Handbook of Microbiological Media, Fourth Edition part 120 ppsx

Mineral Salts Peptonized Milk Agar 1185Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L.. Minimal Agar, Davis 1187Preparation of Growth Suppl

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Mineral Salts Peptonized Milk Agar 1185

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 Rhodococcus

rhodo-chrous.

Mineral Salts Medium with Methanol

Composition per liter:

NaNH4HPO4·4H2O 1.74g

NaH2PO4·H2O 0.54g

MgSO4·7H2O 0.2g

KCl 0.04g

FeSO4·7H2O 5.0mg

Methanol 5.0mL

Trace minerals solution 1.0mL

pH 7.2 ± 0.2 at 25°C

Trace Minerals Solution:

H3BO3 2.86g

MnCl2·4H2O 1.81g

ZnSO4·7H2O 0.22g

CuSO4·5H2O 0.08g

CoCl2·6H2O 0.06g

Na2MoO4·2H2O 25.0mg

Preparation of Trace Minerals Solution: Add components to

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

Preparation of Medium: Add components, except methanol, to

Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–

121°C Cool to 50°C Filter sterilize methanol Aseptically add sterile

methanol to cooled, sterile basal medium

Use: For the cultivation and maintenance of Rhodococcus

rhodo-chrous.

Mineral Salts Medium with Methanol

and Yeast Extract Composition per liter:

NaNH4HPO4·4H2O 1.74g

NaH2PO4·H2O 0.54g

MgSO4·7H2O 0.2g

Yeast extract 0.2g

KCl 0.04g

FeSO4·7H2O 5.0mg

Methanol 5.0mL

pH 7.2 ± 0.2 at 25°C

H3BO3 2.86g

MnCl2·4H2O 1.81g

ZnSO4·7H2O 0.22g

CuSO4·5H2O 0.08g

CoCl2·6H2O 0.06g

Na2MoO4·2H2O 25.0mg

Preparation of Medium: Add components, except methanol, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°C Filter sterilize methanol Aseptically add sterile methanol to cooled, sterile basal medium Aseptically distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Pseudomonas species.

Mineral Salts Medium for Thermophiles Compositionper liter:

NaNO3 0.25g

NH4Cl 0.25g

Na2HPO4 210.0mg MgSO4·7H2O 200.0mg NaH2PO4 90.0mg KCl 40.0mg CaCl2 15.0mg FeSO4 1.0mg Trace minerals solution 10.0mL

n-Heptadecane 1.0mL

Compositionper liter:

ZnSO4·7H2O 7.0mg

H3BO4 1.0mg MoO3 1.0mg CuSO4·5H2O 500.0μg CoSO4·7H2O 18.0μg MnSO4·5H2O 7.0μg

Preparation of Trace Minerals Solution: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly

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 Bacillus thermoleovorans.

Mineral Salts Peptonized Milk Agar

(SPMA) Compositionper liter:

Agar 15.0g Milk, peptonized 1.0g Mineral solution 100.0mL

Mineral Solution:

Compositionper 100.0mL:

MgSO4·7H2O 0.5g CaCl2 0.25g

K2HPO4 0.25g (NH4)2SO4 0.1g FeCl3·6H2O 0.01g MnCl2 0.1mg

Preparation of Mineral Solution: Add components to distilled/ deionized water and bring volume to 100.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except mineral solu-tion, 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 100.0mL of sterile mineral solution Mix thoroughly Pour into sterile Petri dishes

or distribute into sterile tubes

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1186 Mineral Salts for Thermophiles

Use: For the cultivation of freshwater Myxobacterium species.

Mineral Salts for Thermophiles

(L Salts for Thermophiles)

NaNO3 0.25g

NH4Cl 0.25g

Na2HPO4 0.21g

MgSO4·7H2O 0.2g

NaH2PO4 0.09g

KCl 0.04g

CaCl2 0.02g

FeSO4 1.0mg

n-Heptadecane 1.0mL

ZnSO4·7H2O 7.0mg

H3BO4 1.0mg

MoO3 1.0mg

CuSO4·5H2O 500.0μg

CoSO4·7H2O 18.0μg

MnSO4·5H2O 7.0μg

Preparation of Medium: Add components, except n-heptadecane,

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

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

n-heptadecane Mix thoroughly Distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Bacillus

thermoleo-vorans.

Minerals Modified Glutamate Agar

Agar 15.0g

Lactose 10.0g

Sodium glutamate 6.35g

NH4Cl 2.5g

K2HPO4 0.9g

Sodium formate 0.25g

MgSO4·7H2O 0.1g

L-Aspartic acid 0.024g

L-Arginine 0.02g

L-Cystine 0.02g

Bromcresol Purple 0.01g

CaCl2·2H2O 0.01g

Ferric ammonium citrate 0.01g

Nicotinic acid 1.0mg

Pantothenic acid 1.0mg

Thiamine 1.0mg

pH 6.7 ± 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 10 min at 11

psi pressure–116°C Pour into sterile Petri dishes in 20.0mL volumes

Use: For the cultivation of coliform bacteria from foods

Minerals Modified Medium Compositionper liter:

Lactose 20.0g Sodium glutamate 12.7g

NH4Cl 5.0g

K2HPO4 1.8g Sodium formate 0.5g MgSO4·7H2O 0.2g

L-Aspartic acid 0.048g

L-Cystine 0.04g

L-Arginine 0.04g Ferric ammonium citrate 0.02g CaCl2·2H2O 0.02g Bromcresol Purple 0.02g Thiamine 2.0mg Nicotinic acid 2.0mg Pantothenic acid 2.0mg

pH 6.7 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Oxoid Unipath

Preparation of Medium: Add NH4Cl to distilled/deionized water and bring volume to 800.0mL Add remaining components and bring volume to 1.0L Mix thoroughly Adjust pH to 6.7 Distribute into tubes

or flasks Autoclave for 10 min at 10 psi pressure–116°C Check pH af-ter autoclaving This medium is double strength

Use: For the enumeration of coliform bacteria in water

Minimal Agar I Compositionper liter:

Agar 20.0g Glucose 20.0g (NH4)2SO4 5.0g

KH2PO4 1.0g MgSO4·7H2O 0.5g NaCl 0.1g CaCl2·2H2O 0.1g Inositol 2.0mg

KI 1.0mg

H3BO3 0.5mg ZnSO4·7H2O 0.4mg MnSO4·4H2O 0.4mg Thiamine·HCl 0.4mg Pyroxidine·HCl 0.4mg Niacin 0.4mg Calcium pantothenate 0.4mg

p-Aminobenzoic acid 0.2mg

Riboflavin 0.2mg FeCl3 0.2mg

Na2MoO4·4H2O 0.2mg CuSO4·5H2O 0.04mg Folic acid 2.0μg Biotin 2.0μg Growth supplement solution 10.0mL

pH 5.5 ± 0.2 at 25°C

Growth Supplement Solution:

L-Tryptophan 20.0mg Uracil 20.0mg

L-Histidine·HCl 20.0mg

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Minimal Agar, Davis 1187

Preparation of Growth Supplement Solution: Add

compo-nents to distilled/deionized water and bring volume to 10.0mL Mix

thoroughly Filter sterilize

Preparation of Medium: Add components, except growth

supple-ment solution, 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 50°–55°C Aseptically

add 10.0mL of sterile growth supplement solution Mix thoroughly

Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the cultivation of Saccharomyces cerevisiae.

Minimal Agar II Compositionper liter:

Agar 20.0g

Glucose 20.0g

(NH4)2SO4 5.0g

KH2PO4 1.0g

MgSO4·7H2O 0.5g

NaCl 0.1g

CaCl2·2H2O 0.1g

Inositol 2.0mg

KI 1.0mg

H3BO3 0.5mg

ZnSO4·7H2O 0.4mg

MnSO4·4H2O 0.4mg

Thiamine·HCl 0.4mg

Pyroxidine·HCl 0.4mg

Niacin 0.4mg

Calcium pantothenate 0.4mg

Riboflavin 0.2mg

FeCl3 0.2mg

Na2MoO4·4H2O 0.2mg

CuSO4·5H2O 0.04mg

Folic acid 2.0μg

Biotin 2.0μg

Growth supplement solution 10.0mL

pH 5.5 ± 0.2 at 25°C

Adenine sulfate 20.0mg

L-Arginine·HCl 20.0mg

990.0mL Mix thoroughly Gently heat and bring to boiling Autoclave

for 15 min at 15 psi pressure–121°C Cool to 50°–55°C Aseptically

add 10.0mL of sterile growth supplement solution Mix thoroughly

Pour into sterile Petri dishes or distribute into sterile tubes

Minimal Agar III Compositionper liter:

Agar 20.0g

Glucose 20.0g

(NH4)2SO4 5.0g

KI 1.0mg

pH 5.5 ± 0.2 at 25°C

L-Leucine 30.0mg

L-Tryptophan 20.0gm Uracil 20.0mg

Preparation of Growth Supplement Solution: Add compo-nents to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except growth supple-ment solution, 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 50°–55°C Aseptically add 10.0mL of sterile growth supplement solution Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Minimal Agar, Davis Compositionper liter:

Agar 15.0g

K2HPO4 7.0g

KH2PO4 2.0g (NH4)2SO4 1.0g Glucose 1.0g Sodium citrate 0.5g MgSO4·7H2O 0.1g

pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from BD Di-agnostic Systems

Preparation of Medium: Add components to cold distilled/deion-ized 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, cultivation, and characterization of nutritional

mutants of Escherichia coli

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1188 Minimal Broth I

Minimal Broth I Compositionper liter:

Glucose 20.0g

(NH4)2SO4 5.0g

KH2PO4 1.0g

MgSO4·7H2O 0.5g

NaCl 0.1g

CaCl2·2H2O 0.1g

Inositol 2.0mg

KI 1.0mg

H3BO3 0.5mg

ZnSO4·7H2O 0.4mg

MnSO4·4H2O 0.4mg

Thiamine·HCl 0.4mg

Niacin 0.4mg

Riboflavin 0.2mg

FeCl3 0.2mg

Na2MoO4·4H2O 0.2mg

CuSO4·5H2O 0.04mg

Folic acid 2.0μg

Biotin 2.0μg

Growth supplement solution 10.0mL

pH 5.5 ± 0.2 at 25°C

L-Tryptophan 20.0mg

Uracil 20.0mg

990.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–

121°C Aseptically add 10.0mL of sterile growth supplement solution

Mix thoroughly Aseptically distribute into sterile tubes or flasks

Minimal Broth II Compositionper liter:

Glucose 20.0g

(NH4)2SO4 5.0g

KH2PO4 1.0g

MgSO4·7H2O 0.5g

NaCl 0.1g

CaCl2·2H2O 0.1g

Inositol 2.0mg

KI 1.0mg

H3BO3 0.5mg

ZnSO4·7H2O 0.4mg

MnSO4·4H2O 0.4mg

Thiamine·HCl 0.4mg

Niacin 0.4mg

Riboflavin 0.2mg

FeCl3 0.2mg

pH 5.5 ± 0.2 at 25°C

Adenine sulfate 20.0mg

Preparation of Medium: Add components, except growth supple-ment solution, to distilled/deionized water and bring volume to 990.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure– 121°C Aseptically add 10.0mL of sterile growth supplement solution Mix thoroughly Aseptically distribute into sterile tubes or flasks

Minimal Broth III Compositionper liter:

Agar 20.0g Glucose 20.0g (NH4)2SO4 5.0g

KI 1.0mg

pH 5.5 ± 0.2 at 25°C

L-Leucine 30.0mg

L-Tryptophan 20.0mg Uracil 20.0mg

Preparation of Medium: Add components, except growth supple-ment solution, to distilled/deionized water and bring volume to

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Minimum Essential Medium with Bicarbonate, Serum, and Antibiotics 1189

990.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–

121°C Aseptically add 10.0mL of sterile growth supplement solution

Mix thoroughly Aseptically distribute into sterile tubes or flasks

Minimal Broth, Davis Compositionper liter:

K2HPO4 7.0g

KH2PO4 2.0g

(NH4)2SO4 1.0g

Glucose 1.0g

Sodium citrate 0.5g

MgSO4·7H2O 0.1g

pH 7.0 ± 0.2 at 25°C

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

Di-agnostic Systems

Preparation of Medium: Add components to cold

distilled/deion-ized 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, cultivation, and characterization of nutritional

mutants of Escherichia coli Also recommended for the isolation and

characterization of nutritional mutants from wild-type strains of

Bacil-lus subtilis when used in conjunction with minimal agar Davis and

antibiotic medium 3

Minimal F-Top Agar Compositionper liter:

NaCl 8.0g

Agar 4.5g

K2HPO4 2.1g

KH2PO4 0.6g

(NH4)2SO4 0.3g

Glucose 0.3g

Sodium citrate 0.15g

MgSO4·7H2O 0.03g

pH 7.0 ± 0.2 at 25°C

Preparation of Minimal Agar: Add components to cold 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 distribution of bacteriophage or bacterial cells evenly in

a thin layer over the surface of a plate

Minimal Lactate Medium

See: ML Medium Minimal Medium for Denitrifying Bacteria Compositionper liter:

Solution A 980.0mL

Solution B 10.0mL

Solution C 10.0mL

Solution A:

KNO3 5.0g

Carbon source 4.0g

(NH4)2SO4 1.0g

K2HPO4·3H2O 0.87g

KH2PO4 0.54g

Preparation of Solution A: 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 25°C

Solution B:

MgSO4·7H2O 2.0g

Preparation of Solution B: Add MgSO4·7H2O to distilled/deion-ized water and bring volume to 100.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C

Solution C:

CaCl2·2H2O 0.2g FeSO4·7H2O 0.1g MnSO4·H2O 0.05g CuSO4·5H2O 0.01g

Na2MoO4·2H2O 0.01g

HCl (0.1N solution) 100.0mL

Preparation of Solution C: Combine components Mix

thorough-ly Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C

Preparation of Medium: Aseptically combine 980.0mL of cooled sterile solution A, 10.0mL of cooled sterile solution B, and 10.0mL of cooled sterile solution C Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the isolation and cultivation of denitrifying bacteria

Minimal Medium for Penicillium

Interspecific Hybrids Compositionper liter:

Glucose 40.0g NaNO3 3.0g

KH2PO4 1.0g KCl 0.5g MgSO4·7H2O 0.5g FeSO4·7H2O 0.01g

pH 6.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized wa-ter and bring volume to 1.0L Mix thoroughly Adjust pH to 6.0 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation of genetic variants of Penicillium species

Minimum Essential Medium with Bicarbonate, Serum, and Antibiotics Compositionper 1100.0mL:

Minimum essential medium 950.0mL Fetal bovine serum, heat inactivated 100.0mL NaHCO3 solution 40.0mL Penicillin-streptomycin solution 10.0mL

pH 7.2 ± 0.2 at 25°C

Minimum Essential Medium (MEM):

Inorganic salt solution 400.0mL Other component solution 400.0mL Amino acid solution 100.0mL Vitamin solution 100.0mL

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1190 Minimum Salts with HiVeg Acid Hydrolysate

Inorganic Salt Solution:

NaCl 6.8g

KCl 0.4g

CaCl2, anhydrous 0.2g

NaH2PO4·H2O 0.14g

MgSO4, anhydrous 97.67mg

Preparation of Inorganic Salt Solution : Add components to

dis-tilled/deionized water and bring volume to 400.0mL Mix thoroughly

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

Other Component Solution:

D-Glucose 1.0g

Phenol Red 10.0mg

Preparation of Other Component Solution: Add components

to distilled/deionized water and bring volume to 400.0mL Mix

thor-oughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C

Amino Acid Solution:

L-Glutamine 292.0mg

L-Lysine·HCl 72.5mg

L-Isoleucine 52.0mg

L-Leucine 52.0mg

L-Tyrosine, disodium salt 52.0mg

L-Threonine 48.0mg

L-Valine 46.0mg

L-Histidine·HCl·H2O 42.0mg

L-Phenylalanine 32.0mg

L-Cysteine·2HCl 31.0mg

L-Methionine 15.0mg

L-Glutamic acid 14.7mg

L-Aspartic acid 13.3mg

L-Asparagine·H2O 13.2mg

L-Proline 11.5mg

L-Serine 10.5mg

L-Tryptophan 10.0mg

L-Alanine 8.9mg

Glycine 7.5mg

Preparation of Amino Acid Solution : Add components to

Filter sterilize

Vitamin Solution:

i-Inositol 2.0mg

D-Ca pantothenate 1.0mg

Choline chloride 1.0mg

Folic acid 1.0mg

Niacinamide 1.0mg

Pyridoxal·HCl 1.0mg

Thiamine·HCl 1.0mg

Riboflavin 0.1mg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 100.0mL Mix thoroughly Filter

sterilize

Preparation of Minimum Essential Medium (MEM):

Asepti-cally combine 400.0mL of sterile inorganic salt solution, 400.0mL of

sterile other component solution, 100.0mL of sterile amino acid

solu-tion, and 100.0mL of sterile vitamin solution

NaHCO 3 Solution:

NaHCO3 2.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to distilled/de-ionized water and bring volume to 40.0mL Mix thoroughly Filter ster-ilize

Penicillin-Streptomycin Solution Compositionper 10.0mL:

Penicillin 0.01g Streptomycin 0.01g

Preparation of Penicillin-Streptomycin Solution: Add compo-nents to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Aseptically combine 950.0mL of sterile minimum essential medium, 100.0mL of sterile heat inactivated fetal bovine serum, 40.0mL of sterile NaHCO3 solution, and 10.0mL of ster-ile penicillin-streptomycin solution Adjust pH to 7.2 with humidified 10% CO2 in 90% air

Use: For the cultivation of Encephalitozoon cuniculi,

Encephalito-zoon hellem, EncephalitoEncephalito-zoon intestinalis, Naegleria fowleri, and Nosema corneum.

Minimum Salts with HiVeg Acid Hydrolysate Compositionper liter:

Na2HPO4 6.8g Glucose 4.0g Plant acid hydrolysate 4.0g

KH2PO4 3.0g

NH4Cl 1.0g NaCl 0.5g MgSO4 0.24g

pH 6.8 ± 0.2 at 25°C

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

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Heat gently and bring

to boiling Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation of Escherichia coli strains used for genetic

and molecular studies

MIO HiVeg Medium (Motility Indole Ornithine HiVeg Medium) Compositionper liter:

Plant hydrolysate 10.0g Plant peptone 10.0g

L-Ornithine hydrochloride 5.0g Yeast extract 3.0g Agar 2.0g Glucose 1.0g Bromcresol Purple 0.02g

pH 6.6 ± 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 to 1.0L Mix thoroughly Gently heat and bring to

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boil-Mitsuokella dentalis Medium 1191

ing Distribute into tubes or flasks Autoclave for 15 min at 15 psi

pres-sure–121°C

Use: For the differentiation of Gram-negative enteric bacteria based

on their motility, indole production, and ornithine decarboxylase

activ-ity

MIO Medium

See: Motility Indole Ornithine Medium

Mist Agar Compositionper liter:

Cow manure, dry 50.0g

Agar 15.0g

Preparation of Medium: Add cow manure to 1.0L of tap water

Boil for 1 hr Filter through cheesecloth Filter through paper Add agar

to filtrate and bring volume to 1.0L with tap water 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 Streptomyces

fragmen-tosporus.

Mitis Salivarius Agar Compositionper liter:

Sucrose 50.0g

Agar 15.0g

Enzymatic digest of protein 10.0g

Proteose peptone 10.0g

K2HPO4 4.0g

Dextrose 1.0g

Trypan Blue 0.08g

Crystal Violet 0.8mg

Na2TeO3 solution 1.0mL

pH 7.0 ± 0.2 at 25°C

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

Di-agnostic Systems

Na 2 TeO 3 Solution:

Na2TeO3 0.1g

Preparation of Na 2 TeO 3 Solution: Add Na2TeO3 to 10.0mL of

distilled/deionized water Mix thoroughly Filter sterilize

Caution: Potassium tellurite is toxic

water and bring volume to 999.0mL Mix thoroughly Gently heat and

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

medium to 50°–55°C Aseptically add 1.0mL of the sterile Na2TeO3

solution to the cooled basal medium Mix thoroughly Pour into sterile

Petri dishes or distribute into sterile tubes

Use: For the selective isolation of Streptococcus mitis, Streptococcus

salivarius, and other viridans streptococci and enterococci.

Mitis Salivarius HiVeg Agar Base with Tellurite

Sucrose 50.0g

Agar 15.0g

Plant hydrolysate 15.0g

Plant peptone 5.0g

K2HPO4 4.0g Glucose 1.0g Trypan Blue 0.075g Crystal Violet 0.8mg

Na2TeO3 solution 1.0mL

pH 7.0 ± 0.2 at 25°C

Source: This medium, without tellurite, is available as a premixed powder from HiMedia

Na 2 TeO 3 Solution:

Na2TeO3 0.1g

Preparation of Na 2 TeO 3 Solution: Add Na2TeO3 to 10.0mL of distilled/deionized water Mix thoroughly Filter sterilize

Caution: Potassium tellurite is toxic

Preparation of Medium: Add components to distilled/deionized water and bring volume to 999.0mL Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool medium to 50°–55°C Aseptically add 1.0mL of the sterile Na2TeO3 solution to the cooled basal medium Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the selective isolation of Streptococcus mitis, Streptococcus

salivarius, and other viridans streptococci and enterococci.

Mitsuokella dentalis Medium

Yeast extract 10.0g Beef extract 5.0g Glucose 5.0g Trypticase™ 5.0g

L-Cysteine·HCl 0.5g (NH4)2SO4 0.5g Resazurin 1.0mg Bovine serum 50.0mL Mineral solution 40.0mL Hemin solution 1.0mL Vitamin K1 solution 0.2mL

pH 6.9 ± 0.2 at 25°C

Bovine Serum:

Bovine serum 50.0mL

Preparation of Bovine Serum: Incubate 50.0mL of bovine serum

in a GasPak™ container overnight to make anaerobic

Mineral Solution:

NaHCO3 10.0g NaCl 2.0g

K2HPO4 1.0g

KH2PO4 1.0g MgSO4·7H2O 0.48g CaCl2·2H2O 0.3g

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

Hemin Solution:

Hemin 5.0mg NaOH (0.002% solution) 1.0mL

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1192 Mixed Cereal Agar

Preparation of Hemin Solution: Add hemin to 1.0mL of NaOH

solution Mix thoroughly

Vitamin K 1 Solution:

Vitamin K1 1.09g

Preparation of Vitamin K 1 Solution: Add vitamin K1 to 99.0mL

of absolute ethanol Store in the dark at 4°C

Preparation of Medium: Add components, except L-cysteine·HCl

and bovine serum, to distilled/deionized water and bring volume to

950.0mL Mix thoroughly Gently heat and bring to boiling Continue

boiling for 5 min Cool to room temperature while sparging with 100%

CO2 Add L-cysteine·HCl Mix thoroughly Adjust pH to 6.0 with 8N

NaOH After pH has been reached, change sparging gas to 100% N2

Anaerobically distribute into bottles under 100% N2 Autoclave for 15

min at 15 psi pressure–121°C Aseptically and anaerobically add

50.0mL of bovine serum Mix thoroughly

Use: For the cultivation and maintenance of Mitsuokella dentalis.

Mixed Cereal Agar Compositionper liter:

Gerber™ mixed cereal (oats, wheat,

corn, barley) 50.0g

Agar 15.0g

Sucrose 15.0g

Thiamine·HCl 5.0mg

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 Tilletia caries

Mixotrophic Nitrobacter Medium

(DSMZ Medium 756a) Compositionper liter:

NaNO2 2.0g

Yeast extract 1.5g

Peptone 1.5g

Na-pyruvate 0.55g

Stock solution 100.0mL

Trace elements solution 1.0mL

pH 7.4 ± 0.2 at 25°C

Stock Solution:

NaCl 5.0g

KH2PO4 1.5g

MgSO4·7H2O 0.5g

CaCO3 0.07g

Preparation of Stock Solution: Add components to

distilled/deion-ized water and bring volume to 1.0L Mix thoroughly

Trace Elements Solution:

FeSO4·7H2O 97.3mg

H3BO3 49.4mg

ZnSO4·7H2O 43.1mg

(NH4)6Mo7O24·4H2O 37.1mg

MnSO4·2H2O 33.8mg

CuSO4·5H2O 25.0mg

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

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.4 Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C

Use: For the cultivation of Nitrobacter vulgaris.

Mixotrophic Nitrobacter Medium

(LMG Medium 246) Compositionper liter:

NaNO2 2.0g Yeast extract 1.50g Peptone 1.50g Na-pyruvate 0.55g Stock solution 100.0mL Trace elements solution 1.0mL

pH 7.4 ± 0.2 at 25°C

Stock Solution:

NaCl 5.0g

KH2PO4 1.5g MgSO4·7H2O 0.5g CaCO3 0.07g

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

(NH4)Mo7O2 437.10mg FeSO4·7H2O 97.30mg ZnSO4·7H2O 43.10mg

H3BO3 39.40mg MnSO4·H2O 33.80mg CuSO2·5H2O 25.00mg

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

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.4 with NaOH Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation of mixotrophic Nitrobacter spp.

Mixotrophic Nitrobacter Medium, 10%

(DSMZ Medium 756b) Compositionper liter:

NaNO2 2.0g Yeast extract 0.15g Peptone 0.15g Na-pyruvate 0.055g Stock solution 100.0mL Trace elements solution 1.0mL

pH 8.6 ± 0.2 at 25°C

Stock Solution:

NaCl 5.0g

KH2PO4 1.5g

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MJ Medium 1193

MgSO4·7H2O 0.5g

CaCO3 0.07g

Preparation of Stock Solution: Add components to

distilled/deion-ized water and bring volume to 1.0L Mix thoroughly

FeSO4·7H2O 97.3mg

H3BO3 49.4mg

ZnSO4·7H2O 43.1mg

(NH4)6Mo7O24·4H2O 37.1mg

MnSO4·2H2O 33.8mg

CuSO4·5H2O 25.0mg

Preparation of Trace Elements Solution: Add components to

water and bring volume to 1.0L Mix thoroughly Adjust pH to 8.6

Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–

121°C

Use: For the cultivation of Nitrobacter hamburgensis and Nitrobacter

vulgaris.

MJ Medium (DSMZ Medium 1011) Composition per liter:

NaCl 30.0g

MgCl2·6H2O 4.18g

MgSO4·7H2O 3.4g

KCl 0.33g

NH4Cl 0.25g

KH2PO4 0.14g

CaCl2·2H2O 0.014g

Fe(NH4)2(SO4)2·6H2O 0.01g

NiCl2·6H2O 0.5mg

Na2SeO3·5H2O 0.5mg

Vitamin solution 10.0mL

Thiosulfate solution 10.0mL

Bicarbonate solution 10.0mL

pH 6.7 ± 0.2 at 25°C

Bicarbonate Solution :

NaHCO3 1.5g

Preparation of Bicarbonate Solution: Add components to

Filter sterilize

Thiosulfate Solution:

Composition per10.0mL:

NaS2O3·5H2O 1.5g

Preparation of Thiosulfate Solution: Add components to

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

temper-ature

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

Pyridoxine-HCl 10.0mg Thiamine-HCl·2H2O 5.0mg Riboflavin 5.0mg Nicotinic acid 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 thiosulfate, bi-carbonate, and vitamin solutions, to distilled/deionized water and bring volume to 970.0mL Mix thoroughly Gently heat and bring to boiling Boil for 1 min Cool to room temperature while sparging with a gas mix-ture of 80% N2 + 20% CO2 Dispense under the same atmosphere into culture vessels Fill up to a volume of 20% volume of vessel Autoclave for 15 min at 15 psi pressure–121°C Cool to room temperature under

an atmosphere of 0% N2 + 20% CO2 Aseptically add sterile thiosulfate, bicarbonate, and vitamin solutions Mix thoroughly Adjust pH to 6.7 Aseptically dispense into tubes, flasks, or bottles After inoculation pres-surize vessels to 0.5 bar overpressure with 80% N2 + 20% CO2gas mix-ture Add sterile air in an amount that is equivalent to a volume of 20%

of the headspace After inoculation reduce medium with 10–20mg so-dium dithionite per liter meso-dium, added from a 5% solution freshly prepared under N2 and filter sterilized Pressurize vessels to 2 bar over-pressure with 80% H2and 20% CO2

Use: For the cultivation of Sulfurimonas autotrophica, Thiomicrospira

thermophila, and Desulfothermus okinawensis.

MJ Medium (DSMZ Medium 1011) Composition per liter:

NaCl 30.0g MgCl2·6H2O 4.18g MgSO4·7H2O 3.4g KCl 0.33g

NH4Cl 0.25g

KH2PO4 0.14g CaCl2·2H2O 0.014g Fe(NH4)2(SO4)2·6H2O 0.01g

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1194 MJ Medium for Thiobacter subterraneus

NiCl2·6H2O 0.5mg

Na2SeO3·5H2O 0.5mg

Vitamin solution 10.0mL

Thiosulfate solution 10.0mL

Bicarbonate solution 10.0mL

Pyruvate solution 10.0mL

Yeast extract solution 10.0mL

Lactate soltuion 10.0mL

pH 6.7 ± 0.2 at 25°C

Pyruvate Solution :

Sodium pyruvate 0.5g

Preparation of Pyruvate Solution: Add sodium pyruvate to

Filter sterilize

Lactate Solution :

Sodium lactate 0.5g

Preparation of Lactate Solution: Add sodium lactate to distilled/

sterilize

Yeast Extract Solution :

Yeast extract 0.1g

Preparation of Yeast Extract Solution: Add yeast extract to

Filter sterilize

NaHCO3 1.5g

Preparation of Bicarbonate Solution: Add NaHCO3 to distilled/

sterilize

Thiosulfate Solution:

Composition per10.0mL:

NaS2O3·5H2O 1.5g

Preparation of Thiosulfate Solution: Add NaS2O3·5H2O to

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

temper-ature

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

Pyridoxine-HCl 10.0mg Thiamine-HCl·2H2O 5.0mg Riboflavin 5.0mg Nicotinic acid 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 lactate, pyruvate, yeast extract, thiosulfate, bicarbonate, and vitamin solutions, to dis-tilled/deionized water and bring volume to 970.0mL Mix thoroughly Gently heat and bring to boiling Boil for 1 min Cool to room tempera-ture while sparging with a gas mixtempera-ture of 80% H2 + 20% CO2 Dispense under the same atmosphere into culture vessels Fill up to a volume of 20% volume of vessel Autoclave for 15 min at 15 psi pressure–121°C Cool to room temperature under an atmosphere of 80% H2 + 20% CO2 Aseptically add sterile lactate, pyruvate, yeast extract, thiosulfate, bicar-bonate, and vitamin solutions Mix thoroughly Adjust pH to 6.7 Asep-tically dispense into tubes, flasks, or bottles After inoculation pressurize vessels to 0.5 bar overpressure with 80% H2 + 20% CO2gas mixture Add sterile air in an amount that is equivalent to a volume of 20% of the headspace After inoculation reduce medium with 10–20

mg sodium dithionite per liter medium, added from a 5% solution freshly prepared under N2 and filter sterilized Pressurize vessels to 2 bar overpressure with 80% H2and 20% CO2

Use: For the cultivation of Desulfothermus okinawensis.

MJ Medium for Thiobacter subterraneus

(DSMZ Medium 1011a) Composition per liter:

MnCl2·6H2O 4.18g NaCl 3.0g MgSO4·7H2O 0.34g KCl 0.33g

NH4Cl 0.25g

KH2PO4 0.14g CaCl2·2H2O 0.014g Fe(NH4)2(SO4)2·6H2O 0.01g NiCl2·6H2O 0.5mg

Na2SeO3·5H2O 0.5mg Trace elements solution 10.0mL Vitamin solution 10.0mL Thiosulfate solution 10.0mL Bicarbonate solution 10.0mL

pH 6.7 ± 0.2 at 25°C

NaHCO3 1.5g

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