.5mg

Na2SeO3·5H2O 0.5mg

Trace elements solution SL-10 10.0mL

Trace Elements Solution SL-10:

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 SL-10: Add

nitrilotri-acetic acid to 500.0mL of distilled/deionized water Dissolve by

adjust-ing pH to 6.5 with KOH Add remainadjust-ing components Add distilled/

deionized water to 1.0L Mix thoroughly Adjust pH to 7.0

Preparation of DMJ Synthetic Seawater: Add components to

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

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

temper-ature

Pyridoxine-HCl 10.0mg

Thiamine-HCl·2H2O 5.0mg

Riboflavin 5.0mg

D-Ca-pantothenate 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 sulfur, bicar-bonate solution, and vitamin solution to seawater and bring volume to 1.0L Dispense into serum bottles Autoclave for 15 min at 15 psi pres-sure–121°C under an atmosphere of air Sterilize sulfur separately in screw-capped tubes by steaming in a water bath for 3 hr on each of 3 successive days Aseptically add the sterilized sulfur, bicarbonate, and vitamin solutions Mix thoroughly Sparge with 80% H2 + 20% CO2 Seal the serum tubes with butyl rubber stoppers Increase the 80% H2 + 20% CO2 gas phase pressure to 300 kPa

Use: For the cultivation of Nitratiruptor spp and Nitratifactor spp.

Nitriliruptor alkaliphilus Medium

Na2CO3 22.0g

Na2HCO3 8.0g NaCl 6.0g

K2HPO4 0.5g Isobutyroamide solution 10.0mL Trace elements solution 1.0mL Magnesium sulfate solution 1.0mL Vitamin solution 1.0mL Thiosulfate solution 0.01mL

pH 9.5 ± 0.5 at 25°C

Isobutyroamide Solution:

Compositionper 10.0ml:

Isobutyroamide 0.87g

Preparation of Isobutyroamide Solution: Add isobutyroamide

to distilled/deionized water and bring volume to 10.0mL Mix thor-oughly Filter sterilize

Compositionper 10.0ml:

Vitamin B12 1.0mg

Preparation of Vitamin Solution: Add vitamin B12 to distilled/ deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Magnesium Sulfate Solution:

Composition per10.0mL:

MgSO4·7H2O 2.0g

Preparation of Magnesium Sulfate Solution: Add MgSO4·7H2O

to distilled/deionized water and bring volume to 10.0mL Mix thor-oughly Autoclave for 15 min at 15 psi pressure–121°C Cool to room temperature

Thiosulfate Solution:

Composition per10.0mL:

Na2S2O3·5H2O 1.6g

Preparation of Thiosulfate Solution: Add Na2S2O3·5H2O to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Adjust to pH 10.0 Filter sterilize

EDTA 5.0g FeSO4·7H2O 2.0g

H3BO3 0.01g

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1294 Nitrilotriacetate Medium

ZnSO4·7H2O 0.3g

CoCl2·6H2O 0.2g

MnCl2·4H2O 0.03g

NiCl2·2H2O 0.02g

NaMoO4·2H2O 0.02g

CuCl2 0.01g

Preparation of Trace Elements Solution: Add components to

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

Adjust pH to 3-4

Preparation of Medium: Add components, except vitamin, trace

elements, thiosulfate, isobutyroamide, and magnesium sulfate

solu-tions, to distilled/deionized water and bring volume to 987.0mL Mix

thoroughly Dispense into closed bottles Autoclave for 15 min at 15

psi pressure–121°C Cool to room temperature After cooling, there

may be some precipitate on the bottom Decant into sterile bottle to

eliminate precipitate Aseptically add vitamin, trace elements,

thiosul-fate, isobutyroamide, and magnesium sulfate solutions Adjust pH to

9.5 Aseptically dispense into culture vessels

Use: For the cultivation of Nitriliruptor alkaliphilus.

Nitrilotriacetate Medium Compositionper 1002.0mL:

MgSO4·7H2O 1.0g

Nitrilotriacetate 1.0g

Na2HPO4·2H2O 0.41g

KH2PO4 0.26g

CaCl2·2H2O 0.2g

Trace elements solution 1.0mL

Vitamin solution 1.0mL

pH 6.5 ± 0.2 at 25°C

FeCl2·4H2O 1.5g

CoCl2·6H2O 120.0mg

MnCl2·4H2O 100.0mg

ZnCl2 68.0mg

H3BO3 62.0mg

NiCl2·6H2O 24.0mg

CuCl2·2H2O 17.0mg

HCl (0.05M solution) 1.0L

Preparation of Trace Elements Solution: Add FeCl2·4H2O to

1.0L of HCl solution Mix thoroughly Add distilled/deionized water

and bring volume to 1.0L Add remaining components Mix

thorough-ly

Folic acid 20.0g

α-Lipoic acid 50.0mg

Pantothenic acid 50.0mg

Riboflavin 50.0mg

Thamine·HCl 50.0mg

Vitamin B12 50.0mg

Nicotinamide 25.0mg

Biotin 20.0mg

Pyridoxamine·HCl 10.0mg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 1.0L Stir for 2-3 hr Filter steril-ize

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Distribute into tubes

Use: For the cultivation of Chelatobacter heintzii and Chelatococcus

saccharophobus.

Nitrincola Medium

NaCl 17.5g Na-acetate 10.0g

Na2B4O7 4.0g

NH4Cl 0.5g

K2HPO4 0.25g Yeast extract 0.10g

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 Adjust pH to 9.0 Au-toclave for 15 min at 15 psi pressure–121°C. Cool to room tempera-ture

Use: For the cultivation of Nitrincola spp.

Nitrobacter agilis Medium

CaCO3 10.0g NaCl 0.3g

Na2CO3 0.25g KNO2 0.17g

K2HPO4 0.14g MgSO4·7H2O 0.14g FeSO4·7H2O 0.03g MnSO4·4H2O 0.01g Biotin solution 10.0mL

Biotin Solution:

Biotin 0.15g

Preparation of Biotin Solution: Add biotin to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add Na2CO3 to distilled/deionized water and bring volume to 200.0mL Mix thoroughly In a separate flask, add the remaining components, except the biotin solution, to distilled/de-ionized water and bring volume to 790.0mL Autoclave the Na2CO3 solution and salts solution separately for 15 min at 15 psi pressure– 121°C Cool to 25°C Aseptically combine the sterile Na2CO3 solu-tion, sterile salts solusolu-tion, and sterile biotin solution Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of Nitrobacter agilis.

Nitrobacter Medium 203

Solution C 1.0mL Solution A 0.5mL Solution B 0.5mL Solution D 0.5mL

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