Handbook of Microbiological Media, Fourth Edition part 30 pdf

5.0mg Preparation of Selective Supplement Solution: Add components to distilled/deionized water and bring volume to 10.0mL.. Preparation of Medium: Add components, except selective sup-p

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Burk’s Medium 285

Burke’s Modified Nitrogen-Free Medium with Benzoate

Compositionper liter:

Sodium benzoate 0.72g

MgSO4·7H2O 0.2g

Na2HPO4 0.189g

NaHCO3 0.05g

CaSO4·2H2O 0.02g

KH2PO4 0.011g

SrCl2·6H2O 0.01g

NaCl 0.01g

Adenine 0.01g

FeSO4·7H2O 6.0mg

Na2MoO3 0.5mg

pH 7.8 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

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

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

Use: For the cultivation of Pseudomonas species and other

microor-ganisms which can utilize benzoate as sole carbon source

Burkholderia cepacia Agar

Agar 12.0g

Sodium pyruvate 7.0g

Peptone 5.0g

KH2PO4 4.4g

Yeast extract 4.0g

Bile salts 1.5g

Na2HPO4 1.4g

(NH4)2SO4 1.0g

MgSO4 0.2

Phenol Red 0.02g

Fe(NH4)2(SO4)2·6H2O 0.01g

Crystal Violet 0.001g

Selective supplement solution 10.0mL

pH 6.2 ± 0.2 at 25°C

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

Unipath

Selective Supplement Solution:

Compositionper 10.0mL:

Polymyxin B 150,000IU

Ticarcillin 100.0mg

Gentamicin 5.0mg

Preparation of Selective Supplement Solution: Add components

to distilled/deionized water and bring volume to 10.0mL Mix

thor-oughly Filter sterilize

Preparation of Medium: Add components, except selective

sup-plement solution, to distilled/deionized water and bring volume to

990.0mL Mix thoroughly Gently heat while stirring and bring to

boil-ing Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°C

Aseptially add 10.0mL selective supplement solution Mix thoroughly

Pour into sterile Petri dishes

Use: For the selective isolation of Burkholderia cepacia from the

respiratory secretions of patients with cystic fibrosis and for routine

testing of non-sterile inorganic salt solutions containing preservative

Slow growing B cepacia can be missed on conventional media such as

blood or MacConkey agar due to overgrowth caused by other faster

growing organisms found in the respiratory tract of CF patients such as

mucoid Klebsiella species, Pseudomonas aeruginosa, and

Staphylo-coccus species This may lead to the infection being missed or wrongly

diagnosed

Burkholderia pseudomallei Selective Agar

(BPSA)

Composition per liter:

Agar 15.0g Pancreatic Digest of Casein 5.0g Maltose 4.0g Yeast Extract 2.5g Glucose 1.0g Neutral Red 0.1g Gentamicin solution 10.0mL Glycerol 10.0mL Nile Blue solution 1.0mL

pH 7.0 ± 0.2 at 25°C

Gentamicin Solution:

Gentamicin 20.0mg

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

Nile Blue Solution:

Nile Blue 0.2g

Preparation of Nile Blue Solution: Add Nile blue to 10.0mL of a 1% solution of dimethyl sulfoxide Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except gentamcin solu-tion, Nile Blue solusolu-tion, and glycerol, to distilled/deionized water and bring volume to 979.0mL Mix thoroughly Gently heat while stirring and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°C Aseptially add 10.0mL sterile gentamicin solution, 1.0mL sterile Nile blue solution, and 10.0mL filter-sterilized glycerol Mix thoroughly for 5 min on a heated magnetic stirrer at 40°C Pour into sterile Petri dishes

Use: For the cultivation of Burkholderia pseudomallei from clinical

specimens collected from non-sterile sites with improved recovery of

the more easily inhibited strains of B pseudomallei.

Burk’s Medium

Sucrose 20.0g MgSO4·7H2O 0.2g

K2HPO4 0.8g

KH2PO4 0.25g CaSO4 0.13g FeCl3 1.45mg

Na2MoO3 0.253mg

Source: This medium is available from HiMedia

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

Use: For thecultivation of nitrogen fixing bacteria, such as Azotrobacter

spp., from soil

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286 Bushnell-Haas Agar

Bushnell-Haas Agar

Agar 15.0g

KH2PO4 1.0g

K2HPO4 1.0g

NH4NO3 1.0g

MgSO4·7H2O 0.2g

FeCl3 0.05g

CaCl2·2H2O 0.02g

pH 7.0 ± 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 For

use in cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0%

hy-drocarbon on agar surface or aseptically add sterile hyhy-drocarbon to

cooled agar prior to pouring plates

Use: For examining fuels for microbial contamination and for

study-ing hydrocarbon utilization by microorganisms Also for the

cultiva-tion of Nocardia species.

Bushnell-Haas Broth

KH2PO4 1.0g

K2HPO4 1.0g

NH4NO3 1.0g

MgSO4·7H2O 0.2g

FeCl3 0.05g

CaCl2·2H2O 0.02g

pH 7.0 ± 0.2 at 25°C

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

Di-agnostic Systems

or flasks Autoclave for 15 min at 15 psi pressure–121°C For use in

cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0%

hydrocar-bon on broth surface or add directly to broth

Use: For examining fuels for microbial contamination and for studying

the hydrocarbon utilization by microorganisms Also for the cultivation of

Nocardia species.

Bushnell-Haas Medium

KH2PO4 1.0g

K2HPO4 1.0g

NH4NO3 1.0g

Cholesterol 0.3g

MgSO4·7H2O 0.2g

FeCl3 0.05g

CaCl2·2H2O 0.02g

pH 7.0 ± 0.2 at 25°C

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

Di-agnostic Systems

or flasks Autoclave for 15 min at 15 psi pressure–121°C For use in

cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0%

hydrocar-bon on broth surface or add directly to broth

Use: For the cultivation of Nocardia species.

Butanediol Medium

NaH2PO4·H2O 2.1g 1,4-Butanediol 1.0g NaCl 1.0g

NH4Cl 1.0g CaCl2·2H2O 0.5g MgSO4·7H2O 0.5g

K2HPO4 0.3g Yeast extract 0.2g Modified Wolfe’s mineral solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Modified Wolfe’s Mineral Solution:

MgSO4·7H2O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO4·H2O 0.5g CaCl2 0.1g CoCl2·6H2O 0.1g FeSO4·7H2O 0.1g ZnSO4·7H2O 0.1g AlK(SO4)2·12H2O 0.01g CuSO4·5H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Na2SeO3 0.01g NaWO4·2H2O 0.01g NiC12·6H2O 0.01g

Preparation of Modified Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water Adjust pH

to 6.5 with KOH Add remaining components one at a time Add dis-tilled/deionized water to 1.0L Adjust pH to 6.8

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C

Use: For the cultivation of Pseudomonas putida.

Butyrivibrio Species Medium

Na2CO3 4.0g Pancreatic digest of casein 2.0g Yeast extract 2.0g

K2HPO4 0.3g Hemin 1.0mg Resazurin 1.0mg Rumen fluid, clarified 150.0mL Minerals solution 75.0mL Carbohydrate solution 20.0mL

L-Cysteine·HCl·H2O solution 10.0mL

Na2S·9H2O solution 10.0mL Volatile fatty acid mixture 3.1mL

pH 6.7 ± 0.2 at 25°C

Minerals Solution:

NaCl 12.0g

KH2PO4 6.0g (NH4)2SO4 6.0g

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BYE Agar 287

MgSO4·7H2O 2.5g

CaCl2·2H2O 1.6g

Preparation of Minerals Solution: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly

L -Cysteine·HCl·H 2 O Solution:

L-Cysteine·HCl·H2O 0.25g

Preparation of L -Cysteine·HCl·H 2 O Solution: Add L

-cys-teine·HCl·H2O to distilled/deionized water and bring volume to 10.0mL

Mix thoroughly Sparge with 100% CO2 Autoclave for 15 min at 15 psi

pressure–121°C

Na 2 S·9H 2 O Solution:

Na2S·9H2O 0.25g

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% CO2 Autoclave for 15 min at 15 psi pressure–

121°C

Carbohydrate Solution:

Glucose 1.0g

Cellobiose 1.0g

Glycerol 1.0g

Maltose 1.0g

Starch, soluble 1.0g

Preparation of Carbohydrate Solution : Add components to

Sparge under 100% CO2 Autoclave for 15 min at 15 psi pressure–

121°C

Volatile Fatty Acid Mixture:

Acetic acid 4.25mL

Propionic acid 1.50mL

Butyric acid 1.0mL

DL-2-Methyl butyric acid 0.25mL

iso-Butyric acid 0.25mL

iso-Valeric acid 0.25mL

n-Valeric acid 0.25mL

Preparation of Volatile Fatty Acid Mixture: Combine

compo-nents Mix thoroughly

Preparation of Medium: Prepare and dispense medium under

100% CO2 Add components, except carbohydrate solution, Na2CO3,

L-cysteine·HCl·H2O solution, and Na2S·9H2O solution, to

distilled/de-ionized water and bring volume to 960.0mL Mix thoroughly Gently

heat and bring to boiling Continue boiling for 5 min Cool to room

temperature while sparging with 100% CO2 Add Na2CO3 Continue

sparging with 100% CO2 until pH reaches 6.8 Distribute into

rubber-stoppered tubes under 100% CO2 Autoclave for 15 min at 15 psi

pres-sure–121°C Aseptically and anaerobically add 20.0mL of sterile

car-bohydrate solution, 10.0mL of sterile L-cysteine·HCl·H2O solution,

and 10.0mL of sterile Na2S·9H2O solution or, using a syringe, inject

the appropriate amount of sterile carbohydrate solution, sterile

Na2S·9H2O solution, and sterile L-cysteine·HCl·H2O solution into

in-dividual tubes containing medium

Use: For the cultivation of Butyrivibrio species

Butzler Medium

See: Campylobacter Selective Medium, Butzler’s

Butzler’s Campylobacter Medium

See: Campylobacter Selective Medium, Butzler’s

BY Agar Medium (ATCC Medium 2038)

Agar 15.0g Yeast extract 5.0g Pancreatic digest of casein 5.0g Beef extract 5.0g NaCl 2.5g

K2HPO4 0.1g MgSO4·7H2O 0.05g

pH 7.2 ± 0.2 at 25°C

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

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

Use: For the cultivation and maintenance of Paracoccus thiocyanatus.

BY+ Medium

Glucose 5.0g Peptone 1.0g Yeast extract 1.0g Seawater 1.0L

Preparation of Medium: Combine components Mix thoroughly Gently heat and bring to boiling Distribute into flasks or tubes Auto-clave for 15 min at 15 psi pressure–121°C

Use: For the cultivation of Arenariomyces triseptatus, Haliphthoros

milfordensis, Haliphthoros philippinensis, Halosphaeria salina, Japonochytrium species, Lignincola laevis, Lindra thalassiae, Schizochytrium aggregatum, Thraustochytrium species, and Torpe-dospora radiata

BYE Agar

Pancreatic digest of casein 16.0g Agar 13.5g Brain heart, solids from infusion 8.0g Peptic digest of animal tissue 5.0g NaCl 5.0g Glucose 2.0g

Na2HPO4 2.5g Yeast extract 2.0g Blood, human or animal, sterile 150.0mL

pH 7.8–8.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except blood, to dis-tilled/deionized water and bring volume to 850.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add 150.0mL of sterile blood Outdated, citrated, or hepa-rinized blood (blood from a blood bank is acceptable) Pour into sterile Petri dishes

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288 BYE HiVeg Agar with Blood

Use: For the isolation and cultivation of Mycoplasma species and L

-forms of bacteria For the detection of Mycoplasma species in tissue

cul-ture and cell lines

BYE HiVeg Agar with Blood

Agar 13.0g

Plant infusion 10.0g

Plant peptone No 3 10.0g

Plant special infusion 7.5g

NaCl 5.0g

Na2HPO4 2.5g

Glucose 2.0g

Yeast extract 2.0g

Horse or human blood, sterile 150.0mL

pH 7.9 ± 0.2 at 25°C

Source: This medium, without blood, is available as a premixed

pow-der from HiMedia

Preparation of Medium: Add components, except blood, to

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

Aseptically add 150.0mL of sterile blood Outdated, citrated, or

hepa-rinized blood (blood from a blood bank is acceptable) Pour into sterile

Petri dishes

Use: For the isolation and cultivation of Mycoplasma species and L

-forms of bacteria For the detection of Mycoplasma species in tissue

cul-ture and cell lines

BYE HiVeg Broth with Blood

Plant infusion 10.0g

Plant peptone No 3 10.0g

Plant special infusion 7.5g

NaCl 5.0g

Na2HPO4 2.5g

Glucose 2.0g

Yeast extract 2.0g

Horse or human blood, sterile 150.0mL

pH 7.9 ± 0.2 at 25°C

Source: This medium, without blood, is available as a premixed

pow-der from HiMedia

Preparation of Medium: Add components, except blood, to

Aseptically add 150.0mL of sterile blood Outdated, citrated, or

hepa-rinized blood (blood from a blood bank is acceptable)

Use: For the cultivation of Mycoplasma species and L-forms of bacteria

BYEB (Buffered Yeast Extract Broth)

ACES buffer

(2-[(2-amino-2-oxoethyl)-amino]-ethane sulfonic acid) 10.0g

Yeast extract 10.0g

α-Ketoglutarate 1.0g

Fe4(P2O7)3·9H2O 0.25g

pH 6.9 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 6.9 Fil-ter sFil-terilize Aseptically distribute into sFil-terile tubes or flasks

Use: For the cultivation of Legionella pneumophila.

C/10 Agar

Agar 15.0g Pancreatic digest of casein 3.0g CaCl2·2H2O 1.36g

pH 7.2 ± 0.2 at 25°C

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

to boiling Adjust pH t o 7.2 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation of Cytophaga flevensis, Flexibacter filiformis,

Myxococcus

C/10 Medium Reichenbach

Agar 15.0g Pancreatic digest of casein 3.0g CaCl2 1.0g

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except agar, to distilled/ deionized water and bring volume to 1.0L Adjust pH to 7.2 Add agar Mix thoroughly Gently heat to boiling Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation and maintenance of Flexibacter filiformis.

fulvus, and Myxococcus xanthus.

C 3G Spiroplasma Medium

Sucrose 100.0g Phenol Red 10.0mg PPLO broth without Crystal Violet 500.0mL Horse serum 150.0mL Fresh yeast extract solution 50.0mL CMRL-1066 medium 5.0mL

pH 7.5 ± 0.2 at 25°C

Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems

PPLO Broth without Crystal Violet:

Beef heart, infusion from 11.52g Peptone 2.32g NaCl 1.15g

Preparation of PPLO Broth without Crystal Violet: Add components to distilled/deionized water and bring volume to 500.0mL Mix thoroughly

Fresh Yeast Extract Solution:

Baker’s yeast, live, pressed, starch-free 25.0g

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C 3N Spiroplasma Medium 289

Preparation of Fresh Yeast Extract Solution: Add the live

Bak-er’s yeast to 100.0mL of distilled/deionized water Autoclave for 90

min at 15 psi pressure–121°C Allow to stand Remove supernatant

so-lution Adjust pH to 6.6–6.8 Filter sterilize

CMRL-1066 Medium:

NaCl 6.8g

NaHCO3 2.2g

D-Glucose 1.0g

KCl 0.4g

CaCl2, anhydrous 0.2g

MgSO4·7H2O 0.2g

NaH2PO4·H2O 0.14g

L-Glutamine 0.1g

Sodium acetate·3H2O 0.083g

L-Glutamic acid 0.075g

L-Arginine·HCl 0.07g

L-Lysine·HCl 0.07g

L-Leucine 0.06g

Glycine 0.05g

Ascorbic acid 0.05g

L-Proline 0.04g

L-Tyrosine 0.04g

L-Aspartic acid 0.03g

L-Threonine 0.03g

L-Alanine 0.025g

L-Phenylalanine 0.025g

L-Serine 0.025g

L-Valine 0.025g

L-Cystine 0.02g

L-Histidine·HCl·H2O 0.02g

L-Isoleucine 0.02g

Phenol Red 0.02g

L-Methionine 0.015g

Deoxyadenosine 0.01g

Deoxycytidine 0.01g

Deoxyguanosine 0.01g

Glutathione, reduced 0.01g

Thymidine 0.01g

Hydroxy-L-proline 0.01g

L-Tryptophan 0.01g

Nicotinamide adenine dinucleotide 7.0mg

Tween™ 80 5.0mg

Sodium glucoronate·H2O 4.2mg

Coenzyme A 2.5mg

Cocarboxylase 1.0mg

Flavin adenine dinucleotide 1.0mg

Nicotinamide adenine

dinucleotide phosphate 1.0mg

Uridine triphosphate 1.0mg

Choline chloride 0.5mg

Cholesterol 0.2mg

5-Methyldeoxycytidine 0.1mg

Inositol 0.05mg

p-Aminobenzoic acid 0.05mg

Niacin 0.025mg

Niacinamide 0.025mg

Pyridoxine 0.025mg

Pyridoxal·HCl 0.025mg

Biotin 0.01mg

D-Calcium pantothenate 0.01mg Folic acid 0.01mg Riboflavin 0.01mg Thiamine·HCl 0.01mg

Source: CMRL-1066 medium is available as a premixed powder from BD Diagnostics

Preparation of CMRL-1066 Medium: Add components to dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly Ad-just pH to 7.2 Filter sterilize

Preparation of Medium: Add components—except horse serum, fresh yeast extract, and CMRL medium—to distilled/deionized water and bring volume to 795.0mL Adjust pH to 7.5 Autoclave for 15 min

at 15 psi pressure–121°C Aseptically add 150.0mL of sterile horse se-rum, 50.0mL of sterile fresh yeast extract solution, and 5.0mL of sterile CMRL medium Distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Spiroplasma species.

C 3N Spiroplasma Medium

Sucrose 12.0g Phenol Red 10.0mg PPLO broth without Crystal Violet 50.0mL Horse serum 20.0mL Fresh yeast extract solution 5.0mL CMRL-1066 medium 0.5mL

pH 7.5 ± 0.2 at 25°C

PPLO Broth without Crystal Violet:

Beef heart, infusion from 11.52g Peptone 2.32g NaCl 1.15g

Source: PPLO broth without Crystal Violet is available as a premixed powder from BD Diagnostic Systems

Preparation of PPLO Broth without Crystal Violet: Add components to distilled/deionized water and bring volume to 500.0mL Mix thoroughly

Fresh Yeast Extract Solution:

Baker’s yeast, live, pressed, starch-free 25.0g

Preparation of Fresh Yeast Extract Solution: Add the live Bak-er’s yeast to 100.0mL of distilled/deionized water Autoclave for 90 min at 15 psi pressure–121°C Allow to stand Remove supernatant so-lution Adjust pH to 6.6–6.8 Filter sterilize

CMRL-1066 Medium:

NaCl 6.8g NaHCO3 2.2g D-Glucose 1.0g KCl 0.4g L-Cysteine·HCl·H2O 0.26g CaCl2, anhydrous 0.2g MgSO4·7H2O 0.2g NaH2PO4·H2O 0.14g L-Glutamine 0.1g Sodium acetate·3H2O 0.083g L-Glutamic acid 0.075g L-Arginine·HCl 0.07g L-Lysine·HCl 0.07g

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290 CAE Agar Base with Triphenyltetrazolium Chloride

L-Leucine 0.06g

Glycine 0.05g

Ascorbic acid 0.05g

L-Proline 0.04g

L-Tyrosine 0.04g

L-Aspartic acid 0.03g

L-Threonine 0.03g

L-Alanine 0.025g

L-Phenylalanine 0.025g

L-Serine 0.025g

L-Valine 0.025g

L-Cystine 0.02g

L-Histidine·HCl·H2O 0.02g

L-Isoleucine 0.02g

Phenol Red 0.02g

L-Methionine 0.015g

Deoxyadenosine 0.01g

Deoxycytidine 0.01g

Deoxyguanosine 0.01g

Glutathione, reduced 0.01g

Thymidine 0.01g

Hydroxy-L-proline 0.01g

L-Tryptophan 0.01g

Nicotinamide adenine dinucleotide 7.0mg

Tween™ 80 5.0mg

Sodium glucoronate·H2O 4.2mg

Coenzyme A 2.5mg

Cocarboxylase 1.0mg

Flavin adenine dinucleotide 1.0mg

Nicotinamide adenine

dinucleotide phosphate 1.0mg

Uridine triphosphate 1.0mg

Choline chloride 0.5mg

Cholesterol 0.2mg

5-Methyldeoxycytidine 0.1mg

Inositol 0.05mg

Niacin 0.025mg

Niacinamide 0.025mg

Pyridoxine 0.025mg

Pyridoxal·HCl 0.025mg

Biotin 0.01mg

D-Calcium pantothenate 0.01mg

Folic acid 0.01mg

Riboflavin 0.01mg

Thiamine·HCl 0.01mg

Source: CMRL-1066 medium is available as a premixed powder

from BD Diagnostics

Preparation of CMRL-1066 Medium: Add components to

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

Ad-just pH to 7.2 Filter sterilize

Preparation of Medium: Add components—except horse serum,

fresh yeast extract, and CMRL medium—to distilled/deionized water

and bring volume to 75.0mL Adjust pH to 7.5 Autoclave for 15 min

at 15 psi pressure–121°C Aseptically add 20.0mL of sterile horse

se-rum, 5.0mL of sterile yeast extract, and 0.5mL of sterile CMRL

medi-um Distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Spiroplasma kunkelii.

CA

See: Carrot Decoction Agar

CA YE Broth

See: Casamino Acids Yeast Extract Salts Broth, Gorbach

Cadmium Fluoride Acriflavin Tellurite Medium

CAE Agar Base with Triphenyltetrazolium Chloride

(Citrate Azide Enterococcus HiVeg Agar Base)

Agar 15.0g Casein enzymatic hydrolysate 15.0g Sodium citrate 15.0g

KH2PO4 5.0g Yeast extract 5.0g

Na2CO3 2.0g Polysorbate 80 1.0g NaN3 0.4g 2,3,5-Triphenyltetrazolium chloride solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Source: This medium, without triphenyltetrazolium chloride solution,

is available as a premixed powder from HiMedia

Caution: Sodium azide is toxic Azides also react with metals and disposal must be highly diluted

2,3,5-Triphenyltetrazolium Chloride Solution:

2,3,5-Triphenyltetrazolium chloride 0.1g

Preparation of 2,3,5-Triphenyltetrazolium Chloride Solu-tion: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except 2,3,5-triphe-nyltetrazolium chloride solution, to distilled/deionized water and bring volume to 990.0mL Mix thoroughly Gently heat and bring to boiling Autoclave for 10 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add 10.0mL 2,3,5-triphenyltetrazolium chloride solution Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the isolation, cultivation, and enumeration of entercocci in water, sewage, and feces by the membrane filter method For the direct plating of specimens for the detection and enumeration of fecal strep-tococci

CAE HiVeg Agar Base with Triphenyltetrazolium Chloride

(Citrate Azide Enterococcus HiVeg Agar Base)

Agar 15.0g Plant hydrolysate 15.0g Sodium citrate 15.0g

KH2PO4 5.0g Yeast extract 5.0g

Na2CO3 2.0g Polysorbate 80 1.0g NaN3 0.4g 2,3,5-Triphenyltetrazolium chloride solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Source: This medium, without triphenyltetrazolium chloride solution,

is available as a premixed powder from HiMedia

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CAL Broth 291

Caution: Sodium azide is toxic Azides also react with metals and

disposal must be highly diluted

2,3,5-Triphenyltetrazolium Chloride Solution:

2,3,5-Triphenyltetrazolium chloride 0.1g

Preparation of 2,3,5-Triphenyltetrazolium Chloride

Solu-tion: Add 2,3,5-triphenyltetrazolium chloride to distilled/deionized

water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except

2,3,5-triphe-nyltetrazolium chloride solution, to distilled/deionized water and bring

volume to 990.0mL Mix thoroughly Gently heat and bring to boiling

Aseptically add 10.0mL 2,3,5-triphenyltetrazolium chloride solution

Mix thoroughly Pour into sterile Petri dishes or distribute into sterile

tubes

Use: For the isolation, cultivation, and enumeration of entercocci in

water, sewage, and feces by the membrane filter method For the direct

plating of specimens for the detection and enumeration of fecal

strep-tococci

Caffeic Acid Ferric Citrate Test Medium

(CAFC Test Medium) (Caffeic Acid Agar)

Agar 20.0g

(NH4)2SO4 5.0g

Glucose 5.0g

Yeast extract 2.0g

K2HPO4 0.8g

MgSO4·3H2O 0.7g

Caffeic acid·1/2H2O 0.18g

Chloramphenicol 0.05g

Ferric citrate solution 4.0mL

pH 6.5 ± 0.2 at 25°C

Ferric Citrate Solution:

Composition per 20.0mL:

Ferric citrate 100.0mg

Preparation of Ferric Citrate Solution: Add ferric citrate to

20.0mL of distilled/deionized water Mix thoroughly

Preparation of Medium: Add components, except

chlorampheni-col, to distilled/deionized water and bring volume to 1.0L Mix

thor-oughly Heat to boiling Autoclave for 15 min at 15 psi pressure–

121°C Cool to 45°–50°C Aseptically add 0.05g of chloramphenicol

Mix thoroughly Pour into sterile Petri dishes

Use: For the isolation and presumptive identification of Cryptococcus

neoformans Cryptococcus neoformans appears as dark brown

colo-nies All other Cryptococcus species appear as light brown or

nonpig-mented colonies

Caffeine Medium

Agar 15.0g

Solution A 400.0mL

Solution B 400.0mL

Solution C 200.0mL

pH 5.0 ± 0.2 at 25°C

Solution A:

Na2HPO4 7.8g

KH2PO4 3.0g Caffeine 1.0g NaCl 0.58g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 400.0mL Mix thoroughly Adjust pH to 5.0

Solution B:

MgSO4·7H2O 0.12g CaCl2·2H2O 11.0mg

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 400.0mL Mix thoroughly

Solution C:

FeCl3 16.0mg

Preparation of Solution C: Add FeCl3 to distilled/deionized water and bring volume to 200.0mL Mix thoroughly

Preparation of Medium: To 400.0mL of solution A, add 400.0mL

of solution B and 200.0mL of solution C Adjust pH to 5.0 Add agar Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min

at 15 psi pressure–121°C Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the cultivation of Pseudomonas species.

CAGV Medium

CAL Agar (Cellobiose Arginine Lysine Agar)

(Yersinia Isolation Agar)

Agar 20.0g L-Arginine·HCl 6.5g L-Lysine·HCl 6.5g NaCl 5.0g Cellobiose 3.5g Yeast extract 3.0g Sodium deoxycholate 1.5g Neutral Red 0.03g

pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Heat to boiling Do not autoclave Pour into sterile Petri dishes

Use: For the isolation and characterization of Yersinia enterocolitica from fecal specimens and enumeration of Yersinia enterocolitica from

water and other liquid specimens

CAL Broth (Cellobiose Arginine Lysine Broth)

L-Arginine·HCl 6.5g L-Lysine·HCl 6.5g NaCl 5.0g Cellobiose 3.5g Yeast extract 3.0g

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292 CAL HiVeg Agar

Sodium deoxycholate 1.5g

Neutral Red 0.03g

pH 7.3 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Heat to boiling Do

not autoclave Distribute into sterile tubes in 6.0–8.0mL volumes

Use: For the isolation and characterization of Yersinia enterocolitica

from fecal specimens and enumeration of Yersinia enterocolitica from

CAL HiVeg Agar (Cellobiose Arginine Lysine HiVeg Agar)

Agar 20.0g

L-Arginine 6.5g

L-Lysine hydrochloride 6.5g

NaCl 5.0g

Cellobiose 3.5g

Yeast extract 3.0g

Synthetic detergent No III 1.5g

Neutral Red 0.03g

pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from

Hi-Media

not autoclave Pour into sterile Petri dishes

from fecal specimens and enumeration of Y enterocolitica from water.

CAL HiVeg Broth (Cellobiose Arginine Lysine HiVeg Broth)

L-Arginine 6.5g

L-Lysine hydrochloride 6.5g

NaCl 5.0g

Cellobiose 3.5g

Yeast extract 3.0g

Synthetic detergent No III 1.5g

Neutral Red 0.03g

pH 7.1 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from

Hi-Media

not autoclave Distribute into sterile tubes

from fecal specimens and enumeration of Yersinia enterocolitica from

Calcium Caseinate Agar

Agar 13.0g

Peptic digest of animal tissue 4.0g

Calcium caseinate 3.5g

Meat extract 2.0g

Casein enzymic hydrolysate 2.0g

CaCl2·2H2O 0.2g Tri-potassium citrate 0.35g

Na2HPO4 0.105g

KH2PO4 0.035g NaCl 5.0g

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 Distribute into tubes

or flasks Gently heat and bring to boiling Boil for 10 min Autoclave for 15 min at 15 psi pressure–121°C Mix thoroughly while pouring into Petri dishes

Use: For the detection and enumeration of proteolytic microorganisms

in foodstuffs and other materials

Calcium Caseinate Agar with Skim Milk

Agar 13.0g Skim Milk 10.0g Peptic digest of animal tissue 4.0g Calcium caseinate 3.5g Meat extract 2.0g Casein enzymic hydrolysate 2.0g CaCl2·2H2O 0.2g Tri-potassium citrate 0.35g

Na2HPO4 0.105g

KH2PO4 0.035g NaCl 5.0g

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 Distribute into tubes

or flasks Gently heat and bring to boiling Boil for 10 min Autoclave for 15 min at 15 psi pressure–121°C Mix thoroughly while pouring into Petri dishes

Use: For the detection and enumeration of proteolytic microorganisms

in foodstuffs and other materials

Caldicellulosiruptor Medium

Pancreatic digest of casein 2.0g

K2HPO4 1.5g Cellobiose 1.0g Yeast extract 1.0g NaCl 0.9g

NH4Cl 0.9g

KH2PO4 0.75g

L-Cysteine·HCl 0.75g MgCl2·6H2O 0.4g FeCl3·6H2O 2.5mg Resazurin 0.5mg Trace elements solution SL-10 1.0mL

pH 7.2 ± 0.2 at 25°C

Trace Elements Solution SL-10:

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

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Caldisphaera Medium 293

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

compo-nents Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at

15 psi pressure–121°C

100% N2 Add components to distilled/deionized water and bring

vol-ume to 1.0L Mix thoroughly Sparge with 100% N2 Anaerobically

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

121°C

Use: For the cultivation of Caldicellulosiruptor saccharolyticus.

Caldicellulosiruptor Medium

Pancreatic digest of casein 2.0g

K2HPO4 1.5g

Cellulose 1.0g

Cellobiose 1.0g

Yeast extract 1.0g

NaCl 0.9g

NH4Cl 0.9g

KH2PO4 0.75g

MgCl2·6H2O 0.4g

FeCl3·6H2O 2.5mg

L-Cysteine·HCl 0.75g

Resazurin 0.5mg

pH 7.2 ± 0.2 at 25°C

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

100% N2 Add components to distilled/deionized water and bring

vol-ume to 1.0L Mix thoroughly Sparge with 100% N2 Anaerobically

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

121°C

Use: For the cultivation of Caldicellulosiruptor saccharolyticus.

Caldisphaera Medium

(DSMZ Medium 991)

Sulfur, powder 10.0g MnCl2·4H2O 1.8g (NH4)2SO4 1.3g

KH2PO4 0.28g MgSO4·7H2O 0.25g CaCl2·2H2O 0.07g FeCl3·6H2O 0.02g

Na2B4O7·10H2O 4.5mg Resazurin 1.0mg ZnSO4·7H2O 0.22mg CuCl2·2H2O 0.05mg

Na2MoO4·4H2O 0.03mg VOSO4·2H2O 0.03mg

Na3-citrate·2H2O 0.03mg CoSO4 0.01mg Vitamin solution 10.0mL

Na2S·9H2O solution 10.0mL Yeast extract solution 5.0mL

pH 4.3 ± 0.2 at 25°C

Yeast Extract Solution:

Yeast extract 0.5g

Preparation of Yeast Extract Solution : Add yeast extract to

dis-tilled/deionized water and bring volume to 5.0mL Mix thoroughly Sparge under 100% N2 gas for 3 min Autoclave for 15 min at 15 psi pressure–121°C Store under N2 gas

Na 2 S·9H 2 O Solution :

Na2S·9H2O 0.5g

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

to room temperature

Vitamin Solution:

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 vitamin solution, yeast extract solution, sulfur, and Na2S·9H2O solution, to distilled/deion-ized water and bring volume to 975.0mL Mix thoroughly Gently heat and bring to boiling Boil for 3 min Cool to room temperature under 80% N2 + 20% CO2 Adjust pH to 3.5 with 10N H2SO4 Dispense under same gas atmosphere in suitable culture vessels (e.g., 20.0mL of the medium in 120 mL serum bottles) Autoclave for 15 min at 15 psi pres-sure–121°C Steam sulfur for 3 hr on each of 3 successive days

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Asep-294 Calditerrivibrio Medium

tically mix the sterilized sulfur with the medium and add vitamins and

yeast extract from sterile, anaerobic stock solutions Prior to

inocula-tion change atmosphere to 80% H2 + 20% CO2 Aseptically and

anox-ically add Na2S·9H2O Adjust pH to 4.0–4.5 if necessary After

inoculation pressurize vials to 1 bar overpressure with 80% H2 + 20%

CO2 gas mixture

Use: For the cultivation of Caldisphaera spp.

Calditerrivibrio Medium

NaNO3 0.85g

Na-acetate 0.82g

NH4Cl 0.54g

MgCl2·6H2O 0.2g

CaCl2·2H2O 0.15g

KH2PO4 0.14g

Resazurin 0.5mg

NaHCO3 solution 10.0mL

Vitamin solution 10.0mL

Na2S·9H2O solution 10.0mL

Trace element solution SL-10 1.0mL

Selenite/tungstate solution .1.0mL

pH 7.0 ± 0.2 at 25°C

NaHCO 3 Solution :

NaHCO3 2.5g

Preparation of NaHCO 3 Solution: Add components to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Sparge

with 20% CO2 + 80% H2 Autoclave for 15 min at 15 psi pressure–

121°C Cool to room temperature

Na2S·9H2O 0.5g

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

Selenite/Tungstate Solution:

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

Vitamin Solution:

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

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

Preparation of Medium: Add components, except bicarbonate so-lution, sulfide soso-lution, and vitamin soso-lution, to distilled/deionized wa-ter and bring volume to 970.0mL Mix thoroughly Gently heat and bring to boiling Boils for several minutes Cool to room temperature while sparging with 80% N2 + 20% CO2 Distribute into screw-capped tubes or bottles under an atmosphere of 80% N2 + 20% CO2 Autoclave for 15 min at 15 psi pressure–121°C Add the bicarbonate solution, sul-fide solution, and vitamin solution Adjust the final pH to 7.0

Use: For the cultivation of Calditerrivibrio spp.

Caldivirga Medium

(NH4)2SO4 1.3g Sulfur, powdered 10.0g

Na2S·9H2O 0.5 g

KH2PO4 0.28g MgSO4·7H2O 0.25g CaCl2·2H2O 0.07g FeCl3·6H2O 0.02g

Na2B4O7·10H2O 4.5mg MnCl2·4H2O 1.8mg Resazurin 0.5mg ZnSO4·7H2O 0.22mg CuCl2·2H2O 0.05mg

Na2MoO4·2H2O 0.03mg VOSO4·2H2O 0.03mg CoSO4 0.01mg

Na2S·9H2O solution 7.5mL Yeast extract solution 5.0mL

Na3-citrate·2H2O 3.0mL Vitamin solution 1.0mL

pH 4.0± 0.2 at 25°C

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

to room temperature

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