Handbook of Microbiological Media, Fourth Edition part 62 docx

16.5g Preparation of Salts Solution A: Add components to distilled/de-ionized water and bring volume to 1.0L.. 0.4g HCl, concentrated ...0.1mL MnSO4·H2O ...0.1mL Preparation of Salts Sol

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Double Sugar Agar, Russell 605

Dorset Egg Medium Compositionper liter:

Homogenized whole egg 950.0mL

Glycerol 50.0mL

pH 6.8–7.4 at 25°C

Source: This medium is available as a prepared medium from BD

Di-agnostic Systems

Homogenized Whole Egg:

Compositionper liter:

Whole eggs 18–24

Preparation of Homogenized Whole Egg: Use fresh eggs, less

than 1 week old Scrub the shells with soap Let stand in a soap solution

for 30 min Rinse in running water Soak eggs in 70% ethanol for 15

min Break the eggs into a sterile container Homogenize by shaking

Filter through four layers of sterile cheesecloth into a sterile graduated

cylinder Measure out 1.0L

Preparation of Medium: Filter sterilize glycerol Combine

glycer-ol and homogenized whglycer-ole egg Mix thoroughly Distribute into sterile

screw-capped tubes Place tubes in a slanted position Inspissate at

85°C (moist heat) for 45 min

Use: For the maintenance of Mycobacterium species

Double-Strength Crude Lactobacillus Medium

Compositionper 1025.0mL:

Yeast extract (Basamine) 20.0g

Sucrose 20.0g

Casein hydrolysate 15.0g

Potassium acetate 3.0g

Histidine·HCl·H2O 2.0g

Ascorbic acid 1.0g

Pyridoxamine·HCl 33.0μg

Salts solution A 20.0mL

Salts solution B 5.0mL

pH 5.4 ± 0.2 at 25°C

Salts Solution A:

K2HPO4·3H2O 16.5g

KH2PO4·H2O 16.5g

Preparation of Salts Solution A: Add components to

distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Autoclave for

15 min at 15 psi pressure–121°C

Salts Solution B:

MgSO4·7H2O 8.0g

FeSO4·7H2O 0.4g

NaCl 0.4g

HCl, concentrated 0.1mL

MnSO4·H2O 0.1mL

Preparation of Salts Solution B: Add components to

distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Autoclave for

15 min at 15 psi pressure–121°C

Preparation of Medium: Add components, except salts solution A

and salts solution B, to distilled/deionized water and bring volume to

975.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–

121°C Aseptically add 20.0mL of sterile salts solution A and 5.0mL

of sterile salts solution B Mix thoroughly Adjust pH to 5.4 Mix

thor-oughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of Lactobacillus species.

Double-Strength Crude Medium for Lactobacillus

Sucrose 20.0g Yeast extract 20.0g Casein hydrolysate 15.0g Potassium acetate 3.0g Histidine·HCl 2.0g Ascorbic acid 1.0g Pyridoxamine·HCl 33.0μg Salts solution A 20.0mL Salts solution B 5.0mL

pH 5.4 ± 0.2 at 25°C

Salts Solution A:

KH2PO4·H2O 16.5g

K2HPO4·3H2O 16.5g

Preparation of Salts Solution A: Add components to distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly

Salts Solution B:

MgSO4·7H2O 8.0g FeSO4·7H2O 0.4g MnSO4·H2O 0.4g NaCl 0.4g HCl, concentrated 0.1mL

Preparation of Salts Solution B: Add components to distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly

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 to 5.4 with acetic acid Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation and maintenance of Lactobacillus species.

Double Sugar Agar, Russell (Russell Double Sugar Agar) Composition per liter:

Agar 15.0g Lactose 10.0g Casein enzymic hydrolysate 7.5g NaCl 5.0g Beef extract 3.0g Peptic digest of animal tissue 2.5g Glucose 1.0g Phenol Red 0.025g

pH 7.3 ± 0.2 at 25°C

Source: This medium is available from HiMedia

to boiling Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Allow the tubes to solidify in slanting position to form a generous butt

Use: For the differentiation of Gram-negative enteric bacilli on the basis of their ability to ferment glucose and lactose with or without gas formation

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606 Double Sugar HiVeg Agar

Double Sugar HiVeg Agar

Agar 15.0g

Lactose 10.0g

Plant hydrolysate 7.5g

NaCl 5.0g

Plant extract 3.0g

Plant peptone 2.5g

Glucose 1.0g

Phenol Red 0.025g

pH 7.3 ± 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

wa-ter and bring volume to 1.0L Mix thoroughly Gently heat and bring to

boiling Distribute into tubes Autoclave for 15 min at 15 psi pressure–

121°C Allow tubes to cool in a slanted position

Use: For the identification of Gram-negative enteric bacilli based on

their fermentation of glucose and lactose Bacteria that ferment both

glucose and lactose produce a yellow slant and yellow butt Bacteria

that ferment glucose but do not ferment lactose produce a red slant and

a yellow butt Bacteria that ferment neither glucose nor lactose produce

an unchanged pink-orange color

Doyle and Roman Enrichment Medium

Pancreatic digest of casein 10.0g

Peptic digest of animal tissue 10.0g

NaCl 5.0g

Sodium succinate 3.0g

Yeast extract 2.0g

Glucose 1.0g

NaHSO3 0.1g

L-Cysteine·HCl·H2O 0.1g

Horse blood, lysed 70.0mL

Antibiotic solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Antibiotic Solution:

Cycloheximide 0.05g

Vancomycin 0.015g

Trimethoprim lactate 5.0mg

Polymyxin B 200,000U

Preparation of Antibiotic Solution: Add components to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Filter

sterilize

Caution: Cycloheximide is toxic Avoid skin contact or aerosol

for-mation and inhalation

Preparation of Medium: Add components, except antibiotic

solu-tion and horse blood, to distilled/deionized water and bring volume to

920.0mL Mix thoroughly Gently heat and bring to boiling Autoclave

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

add sterile antibiotic solution and horse blood Mix thoroughly

Asep-tically distribute into sterile flasks in 90.0–100.0mL volumes

Use: For the cultivation and enrichment of Campylobacter species

from foods

Doyle's Enrichment Broth Base with Antibiotic Solution Compositionper liter:

Casein enzymatic hydrolysate 10.0g Peptic digest of animal tissue 10.0g NaCl 5.0g Sodium succinate 3.0g Yeast extract 2.0g Glucose 1.0g

L-Cysteine·HCl 0.1g NaHSO3 0.1g Antibiotic solution 10.0mL

pH 7.0 ± 0.2 at 25°C

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

Cycloheximide 0.05g Vancomycin 0.015g Trimethoprim lactate 5.0mg Polymyxin B 200,000U

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

Caution: Cycloheximide is toxic Avoid skin contact or aerosol for-mation and inhalation

Preparation of Medium: Add components, except antibiotic 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 a selective sterile antibiotic solution Generally 50.0mL of defibrinated horse blood is also added as an enrichment Mix thoroughly

from foods

Doyle's Enrichment HiVeg Broth Base

with Antibiotic Solution Compositionper liter:

Plant hydrolysate 10.0g Plant peptone 10.0g NaCl 5.0g Sodium succinate 3.0g Yeast extract 2.0g Glucose 1.0g

L-Cysteine·HCl 0.1g NaHSO3 0.1g Antibiotic solution 10.0mL

pH 7.0 ± 0.2 at 25°C

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

Cycloheximide 0.05g Vancomycin 0.015g Trimethoprim lactate 5.0mg Polymyxin B 200,000U

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Drigalski Litmus Lactose Agar 607

Preparation of Antibiotic Solution: Add components to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Filter

sterilize

Caution: Cycloheximide is toxic Avoid skin contact or aerosol

for-mation and inhalation

Preparation of Medium: Add components, except antibiotic

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 a selective

sterile antibiotic solution Generally 50.0mL of defibrinated horse

blood is also added as an enrichment Mix thoroughly

from foods

DP Agar Compositionper liter:

Agar 20.0g

Yeast extract 10.0g

K2HPO4 7.0g

Glucose 5.0g

KH2PO4 3.0g

Trisodium citrate·3H2O 0.5g

MgSO4·7H2O 0.1g

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 of Escherichia coli.

DPA with Calcium Carbonate

Agar 20.0g

Glucose 20.0g

Peptone 10.0g

CaCO3 5.0g

Use: For the cultivation and maintenance of Dekkera species

DPM Medium (DSMZ Medium 737) Compositionper liter:

Na-propionate 1.20g

KH2PO4 1.0g

MgSO4·7H2O 0.1g

CaCl2·2H2O 0.01g

Chelated iron solution 1.8mL

Trace elements solution 1.0mL

pH 6.8 ± 0.2 at 25°C

Chelated Iron Solution:

Na2-EDTA 7.56g

FeSO4·5H2O 5.54g

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

Trace Elements Solution:

H3BO3 2.860 MnCl2·4H2O 1.81g ZnSO4·7H2O 0.22g CuSO4·5H2O 0.08g

Na2MoO4·4H2O 0.025g CoCl2 0.025g

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 Distribute into tubes

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

Use: For the cultivation of Frankia sp.

DRBC Agar (Dichloran Rose Bengal Chloramphenicol Agar) Compositionper liter:

Agar 15.0g Glucose 10.0g Peptone 5.0g

KH2PO4 1.0g MgSO4·7H2O 0.5g Rose Bengal 0.025g Dichloran 0.002g Chloramphenicol solution 10.0mL

pH 5.6 ± 0.2 at 25°C

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

Chloramphenicol Solution:

Chloramphenicol 0.1g

Preparation of Chloramphenicol Solution: Add chlorampheni-col to distilled/deionized water and bring volume to 10.0mL Mix thor-oughly Filter sterilize

Preparation of Medium: Add components, except chlorampheni-col 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 45°–50°C Aseptically add sterile chloramphenicol solution Mix thoroughly Pour into sterile Petri

dish-es or distribute into sterile tubdish-es

Use: For the isolation, cultivation, and enumeration of yeasts and molds associated with food spoilage

Drigalski Litmus Lactose Agar Compositionper liter:

Agar 10.0g Lactose 10.0g Meat peptone 5.0g Beef extract 3.0g Litmus 1.0g

pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from Sigma Aldrich

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608 Drigalski Litmus Lactose Agar

Use: For the maintenance of lactic acid bacteria and differentiation of

bacteria on the basis of lactose fermentation Bacteria that ferment

lac-tose appear as red colonies and others as dark blue-purple colonies

Drigalski Litmus Lactose Agar

Lactose 15.0g

Agar 13.0g

NaCl 5.0g

Litmus 1.2g

pH 7.4 ± 0.2 at 25°C

Hi-Media

Drigalski Litmus Lactose Agar, Modified

Agar 16.0g

Lactose 10.0g

Beef extract 4.0g

Bromthymol Blue 0.04g

pH 7.4 ± 0.2 at 25°C

Hi-Media

Drigalski Litmus Lactose HiVeg Agar

Lactose 15.0g

Agar 13.0g

Plant peptone 7.0g

NaCl 5.0g

Litmus 1.2g

pH 7.4 ± 0.2 at 25°C

Hi-Media

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 maintenance of lactic acid bacteria and differentiation of bacteria on the basis of lactose fermentation Bacteria that ferment lac-tose appear as red colonies and others as dark blue-purple colonies

DS Sporulation Medium, Modified

DSA

See: Dextrose Soil Agar

DSA Cellulose

See: Dextrose Sucrose Cellulose Agar

DSIC Medium, Modified (DSMZ Medium 747) Compositionper 994.0mL:

Solution A 910.0mL Solution B 70.0mL Solution C 14.0mL

pH 7.0–7.1 at 25°C

Solution A:

NaCl 125.0g

K2SO4 2.5g Na-acetate 2.0g Yeast extract 0.75g

KH2PO4 0.6g

NH4Cl 0.5g

Na2S2O3·5H2O 0.1g MOPS buffer 10.0mL Vitamin B12 solution 1.0mL Trace elements solution SL-10 1.0mL

Vitamin B 12 Solution:

Vitamin B12 10.0mg

Vitamin B 12 Solution: Add vitamin B12 to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Sparge under 100% N2 gas for 3 min

Trace Elements Solution SL-10:

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

Na2MoO4·2H2O 36.0mg NiCl2·6H2O 24.0mg

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

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

to 10.0mL of HCl solution Mix thoroughly Add distilled/deionized water and bring volume to 1.0L Add remaining components Mix thor-oughly Sparge with 80% N2 + 20% CO2 Autoclave for 15 min at 15 psi pressure–121°C

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Dubos Broth 609

MOPS Buffer:

MOPS [3-(N-morpholino) propane

sulfonic acid] 2.1g

Na-acetate 0.3g

EDTA 0.1g

Preparation of MOPS Buffer: Add components to

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

with 100% N2 Adjust to pH 7.2 Filter sterilize

Preparation of Solution A: Add components to distilled/deionized

water and bring volume to 960.0mL Mix thoroughly Sparge with

100% N2 Gently heat and bring to boiling while continuing to sparge

with 100% N2 Distribute about 13mL aliquots in 15mL Hungate tubes

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

Solution B:

MgCl2·6H2O 10.0g

CaCl2·2H2O 0.2g

Preparation of Solution B: Add components to distilled/deionized

water and bring volume to 70.0mL Mix thoroughly Adjust pH to 7.0

Sparge with 100% N2 Gently heat and bring to boiling while continuing

to sparge with 100% N2 Distribute into a screw-capped bottle

Auto-clave for 15 min at 15 psi pressure–121°C Cool to 25°C

Solution C:

NaHCO3 1.0g

Preparation of Solution C: Add NaHCO3 to distilled/deionized

water and bring volume to 14.0mL Mix thoroughly Sparge with 80%

N2 + 20% CO2 Filter sterilize

Preparation of Medium: Inject 1.0 ml of solution B and 0.2 ml of

solution C in each tube of solution A

Use: For the cultivation of Rhodovibrio sodomensis=Rhodospirillum

sodomense.

DSM 134

See: Haliscomenobacter Medium

DST Agar

See: Diagnostic Sensitivity Test Agar

DTC Agar Compositionper liter:

Agar 20.0g

NaCl 5.0g

Papaic digest of soybean meal 5.0g

Deoxyribonucleic acid 2.0g

Toluidine Blue O 0.1g

Cephalothin solution 10.0mL

Cephalothin Solution:

Cephalothin 1.0g

Preparation of Cephalothin Solution: Add cephalothin to

dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly

Filter sterilize

Preparation of Medium: Add components, except cephalothin

so-lution, 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 sterile ce-phalothin solution Mix thoroughly Pour into sterile Petri dishes

Use: For the isolation and cultivation of Serratia species Serratia

appear as colonies with red halos

DTM Agar (Dermatophyte Test Medium Agar) Composition per liter:

Agar 20.0g Enzymatic digest of soybean meal 10.0g Glucose 10.0g Cycloheximide 0.5g Phenol Red 0.2g Chlortetracycline 0.1g Gentamicin 0.1g

pH 7.3 ± 0.2 at 25°C

Source: Available as a prepared medium from BD Diagnostic Sys-tems

Caution: Cycloheximide is toxic Avoid skin contact or aerosol for-mation and inhalation

Use: For the isolation and cultivation of dermatophytic fungi

Dubos Agar with Filter Paper Compositionper liter:

Agar 15.0g

K2HPO4 1.0g KCl 0.5g MgSO4·7H2O 0.5g NaNO3 0.5g FeSO4·7H2O 0.01g

pH 7.2 ± 0.2 at 25°C

to boiling Adjust pH to 7.2 Autoclave for 15 min at 15 psi pressure– 121°C Pour into sterile Petri dishes Lay sterile strips of Whatman #1 fil-ter paper on the surface of the agar

Use: For the cultivation and maintenance of Cytophaga hutchinsonii.

Dubos Broth Compositionper liter:

Na2HPO4 2.5g

L-Asparagine 2.0g

KH2PO4 1.0g Pancreatic digest of casein 0.5g Tween™ 80 0.2g CaCl2·2H2O 0.5mg CuSO4 0.1mg ZnSO4·7H2O 0.1mg Ferric ammonium citrate 0.05g MgSO4·7H2O 0.01g Bovine serum albumin or bovine serum 20.0mL

pH 6.5 ± 0.2 at 25°C

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610 Dubos Broth Base with Serum and Glycerol

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

Di-agnostic Systems

Preparation of Medium: Add components, except bovine serum or

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

to 980.0mL Mix thoroughly Gently heat and bring to boiling

Auto-clave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Asepti-cally add sterile bovine serum or bovine serum albumin Mix

thoroughly Aseptically distribute into sterile tubes

Use: For the cultivation of Mycobacterium tuberculosis and other

Mycobacterium species.

Dubos Broth Base with Serum and Glycerol

Na2HPO4 2.5g

L-Asparagine 2.0g

KH2PO4 1.0g

Casein enzymatic hydrolysate 0.5g

Polysorbate 80 0.2g

Ferric ammonium citrate 0.05g

MgSO4 0.01g

CaCl2 0.5mg

CuSO4 0.1mg

ZnSO4 0.1mg

Glycerol 50.0mL

Bovine serum or bovine albumin V 20.0mL

pH 6.5 ± 0.2 at 25°C

Source: This medium, without bovine serum or glycerol, is available

as a premixed powder from HiMedia

Preparation of Medium: Add components, except bovine serum,

to distilled/deionized water and bring volume to 980.0mL Mix

thor-oughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi

pressure–121°C Cool to 45°–50°C Aseptically add 20.0mL sterile

bo-vine serum or bobo-vine serum albumin Mix thoroughly Aseptically

dis-tribute into sterile tubes

Dubos Broth with Horse Serum

Na2HPO4 2.5g

L-Asparagine 2.0g

KH2PO4 1.0g

Tween™ 80 0.2g

CaCl2·2H2O 0.5mg

CuSO4 0.1mg

ZnSO4·7H2O 0.1mg

MgSO4·7H2O 0.01g

Horse serum 50.0mL

pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components, except horse serum, to

distilled/deionized water and bring volume to 950.0mL Mix

thorough-ly Gently heat and bring to boiling Autoclave for 15 min at 15 psi

pressure–121°C Cool to 45°–50°C Aseptically add sterile horse

se-rum Mix thoroughly Aseptically distribute into sterile tubes

Use: For the cultivation and maintenance of Corynebacterium species.

Dubos HiVeg Broth Base with Serum and Glycerol Compositionper liter:

Na2HPO4 2.5g

L-Asparagine 2.0g

KH2PO4 1.0g Plant hydrolysate 0.5g Polysorbate 80 0.2g Ferric ammonium citrate 0.05g MgSO4 0.01g CaCl2 0.5mg CuSO4 0.1mg ZnSO4 0.1mg Glycerol 50.0mL Bovine serum or bovine albumin V 20.0mL

pH 6.5 ± 0.2 at 25°C

to distilled/deionized water and bring volume to 980.0mL Mix thor-oughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add 20.0mL sterile bo-vine serum or bobo-vine serum albumin Mix thoroughly Aseptically dis-tribute into sterile tubes

Dubos Mineral Medium Composition per liter:

K2HPO4 1.0g KCl 0.5g MgSO4·7H2O 0.5g NaNO3 0.5g FeSO4·7H2O 0.01g

pH 7.2 ± 0.2 at 25°C

Use: For the isolation and cultivation of Cytophaga species,

Herpeto-siphon species, Saprospira species, and Flexithrix species.

Dubos Oleic Agar Compositionper liter:

Agar 15.0g

Na2HPO4 2.5g

KH2PO4 1.0g

L-Asparagine 1.0g Pancreatic digest of casein 0.5g Ferric ammonium citrate 0.05g MgSO4·7H2O 0.01g CaCl2·2H2O 0.5mg CuSO4 0.1mg ZnSO4·7H2O 0.1mg Dubos oleic albumin complex 20.0mL Penicillin solution 10.0mL

pH 6.6 ± 0.2 at 25°C

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

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Dubos Salts Agar with 1% Sodium Chloride 611

Dubos Oleic Albumin Complex:

Bovine serum albumin, fraction V 5.0g

Oleic acid, sodium salt 0.05g

NaCl (0.85% solution) 100.0mL

Preparation of Dubos Oleic Albumin Complex: Add bovine

serum albumin and oleic acid to 100.0mL of NaCl solution Mix

thor-oughly Filter sterilize

Penicillin Solution:

Penicillin 10,000U

Preparation of Penicillin Solution: Add penicillin to

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

ster-ilize

Preparation of Medium: Add components, except Dubos oleic

al-bumin complex and penicillin solution, to distilled/deionized water and

bring volume to 970.0mL Mix thoroughly Gently heat and bring to

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

50°C Aseptically add sterile Dubos oleic albumin complex and

peni-cillin solution Mix thoroughly Pour into sterile Petri dishes or

distrib-ute into sterile tubes Allow tubes to cool in a slanted position

Use: For the isolation of Mycobacterium tuberculosis and determining

its sensitivity to chemotherapeutic agents

Dubos Oleic HiVeg Agar Base

with Serum and Glycerol Compositionper liter:

Agar 15.0g

Na2HPO4 2.5g

L-Asparagine 1.0g

KH2PO4 1.0g

MgSO4 0.01g

CaCl2 0.5mg

CuSO4 0.1mg

ZnSO4 0.1mg

Glycerol 50.0mL

Bovine serum or bovine albumin V 20.0mL

pH 6.5 ± 0.2 at 25°C

to distilled/deionized water and bring volume to 980.0mL Mix

pressure–121°C Cool to 45°–50°C Aseptically add 20.0mL sterile

bo-vine serum or bobo-vine serum albumin Mix thoroughly Aseptically

dis-tribute into sterile tubes

Dubos Oleic HiVeg Broth Base

with Antibiotic and Oleic Albumin Supplement

Na2HPO4 2.5g

Asparagine 1.0g

KH2PO4 1.0g

Ferric ammonium citrate 0.05g MgSO4 0.01g CaCl2 0.5mg CuSO4 0.1mg ZnSO4 0.1mg Penicllin solution 10.0mL

pH 6.5 ± 0.2 at 25°C

Source: This medium, without penicillin and oleic albumin supple-ment, is available as a premixed powder from HiMedia

Penicillin Solution:

Penicillin 10,000U

Preparation of Penicllin Solution: Add penicillin to distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Filter ster-ilize

Oleic Albumin Supplement:

Oleic acid 10.0g Albumin fraction IV 4.0g NaCl 0.68g NaOH 8.0mg

Preparation of Oleic Albumin Supplement: Add components

to distilled/deionized water and bring volume to 100.0mL Mix thor-oughly Filter sterilize

Preparation of Medium: Add components, except penicllin solu-tion and oleic albumin supplement, to distilled/deionized water and bring volume to 880.0mL 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 sterile penicillin solution and 100.0mL sterile oleic albumin solution Mix thoroughly Aseptically distribute into sterile tubes

Dubos Salts Agar Compositionper liter:

Agar 15.0g

K2HPO4 1.0g KCl 0.5g MgSO4·7H2O 0.5g NaNO3 0.5g FeSO4·7H2O 0.01g Filter paper strips, sterile variable

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except filter paper strips, to distilled/deionized water and bring volume to 1.0L Mix thor-oughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes Aseptically place sterile filter paper strips onto the surface of the solidified medium

Use: For the cultivation and maintenance of Alteromonas species, Cellvibrio mixtus, Cellvibrio species, Cytophaga aurantiaca, Cyto-phaga hutchinsonii, Pseudomonas species, and SporocytoCyto-phaga myxo-coccoides.

Dubos Salts Agar with 1% Sodium Chloride Compositionper liter:

Agar 15.0g NaCl 10.0g

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612 Dubos Salts Agar with Yeast Extract

K2HPO4 1.0g

KCl 0.5g

MgSO4·7H2O 0.5g

NaNO3 0.5g

FeSO4·7H2O 10.0mg

Filter paper strips 1 strip per tube

pH 7.2 ± 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 Aseptically add

a strip of sterile filter paper to the surface of each slant

Use: For the cultivation of Cytophaga species.

Dubos Salts Agar with Yeast Extract

Agar 15.0g

K2HPO4 1.0g

Yeast extract 0.5g

KCl 0.5g

MgSO4·7H2O 0.5g

NaNO3 0.5g

FeSO4·7H2O 0.01g

Filter paper strips, sterile variable

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except filter paper

strips, to distilled/deionized water and bring volume to 1.0L Mix

pressure–121°C Pour into sterile Petri dishes Aseptically place sterile

filter paper strips onto the surface of the solidified medium

Use: For the cultivation and maintenance ofCellulomonas species and

Cellvibrio species.

Dubos Salts Broth Compositionper liter:

K2HPO4 1.0g

KCl 0.5g

MgSO4·7H2O 0.5g

NaNO3 0.5g

FeSO4·7H2O 0.01g

Filter paper strips, sterile variable

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except filter paper

strips, to distilled/deionized water and bring volume to 1.0L Mix

thor-oughly Distribute into tubes containing a filter paper strip (filter paper

strip should protrude above the surface of the broth) Autoclave for 15

min at 15 psi pressure–121°C

Use: For the cultivation ofCytophaga aurantiaca and Pseudomonas

species.

Dubos Salts Broth with Yeast Extract

K2HPO4 1.0g

Yeast extract 0.5g

KCl 0.5g

MgSO4·7H2O 0.5g

NaNO3 0.5g FeSO4·7H2O 0.01g

pH 7.2 ± 0.2 at 25°C

Use: For the cultivation of Cytophaga aurantiaca.

Dubos Salts Broth with Yeast Extract Compositionper liter:

K2HPO4 1.0g Yeast extract 0.5g KCl 0.5g MgSO4·7H2O 0.5g NaNO3 0.5g FeSO4·7H2O 0.01g Filter paper strips, sterile variable

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except filter paper strips, to distilled/deionized water and bring volume to 1.0L Mix thor-oughly Distribute into tubes containing a filter paper strip (filter paper strip should protrude above the surface of the broth) Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation ofCellulomonas species and Cellvibrio

spe-cies.

Dubos Salts Medium (DSMZ Medium 1161) Composition per liter:

Agar 15.0g

K2HPO4 1.0g MgSO4·7H2O 0.5g KCl 0.5g NaNO3 0.5g FeSO4·7H2O 10.0mg Filter paper Variable

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components, except filter paper, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.0 Gently heat while stirring and bring to boiling Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C Pour into sterile Petri dishes or leave in tubes For tubes allow

to solidify on a slant When the agar has solidified, place a strip of ster-ile filter paper on to the surface of each slope Inoculate on to the filter paper

Use: For the cultivation of Cellvibrio spp.

Ducreyi Medium, Revised

See: Haemophilus ducreyi Medium, Revised

Dulaney Slants Compositionper liter:

Egg yolks 50.0mL Locke solution 50.0mL

Locke Solution:

NaCl 0.9g Glucose 0.25g KCl 0.042g

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Dunkelberg Maintenance Medium 613

CaCl2·2H2O 0.024g

Na2CO3 0.02g

Preparation of Locke Solution: Add components to

distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly Filter

sterilize

Preparation of Medium: Aseptically remove the yolks from 5–8

day old hen egg embryos Add an equal volume of sterile Locke solution

containing sterile glass beads Mix thoroughly to homogenize

Asepti-cally distribute into sterile tubes Inspissate tubes in a slanted position at

80°C (moist heat) for 15 min

Use: For the cultivation of Calymmatobacter granulomatis from

clin-ical specimens

Dulcitol Selenite Broth (Selenite-F Broth with Dulcitol)

NaH2PO4 10.0g

Dulcitol 4.0g

HNaO3Se 4.0g

pH 7.0 ± 0.2 at 25°C

Source: This medium is available from HiMedia

Caution: Sodium hydrogen selenite is a very toxic, corrosive agent

and causes teratogenicity Upon contact with skin, wash immediately

with a lot of water

Preparation of Medium: Add sodium hydrogen selenite to

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

remaining components Mix thoroughly Gently heat if needed to get

all compoents to dissolve Distribute into tubes or flasks Sterilize in a

boiling water bath or free flowing steam for 10 min Excessive heating

is detrimental Do not autoclave

Use: For the selective enrichment of Salmonella species.

Duncan-Strong Sporulation Medium, Modified

(DS Sporulation Medium, Modified)

(Sporulation Medium, Modified)

Proteose peptone 15.0g

Na2HPO4·7H2O 10.0g

Raffinose 4.0g

Yeast extract 4.0g

Sodium thioglycolate 1.0g

pH 7.8 ± 0.2 at 25°C

psi pressure–121°C Adjust pH to 7.8 with filter-sterilized 0.66M

Na2CO3 Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation and induction of sporulation of Clostridium

perfringens.

Dung Extract Agar (DSMZ Medium 781) Compositionper liter:

Agar 15.0g

Malt extract 5.0g

(NO3)2·4H2O 0.72g MgSO4·7H2O 0.5g

K2HPO4 0.25g Peptone 0.1g Dung extract 100.0mL

pH 6.9 ± 0.2 at 25°C

Dung Extract:

Horse dung variable

Preparation of Dung Extract: Add an average sized piece of horse dung to150.0mL water Gently heat and bring to boiling Boil for

2 hr in a water bath Filter Use immediately

Use: For the cultivation and maintenance of Panaeolus cyanescens.

Dunkelberg Agar

See: Peptone Starch Dextrose Agar

Dunkelberg Carbohydrate Medium, Modified Composition per 100.0mL:

Proteose peptone No 3 1.5g Carbohydrate 1.0g

Na2HPO4·2H2O 0.207g Phenol Red 0.055g NaH2PO4·H2O 0.038g Horse serum 5.0mL

pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 95.0mL For carbohydrate, use glucose, maltose, or starch Mix thoroughly Filter sterilize Asepti-cally add sterile horse serum Mix thoroughly AseptiAsepti-cally distribute into sterile tubes or flasks

Use: For the cultivation and differentiation of Gardnerella vaginalis

based on its ability to ferment glucose, maltose, or starch

Dunkelberg Maintenance Medium Composition per liter:

Proteose peptone No 3 20.0g Soluble starch 10.0g Agar 8.0g Glucose 2.0g

Na2HPO4 1.0g NaH2PO4 1.0g

pH 6.8 ± 0.2 at 25°C

Preparation of Medium: Add starch to approximately 100.0mL of cold distilled/deionized water Mix thoroughly Add starch solution to 400.0mL of boiling distilled/deionized water Add remaining compo-nents Mix thoroughly Bring volume to 1.0L with distilled/deionized water Distribute into screw-capped tubes Autoclave for 12 min at 8 psi pressure–112°C

Use: For the cultivation and maintenance of Gardnerella vaginalis

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614 Dunkelberg Semisolid Carbohydrate Fermentation Medium

Dunkelberg Semisolid Carbohydrate

Fermentation Medium Composition per liter:

Proteose peptone No 3 20.0g

Carbohydrate 10.0g

Agar 5.0g

Bromcresol Purple solution 1.0mL

pH 7.4 ± 0.2 at 25°C

Bromcresol Purple Solution:

Bromcresol Purple 0.16g

Ethanol (95% solution) 10.0mL

Preparation of Bromcresol Purple Solution: Add Bromcresol

Purple to 10.0mL of ethanol Mix thoroughly Filter sterilize

water and bring volume to 1.0L For carbohydrate, use glucose,

malt-ose, or starch Mix thoroughly Gently heat and bring to boiling Filter

sterilize Aseptically distribute into sterile tubes or flasks

Use: For the cultivation and differentiation of Gardnerella vaginalis

based on its ability to ferment glucose, maltose, or starch

DV Medium Compositionper 100.1mL:

NaCl 1.8g

MgSO4·7H2O 0.5g

Tris-HCl 100.0mg

KCl 60.0mg

NaNO3 50.0mg

Na2SiO3·9H2O 20.0mg

CaCl2 10.0mg

Nitrilotriacetic acid (NTA) 10.0mg

K2HPO4 3.0mg

FeCl2 0.01mg

Vitamin B12 0.3 μg

Metal solution 3.0mL

Vitamin solution 0.1mL

Seawater, charcoal filtered 100.0mL

Metal Solution:

EDTA 25.0mg

MnCl2 1.0mg

H3BO3 0.5mg

FeCl2 0.25mg

ZnCl2 125.0μg

CoCl2 25.0μg

Preparation of Metal Solution: Add components to

distilled/de-ionized water and bring volume to 25.0mL Mix thoroughly

Vitamin Solution:

Inositol 100.0mg

Thymine 50.0mg

Orotic acid 26.0mg

Thiamine·HCl 20.0mg

Calcium D-(+)-pantothenate 10.0mg

Nicotinic acid 10.0mg

Putrescine·2HCl 4.0mg

Pyridoxine·2HCl 4.0mg

Pyridoxamine·2HCl 2.0mg

p-Aminobenzoic acid 1.0mg

Riboflavin 0.5mg Folic acid 0.025mg Biotin 50.0μg Folinic acid 20.0μg Vitamin B12 5.0μg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 25.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except vitamin solu-tion, to charcoal-filtered seawater and bring volume to 100.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Aseptical-ly add 0.1mL of sterile vitamin solution Mix thoroughAseptical-ly AsepticalAseptical-ly distribute into sterile, screw-capped tubes or flasks

Use: For the cultivation of Dunaliella tertiolecta and

Trichosphaer-ium species.

DYA with Calcium Carbonate Compositionper liter:

Glucose 50.0g Agar 20.0g CaCO3 5.0g Yeast extract 5.0g

Use: For the cultivation of Dekkera intermedia, Dekkera bruxellensis,

Dekkera abstinens, Dekkera anomala, Dekkera custersiana, Dekkera lambica, and Dekkera naardenensis

DYAA

See: Dextrose Yeast Asparagine Agar

DYAA Cellulose Compositionper liter:

Agar 20.0g Cellulose 5.0g Glucose 5.0g

K2HPO4 1.0g

L-Asparagine 0.5g MgSO4·7H2O 0.5g (NH4)2SO4 0.5g Yeast extract 0.5g CaCl2 0.1g

Use: For the cultivation of Bipolaris sacchari and Drechslera

biseptata.

DYAA Cellulose Malt Compositionper liter:

Agar 20.0g Malt extract 10.0g Cellulose 5.0g Glucose 5.0g

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