Handbook of Microbiological Media, Fourth Edition part 128 pps

25.0g Preparation of Fresh Yeast Extract Solution : Add the live Bak-er’s yeast to 100.0mL of distilled/deionized water.. 25.0g Preparation of Fresh Yeast Extract Solution : Add the liv

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Mycoplasma Broth 1265

Basal Medium:

Composition per 700.0mL:

Sorbitol 50.0g

Beef heart, solids from infusion 16.2g

Peptone 3.26g

NaCl 1.62g

Fructose 1.0g

Glucose 1.0g

Sucrose 1.0g

Pancreatic digest of casein 1.0g

Preparation of Basal Medium: Add components to

distilled/de-ionized water and bring volume to 700.0mL Mix thoroughly Adjust

pH to 7.5–7.8 Autoclave for 15 min at 15 psi pressure–121°C Cool to

50°C

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

Preparation of Medium: Filter sterilize horse serum and fresh

yeast extract solution Aseptically add to cooled, sterile basal medium

Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Mycoplasma mycoides,

Spiroplasma apis, Spiroplasma citri, and Spiroplasma melliferum.

Mycoplasma Broth

Composition per 950.0mL:

Glucose 1.0g

Nicotinamide adenine dinucleotide 0.1g

PPLO broth without Crystal Violet 680.0mL

Swine serum (56°C, 30 min) 150.0mL

Fresh yeast extract solution 100.0mL

Phenol Red (0.1% w/v solution) 20.0mL

pH 7.8 ± 0.2 at 25°C

PPLO Broth without Crystal Violet:

Peptone 2.28g

NaCl 1.13g

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 680.0mL

Autoclave for 15 min at 15 psi pressure–121°C Cool to 56°C

Preparation of Medium: Mix glucose, nicotinamide adenine

dinu-cleotide, swine serum, fresh yeast extract solution, and Phenol Red

Mix thoroughly Heat to 56°C Add to cooled, sterile PPLO broth

with-out Crystal Violet Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Mycoplasma anseris and

Mycoplasma lipofaciens.

Mycoplasma Broth

(ATCC Medium 555) Composition per 103.0mL:

Hartley’s digest broth 30.0mL Pig serum 20.0mL Enzymatic hydrolysate of lactalbumin 10.0mL Hanks’ balanced salt solution, 10X 4.0mL Fresh yeast extract solution 2.0mL Phenol Red (0.25% solution) 1.0mL

pH 7.4 ± 0.2 at 25°C

Hartley’s Digest Broth:

Composition per 10.0L:

Ox heart 3,000.0g Pancreatin 50.0g

Na2CO3, anhydrous (0.8% solution) 5.0L HCl, concentrated 80.0mL

pH 7.5 ± 0.2 at 25°C

Preparation of Hartley’s Digest Broth: Finely mince the ox heart Add the meat to 5.0L of distilled/deionized water Gently heat and bring to 80°C Add the 5.0L of Na2CO3 solution Cool to 45°C Add pancreatin and maintain at 45°C for 4 hr while stirring Add the HCl and steam at 100°C for 30 min Cool to room temperature Adjust

pH to 8.0 with 1N NaOH Gently heat and bring to boiling Continue

boiling for 25 min Filter while hot Cool to room temperature Adjust

pH to 7.5 Autoclave for 15 min at 15 psi pressure–121°C

Pig Serum:

Pig serum 100.0mL

Preparation of Pig Serum: Adjust pH of pig serum to 4.4 with

sterile 1N HCl Do not let pH go below 4.2 Let serum stand at 4°C for 18-20 hr Adjust pH to 7.0 with sterile 1N NaOH Centrifuge at 9000

rpm for 20 min Discard pellet Filter supernatant solution through a 0.2μm membrane Store at −70°C

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

Enzymatic Hydrolysate of Lactalbumin:

Enzymatic hydrolysate of lactalbumin 5.0g

Preparation of Enzymatic Hydrolysate of Lactalbumin: Add enzymatic hydrolysate of lactalbumin to 100.0mL of phosphate buff-ered saline, 1X, pH 7.0

Phosphate Buffered Saline Solution, 1X:

Composition per liter:

NaCl 8.0g

Na2HPO4·7H2O 2.16g KCl 0.2g

KH2PO4 0.2g

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1266 Mycoplasma Broth Base

MgCl2·6H2O 0.1g

CaCl2 0.1g

Hanks’ Balanced Salt Solution, 10X:

NaCl 80.0g

Glucose 10.0g

KCl 4.0g

CaCl2 1.4g

MgCl2·6H2O 1.0g

MgSO4·7H2O 1.0g

Na2HPO4·7H2O 0.9g

KH2PO4 0.6g

Preparation of Hanks’ Balanced Salt Solution, 10X: Add

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

thoroughly

Preparation of Medium: Combine components in the following

order: Hanks’ balanced salt solution, 10X, Phenol Red, Hartley’s

di-gest broth, pig serum, enzymatic hydrolysate of lactalbumin, and fresh

yeast extract solution Mix thoroughly Add 36.0mL of

distilled/deion-ized water Adjust pH to 7.4 with 1N NaOH Filter sterilize through a

0.2μm membrane Store at 4°C for up to 3 weeks

Use: For the cultivation of Mycoplasma species.

Mycoplasma Broth Base

(PPLO Broth Base without Crystal Violet)

NaCl 5.0g

Beef extract 3.0g

Yeast extract 3.0g

pH 7.8 ± 0.2 at 25°C

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

Di-agnostic Systems

Preparation of Medium: Add components to distilled/deionized

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

thoroughly Distribute into tubes or flasks Autoclave for 15 min at 15

psi pressure–121°C

Use: Used as a basal medium that should be enriched for the isolation and

cultivation of Mycoplasma species.

Mycoplasma Broth Base, Frey with Horse Serum

Papaic digest of soybean meal 2.5g

KCl 0.4g

MgSO4 0.2g

Na2PO4 1.6g

KH2PO4 0.1g

Horse serum 100.0mL

pH 7.7 ± 0.2 at 25°C

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

Di-agnostic Systems

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

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

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

pres-sure–121°C Cool to 50°C Add sterile, heat-inactivated horse serum Mix thoroughly Aseptically distribute into sterile tubes

Use: For the cultivation of avian mycoplasmas

Mycoplasma Broth Base, Frey with Horse Serum

Pancreatic digest 7.5g Yeast extract 5.0g NaCl 5.0g Papaic digest of soybean meal 2.5g

Na2HPO4 1.6g KCl 0.4g MgSO4·7H2O 0.2g

KH2PO4 0.1g Horse serum 100.0mL

pH 7.7 ± 0.2 at 25°C

Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pres-sure–121°C Cool to 50°C Add sterile, heat-inactivated horse serum Mix thoroughly Aseptically distribute into sterile tubes

Use: For the cultivation of avian mycoplasmas

Mycoplasma Broth, Supplemented

Pancreatic digest of casein 7.0g NaCl 5.0g Beef extract 3.0g Yeast extract 3.0g Beef heart, solids from infusion 2.0g Horse serum 260.0mL Fresh yeast extract solution 65.0mL

pH 7.8 ± 0.2 at 25°C

Preparation of Medium: Add components, except horse serum and fresh yeast extract solution, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring to boiling Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C Cool to 50°C To each 75.0mL of cooled, sterile basal medium, add 20.0mL of sterile horse serum and 5.0mL of fresh yeast extract so-lution Mix thoroughly Aseptically distribute into sterile tubes

Use: For the isolation and cultivation of Mycoplasma species.

Mycoplasma Broth with Supplement G

Bacteriological peptone 10.0g Beef extract 10.0g NaCl 5.0g Special mineral supplement, Oxoid Unipath 0.5g

Mycoplasma supplement G 250.0mL

pH 7.8 ± 0.2 at 25°C

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

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Mycoplasma HiVeg Broth Base with Crystal Violet and Tellurite 1267

Mycoplasma Supplement G:

Thallous acetate 25.0mg

Horse serum 20.0mL

Yeast extract (25% solution) 10.0mL

Penicillin 20,000U

Preparation of Mycoplasma Supplement G: Add components

to distilled/deionized water and bring volume to 20.0mL Mix

thor-oughly Filter sterilize

Caution: Thallous acetate is a poison

Preparation of Medium: Add components, except Mycoplasma

supplement G, to distilled/deionized water and bring volume to 1.0L

Mix thoroughly Gently heat and bring to boiling Distribute into flasks

in 80.0mL volumes Autoclave for 15 min at 15 psi pressure–121°C

Cool to 50°C Aseptically add 20.0mL of sterile Mycoplasma

supple-ment G to each 80.0mL of basal medium Mix thoroughly

Use: For the growth of Mycoplasma species

Mycoplasma Broth with Supplement P

Bacteriological peptone 10.0g

Beef extract 10.0g

NaCl 5.0g

Special mineral supplement, Oxoid Unipath 0.5g

Mycoplasma supplement P 250.0mL

pH 7.8 ± 0.2 at 25°C

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

Unipath

Mycoplasma Supplement P:

Glucose 0.3g

Mycoplasma broth base 0.145g

Thallous acetate 8.0mg

Phenol Red 1.2mg

Methylene Blue chloride 0.3mg

Penicillin 12,000U

Horse serum 6.0mL

Yeast extract (25% solution) 3.0mL

Preparation of Mycoplasma Supplement P: Add components to

distilled/deionized water and bring volume to 20.0mL Mix

thorough-ly Filter sterilize

Caution: Thallous acetate is a poison

Preparation of Medium: Add components, except Mycoplasma

supplement P, to distilled/deionized water and bring volume to 1.0L

Mix thoroughly Gently heat and bring to boiling Distribute into

bot-tles in 1.0mL volumes Autoclave for 15 min at 15 psi pressure–121°C

Cool to room temperature Aseptically add 2.0mL of sterile

Mycoplas-ma supplement P to each bottle

Use: For the cultivation of Mycoplasma species

Mycoplasma Broth with 10% Swine Serum

Composition per liter:

NaCl 4.0g

Yeast extract 2.6g

Beef extract 2.4g

Swine serum, heat inactivated 100.0mL Fresh yeast extract solution 100.0mL

pH 7.8 ± 0.2 at 25°C

Preparation of Medium: Add components, except swine serum and fresh yeast extract solution, to distilled/deionized water and bring volume

to 800.0mL Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°C Add sterile swine se-rum and fresh yeast extract solution Mix thoroughly Aseptically distrib-ute into sterile tubes

Use: For the cultivation and maintenance of Mycoplasma columbinum and Mycoplasma columborale.

Mycoplasma HiVeg Agar Base with

Horse Serum and Yeast Extract (PPLO HiVeg Agar Base) Composition per liter:

Agar 15.0g Plant peptone 10.0g Plant infusion 6.0g NaCl 5.0g Horse serum 260.0mL Fresh yeast extract solution 65.0mL

pH 7.8 ± 0.2 at 25°C

Source: This medium, without horse serum and yeast extract solution,

is available as a premixed powder from HiMedia

Preparation of Medium: Add components, except horse serum and fresh yeast extract solution, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring to boiling Dis-tribute into tubes or flasks Autoclave for 15 min at 15 psi pressure– 121°C Cool to 50°C To each 75.0mL of cooled, sterile basal medium, add 20.0mL of sterile horse serum and 5.0mL of special yeast extract solution Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the preparation of media for the cultivation of Mycoplasma.

Mycoplasma HiVeg Broth Base

with Crystal Violet and Tellurite (PPLO HiVeg Broth Base with CV) Composition per liter:

Plant peptone 10.0g Plant infusion 6.0g NaCl 5.0g Crystal Violet 0.01g

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1268 Mycoplasma HiVeg Broth Base without Crystal Violet and with Ascitic Fluid

Chapman tellurite solution 2.85mL

Ascitic fluid 250.0mL

pH 7.8 ± 0.2 at 25°C

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

powder from HiMedia

Chapman Tellurite Solution:

K2TeO3 1.0g

Preparation of Chapman Tellurite Solution: Add K2TeO3 to

thorough-ly Filter sterilize

Caution: Potassium tellurite is toxic

Preparation of Medium: Add components, except ascitic fluid and

Chapman tellurite solution, to distilled/deionized water and bring

vol-ume to 747.15mL Mix thoroughly Autoclave for 15 min at 15 psi

pressure–121°C Cool to less than 37°C Aseptically add sterile ascitic

fluid and 2.85mL of Chapman tellurite solution Mix thoroughly

Asep-tically distribute into sterile tubes or flasks

Use: For the isolation of Mycoplasma species from clinical specimens.

Mycoplasma HiVeg Broth Base

without Crystal Violet and with Ascitic Fluid

(PPLO HiVeg Broth Base without CV)

Plant peptone 10.0g

Plant infusion 6.0g

NaCl 5.0g

Ascitic fluid 250.0mL

pH 7.8 ± 0.2 at 25°C

Source: This medium, without ascitic fluid, is available as a premixed

powder from HiMedia

Preparation of Medium: Add components, except ascitic fluid, to

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

37°C Aseptically add sterile ascitic fluid If desired, 0.5g of thallium

acetate or 100,000U of penicillin may be added for a more selective

medium Mix thoroughly Aseptically distribute into sterile tubes or

flasks

Use: For the enrichment of pleuro-pneumonia-like organisms (PPLOs)

and Mycoplasma species from clinical specimens.

Mycoplasma Horse Serum Broth

(ATCC Medium 1959)

Mycoplasma broth base 660.0mL

Horse serum 200.0mL

Fresh yeast extract solution 100.0mL

Phenol Red (0.1%) 20.0mL

Glucose solution 10.0mL

NaOH (1N solution) 6.25mL

Arginine solution 5.0mL

Mycoplasma Broth Base:

NaCl 5.0g

Beef extract 3.0g

Yeast extract 3.0g

Preparation of Mycoplasma Broth Base: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly

Glucose Solution:

Glucose 5.0g

Preparation of Glucose Solution : Add glucose to 10.0mL of

dis-tilled/deionized water Mix thoroughly Filter sterilize

Arginine Solution:

L-Arginine 4.2g

Preparation of Arginine Solution : Add arginine to 10.0mL of

distilled/deionized water Mix thoroughly Filter sterilize

Preparation of Medium: Combine 660.0mL Mycoplasma broth base, 20.0mL Phenol Red, and 6.25mL 1N NaOH Mix thoroughly.

Autoclave for 15 min at 15 psi pressure–121°C Cool to 25°C Asepti-cally add 5.0mL sterile arginine solution, 100.0mL sterile fresh yeast extract solution, 10.0mL sterile glucose solution, and 200.0mL filter sterilized horse serum Mix thoroughly Aseptically distribute into ster-ile tubes or flasks

Use: For the preparation of media for the cultivation of Mycoplasma spp.

Mycoplasma Liquid Medium

Arginine 1.0g Glucose 1.0g

L-Cysteine·HCl·H2O 1.0g

Mycoplasma broth base 850.0mL

Horse serum, not inactivated 100.0mL Fresh yeast extract (25% solution) 50.0mL Phenol Red (1.0% solution) 2.0mL DNA calf thymus solution 2.0mL

pH 7.8 ± 0.2 at 25°C

Mycoplasma Broth Base:

Pancreatic digest of casein 7.0g NaCl 5.0g Beef extract 3.0g Yeast extract 3.0g Beef heart, solids from infusion 2.0g

Preparation of Mycoplasma Broth Base: Add components to distilled/deionized water and bring volume to 850.0mL Mix

thorough-ly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°C

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Mycoplasma Medium, Revised 1269

DNA Calf Thymus Solution:

DNA calf thymus 1.0g

Preparation of DNA Calf Thymus Solution: Add DNA calf

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

thoroughly Filter sterilize

Preparation of Medium: Combine components, except Mycoplasma

broth base and DNA calf thymus solution, and mix thoroughly Filter

ster-ilize through a 0.2μm membrane Add sterile solution to 850.0mL of

cooled, sterile Mycoplasma broth base Aseptically add 2.0mL of sterile

DNA calf thymus solution Mix thoroughly Aseptically distribute into

sterile tubes or flasks

Use: For the cultivation and maintenance of Mycoplasma lipophilum

and Mycoplasma species.

Mycoplasma Medium

Heart infusion broth 25.0g

Mucin, bacteriological grade 5.0g

Agar, purified (optional) 7.0g

Hemoglobin 2.0g

Turkey serum, sterile inactivated 100.0mL

pH 7.8 ± 0.2 at 25°C

Preparation of Medium: Add components, except turkey serum and

agar, to 850.0mL distilled water Adjust pH to 7.8 Heat mixture at 93°–

95°C for 30 min in a water bath Restore to original volume Add 0.5%

di-atomaceous earth Mix thoroughly Filter through Whatman GFA (glass

fi-ber paper) in Buchner filter Clarify using 0.45μm Millipore filter Add

15% inactivated turkey serum Sterilize using S3 (0.1μm) Seitz filter Use

positive pressure For solid medium: Prepare 42.5mL of double-strength

broth and 42.5mL of distilled water containing 0.7g of purified agar

Ster-ilize the solutions separately and combine aseptically at 56°C with 15.0mL

of sterile inactivated turkey serum for a final volume of 150.0mL

Use: For the cultivation and maintenance of Mycoplasma

hyosyn-oviae.

Mycoplasma Medium

(CIP Medium 89) (DSMZ Medium 1080) Composition per liter:

NaCl 5.0g

Beef extract 3.0g

Yeast extract 3.0g

Beef heart, infusion from (solids) 2.0g

Selective supplement solution 210.0mL

Yeast extract solution 100.0mL

Phenol Red solution 20.0mL

Yeast Extract Solution :

Yeast extract 25.0g

Preparation of Yeast Extract Solution: Add yeast extract to

dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly

Adjust pH to 7.0 Autoclave for 15 min at 15 psi pressure–121°C

Phenol Red Solution : Composition per 100.0mL:

Phenol Red 1.0g

Preparation of Phenol Red Solution: Add Phenol Red to dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly Adjust pH to 7.0 Autoclave for 15 min at 15 psi pressure–121°C

Selective Supplement Solution:

Ampicillin 1.0g Horse serum 200.0mL Arginine solution 10.0mL

Arginine Solution : Composition per 100.0mL:

L-Arginine 50.0g

Preparation of Arginine Solution: Add L-arginine to distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly Adjust

pH to 7.0

Preparation of Selective Supplement Solution: Add ampicillin

to 10.0mL arginine solution Add horse serum Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except yeast extract, Phenol Red, and selective supplement solutions, to distilled/deionized water and bring volume to 670.0mL Mix thoroughly Adjust pH to 7.2 Distribute into tubes or flasks Gently heat while stirring and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to room temperature Aseptically add yeast extract, Phenol Red, and selective supplement solutions Mix thoroughly Aseptically distribute into tubes

or flasks

Use: For the cultivation of Mycoplasma spp.

Mycoplasma Medium, Revised

Noble agar 10.0g Distilled water 360.0mL Heart infusion broth 300.0mL Pig serum, heat inactivated 200.0mL Enzymatic hydrolysate of lactalbumin 100.0mL Hanks’ balanced salt solution, 10X 40.0mL Fresh yeast extract solution 20.0mL Phenol Red (0.25% solution) 10.0mL

Heart Infusion Broth:

Beef heart, infusion from 500.0g Tryptose 10.0g NaCl 5.0g

Preparation of Heart Infusion Broth: Add components to dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly Gen-tly heat and bring to boiling

Enzymatic Hydrolysate of Lactalbumin:

Enzymatic hydrolysate of lactalbumin 5.0g Phosphate buffered saline, 1X, pH7.0 100.0mL

Preparation of Enzymatic Hydrolysate of Lactalbumin: Add enzymatic hydrolysate of lactalbumin to 100.0mL of phosphate buff-ered saline, 1X, pH 7.0

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1270 Mycoplasma pneumoniae Isolation Medium

Phosphate Buffered Saline Solution, 1X:

NaCl 8.0g

Na2HPO4·7H2O 2.16

KCl 0.2g

KH2PO4 0.2g

MgCl2·6H2O 0.1g

CaCl2 0.1g

Preparation of Phosphate Buffered Saline Solution, 1X: Add

thoroughly

Hanks’ Balanced Salt Solution, 10X:

Na2Cl 80.0g

Glucose 10.0g

KCl 4.0g

CaCl2 1.4g

MgCl2·6H2O 1.0g

MgSO4·7H2O 1.0g

Na2HPO4·7H2O 0.9g

KH2PO4 0.6g

Preparation of Hanks’ Balanced Salt Solution, 10X: Add

thoroughly

Preparation of Medium: Add agar and heart infusion broth to

Ad-just pH to 7.4 Autoclave for 15 min at 15 psi pressure–121°C Aseptically

add pig serum, enzymatic hydrolysate of lactalbumin, Hanks’ balanced

salt solution, 10X, fresh yeast extract solution, and Phenol Red solution

Mix thoroughly Aseptically distribute into sterile tubes

Use: For the cultivation and maintenance of Mycoplasma dispar,

Mycoplasma flocculare, and Mycoplasma hyopneumoniae.

Mycoplasma pneumoniae Isolation Medium

Beef heart for infusion 50.0g

Peptone 10.0g

NaCl 5.0g

Water 900.0mL

Yeast extract solution 100.0mL

α-Gamma horse serum, unheated 200.0mL

pH 7.6–7.8 at 25°C

Yeast Extract Solution:

Yeast, active, dry, Baker’s 250.0g

Preparation of Yeast Extract Solution: Add yeast to 1.0L of

dis-tilled/deionized water Mix thoroughly Gently heat and bring to

boil-ing Filter through Whatman #2 filter paper Adjust the pH of the

filtrate to 8.0 with NaOH Distribute into tubes in 10.0mL volumes

Autoclave for 15 min at 15 psi pressure–121°C Store at −20°C

Preparation of Medium: Add components, except yeast extract so-lution and α-gamma horse serum, 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 yeast extract solution and α-gamma horse serum Mix thoroughly Aseptically distribute into sterile tubes

Use: For the isolation and cultivation of Mycoplasma pneumoniae.

Mycoplasmal Agar Composition per liter:

Papaic digest of soy meal 20.0g Agarose 10.0g NaCl 5.0g Phenol Red (2% solution) 1.0mL

pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components, except agarose, to dis-tilled/deionized water and bring volume to 1.0L Mix thoroughly

Ad-just pH to 7.3 with 1N NaOH Add agarose 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 and cultivation of human mycoplasmas and ure-aplasmas

Mycorrhiza Medium Composition per liter:

Agar 15.0g Glucose 4.0g Ammonium tartrate 1.0g Malt extract 1.0g

KH2PO4 0.2g MgSO4·7H2O 0.1g CaCl2·2H2O 26.0mg NaCl 20.0mg Inositol 10.0mg ZnSO4·7H2O 0.88mg MnSO4·4H2O 0.81mg FeCl3·6H2O 0.8mg Nicotinamide 100.0μg

p-Aminobenzoic acid 100.0μg Pantothenic acid 100.0μg Pyridoxine 100.0μg Thiamine 100.0μg Biotin 25.0μg Riboflavin 25.0μg

Preparation of Medium: Add components to distilled/deionized wa-ter and bring volume to 1.0L Mix thoroughly Gently heat until boiling Distribute into tubes or flasks Autoclave for 10 min at 15 psi pressure– 121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation and maintenance of Thelephora terrestris

Mycosel™ Agar (Cycloheximide Chloramphenicol Agar) Composition per liter:

Agar 15.5g Papaic digest of soybean meal 10.0g Glucose 10.0g

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MYX Agar 1271

Cycloheximide 0.4g

Chloramphenicol 0.05g

pH 6.9 ± 0.2 at 25°C

Caution: Cycloheximide is toxic Avoid skin contact or aerosol

for-mation and inhalation

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

stirring and bring to boiling Autoclave for 15 min at 14 psi pressure–

118°C Avoid overheating Pour into sterile Petri dishes or distribute

into sterile tubes

Use: For the selective isolation of pathogenic fungi from specimens

with other fungi and bacteria

MYCT Medium (DSMZ Medium 972) Composition per liter:

KH2PO4 13.6g

Cyclomaltoheptaose (ß-cyclodextrin) 7.0g

(NH4)2SO4 4.0g

Yeast extract 3.0g

Casein hydrolysate 3.0g

Tween™ 80 1.0g

MgCl2 0.2g

Sodium citrate 0.25g

FeSO4·7H2O 25.0mg

MnSO4·4H2O 25.0mg

pH 6.5 ± 0.2 at 25°C

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 Lactobacillus sp.

Mykorrhiza Agar Composition per liter:

Agar 15.0g

Malt extract 8.0g

Glucose 7.0g

Casein hydrolysate 1.0g

Yeast extract 1.0g

Asparagine 0.5g

KH2PO4 0.5g

MgSO4·7H2O 0.5g

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 Klebsiella pneumoniae.

MYP Agar

See: Mannitol Yolk Polymyxin Agar

MYP Agar Base, HiVeg with Egg Yolk and Polymyxin B

(Phenol Red Egg Yolk Polymyxin Agar Base, HiVeg)

Agar 15.0g

D-Mannitol 10.0g

Plant peptone 10.0g

NaCl 10.0g Plant extract No 1 1.0g Phenol Red 0.025g Egg yolk emulsion, 20% 10.0mL Polymyxin B solution 1.0mL

pH 7.1 ± 0.2 at 25°C

Source: This medium, without egg yolk and polymyxin B, is avail-able as a premixed powder from HiMedia

Egg Yolk Emulsion, 20%:

Chicken egg yolks 11 Whole chicken egg 1 NaCl (0.9% solution) 80.0mL

Preparation of Egg Yolk Emulsion, 20%: Soak eggs with 1:100 dilution of saturated mercuric chloride solution for 1 min Crack eggs and separate yolks from whites Mix egg yolks with 1 chicken egg Measure 20.0mL of egg yolk emulsion and add to 80.0mL of 0.9% NaCl solution Mix thoroughly Filter sterilize Warm to 45°–50°C

Polymyxin B Solution:

Polymyxin B 1.0mg

Preparation of Polymyxin B Solution: Add polymyxin B to dis-tilled/deionized water and bring volume to 1.0mL Mix thoroughly Fil-ter sFil-terilize

Preparation of Medium: Add components—except egg yolk emul-sion, 20%, and polymyxin B solution—to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Gently heat and bring to boil-ing Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add 10.0mL of sterile egg yolk emulsion, 20%, and 1.0mL

of sterile polymyxin B solution Mix thoroughly Pour into sterile Petri dishes

Mysorens Medium Composition per liter:

Peptone 10.0g Meat extract 10.0g Yeast extract 5.0g NaCl 3.0g

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

Use: For the cultivation and maintenance of Arthrobacter mysorens.

MYX Agar (DSMZ Medium 729) Composition per liter:

Na2-glutamate 5.0g Yeast extract 1.0g MgSO4·7H2O 1.0g Glucose solution 10.0mL

pH 7.2 ± 0.2 at 25°C

Composition per10.0mL:

Glucose 2.0g

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

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1272 Myxobacteria Medium

Preparation of Medium: Add components, except glucose solution,

to distilled/deionized water and bring volume to 990.0mL Mix

thor-oughly Adjust pH to 7.2 Aseptically add 10.0mL glucose solution Mix

thoroughly Aseptically distribute into sterile tubes or bottles

Use: For the cultivation of Taxeobacter spp.

Myxobacteria Medium Composition per liter:

Agar 15.0g

Skim milk powder 5.0g

Yeast extract 0.5g

water and bring volume to 1.0L Mix thoroughly Do not adjust pH

Gently heat and bring to boiling Distribute into tubes or flasks

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

Use: For the cultivation and maintenance of Archangium

primige-nium, Chondrococcus macrosporus, and Myxococcus coralloides.

Myxococcus flavescens Medium

Agar 15.0g

Soluble starch 5.0g

Casitone 2.5g

Galactose 1.0g

Raffinose 1.0g

Sucrose 1.0g

Yeast extract 1.0g

MgSO4·7H2O 0.5g

K2HPO4 0.25g

pH 6.0 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Adjust pH to 6.0

Gently heat and bring to boiling Distribute into tubes or flasks

Auto-clave 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 Myxococcus flavescens.

Myxococcus Medium

Agar 12.0g

Meat extract 1.0g

Glucose solution 50.0mL

pH 7.2 ± 0.2 at 25°C

Glucose 1.0g

Preparation of Glucose Solution: Add glucose to

distilled/deion-ized water and bring volume to 50.0mL Mix thoroughly Autoclave for

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

Preparation of Medium: Add components, except glucose

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

thoroughly Adjust pH to 7.2 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 glucose solution Mix thoroughly Pour into sterile

Petri dishes or distribute into sterile tubes or bottles Allow tubes or

bottles to cool in a slanted position

Use: For the cultivation of Myxococcus species.

Myxococcus xanthus Medium

Agar 20.0g Pancratic digest of casein 10.0g MgSO4·7H2O 0.5g

K2HPO4 0.148g

KH2PO4 0.017g

pH 7.6 ± 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 Myxococcus xanthus.

N plus C Medium Composition per liter:

Pancreatic digest of casein 10.0g Glucose 10.0g Citric acid·H2O 4.04g

KH2PO4 2.0g Yeast extract 1.5g CaCl2·2H2O 0.6g MgSO4·7H2O 0.6g FeCl2·4H2O 0.06g ZnSO4·7H2O 0.034g Hemin solution 1.0mL

pH 4.6 ± 0.2 at 25°C

Hemin Solution:

Compositionper 100.0mL:

NaOH 1.0g Hemin 250.0mg

Preparation of Hemin Solution: Add components to distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly Auto-clave for 15 min at 15 psi pressure–121°C

Preparation of Medium: Add components, except hemin solution, to distilled/deionized water and bring volume to 1.0L Mix thoroughly Ad-just pH to 4.6 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Prior to inoculation, add 0.1mL of hemin solution per 100.0mL of medium

Use: For the cultivation and maintenance of Physarum polycephalum.

N DeVogel Medium (Vogel N Medium) Composition per liter:

Sucrose 15.0g

KH2PO4 5.0g Trisodium citrate·2H2O 3.0g

NH4NO3 2.0g MgSO4·7H2O 0.2g CaCl2·H2O solution 20.0mL Biotin solution 5.0mL Trace elements solution 5.0mL

CaCl 2 ·H 2 O Solution:

CaCl2·H2O 0.1g

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NAM Medium 1273

Preparation of CaCl2·H2O Solution: Add CaCl2·H2O to

Biotin Solution:

Biotin 5.0mg

Preparation of Biotin Solution: Add biotin to 50% ethanol and

bring volume to 100.0mL Mix thoroughly Filter sterilize Store at 5°C

Trace Elements Solution:

Citric acid·H2O 5.0g

ZnSO4·7H2O 5.0g

Fe(NH4)2(SO4)2·6H2O 1.0g

CuSO4·5H2O 0.25g

H3BO3, anhydrous 0.05g

MnSO4·H2O 0.05g

Na2MoO4·2H2O 0.05g

Preparation of Trace Elements Solutions: Add components

successively to distilled/deionized water and bring volume to

100.0mL Mix thoroughly after addition of each component Filter

sterilize Add 2–3.0mL of chloroform as a preservative Store at 25°C

Preparation of Medium: Add components, except biotin solution

and trace elements solution, to distilled/deionized water and bring

vol-ume to 990.0mL Mix thoroughly Autoclave for 15 min at 15 psi

pres-sure–121°C Aseptically add 5.0mL of sterile biotin solution and

5.0mL of sterile trace elements solution Mix thoroughly Aseptically

distribute into sterile tubes or flasks

Use: For the cultivation and maintenance ofNeurospora crassa.

NAG Medium (DSMZ Medium 366) Composition per liter:

Agar 15.0g

Glucose 10.0g

Peptone 5.0g

Meat extract 3.0g

pH 7.0 ± 0.2 at 25°C

to boiling Distribute into tubes or flasks Autoclave for 15 min at 15

psi pressure–121°C Do not overheat Pour into sterile Petri dishes or

leave in tubes

Use: For the isolation and cultivation of Xanthomonas fragariae.

Nakayama Glucose Agar Composition per 1001.0mL:

Yeast extract 15.0g

Agar 10.0g

Glucose 10.0g

Peptone 10.0g

Solution A 10.0mL

Solution B 10.0mL

Solution C 1.0mL

pH 6.8 ± 0.2 at 25°C

Solution A:

K2HPO4 0.5g

KH2PO4 0.5g

Preparation of Solution A: Add components to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Solution B:

MgSO4·7H2O 3.0g MnSO4·5H2O 0.1g NaCl 0.1g CuSO4·5H2O 0.01g

Preparation of Solution B: Add components to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Solution C:

Trisodium citrate 2.0g FeSO4·7H2O 0.1g

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Preparation of Medium: Add components, except solution A, so-lution B, and soso-lution C, 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 50°–55°C Asepti-cally add 10.0mL of sterile solution A, 10.0mL of sterile solution B, and 1.0mL of sterile solution C Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the cultivation and maintenance of Bacillus laevolacticus.

NAM Medium Composition per liter:

Pancreatic digest of casein 15.0g Agar 15.0g Papaic digest of soybean meal 5.0g NaCl 5.0g Sheep blood, defibrinated 50.0mL Hemin solution 10.0mL

N-Acetyl muramic acid (NAM) solution 1.0mL

pH 7.3 ± 0.2 at 25°C

Hemin Solution:

Hemin 0.050g

NaOH (1N solution) 1.0mL

Preparation of Hemin Solution: Add hemin to NaOH solution Mix thoroughly Adjust volume to 100.0mL with distilled/deionized wa-ter Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

N-Acetyl Muramic Acid (NAM) Solution:

N-Acetyl muramic acid 100.0mg

Preparation of N-Acetyl Muramic Acid (NAM) Solution:

Add N-acetyl muramic acid to distilled/deionized water and bring

vol-ume to 10.0mL Filter sterilize

Preparation of Medium: Add components, except sheep blood,

hemin solution, and N-acetyl muramic acid (NAM) solution, to

dis-tilled/deionized water and bring volume to 49.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°–55°C Aseptically add 50.0mL of sterile sheep blood, 10.0mL of sterile hemin

solution, and 1.0mL of sterile N-acetyl muramic acid (NAM) solution.

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1274 NANAT Agar

Mix thoroughly Aseptically and anaerobically distribute into sterile

tubes under a gas phase of 80% N2 + 10% CO2 + 10% H2

Use: For the cultivation of Bacteroides forsythus

NAMn

See: Nutrient Agar with Manganese

NANAT Agar (Nalidixic Acid Novobiocin Actidione Tellurite Agar)

Agar 15.0g

NaCl 5.0g

Tween™ 80 5.0g

Papaic digest of soybean meal 3.0g

K2HPO4 2.5g

Glucose 2.5g

Yeast extract 1.0g

Tellurite solution 10.0mL

Antibiotic solution 10.0mL

pH 7.2 ± 0.2 at 25°C

Tellurite Solution:

K2TeO3 0.05g

Preparation of Tellurite Solution: Add K2TeO3 to

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

sterilize

Antibiotic Solution:

Actidione (cycloheximide) 0.04g

Polymyxin B (optional) 0.03g

Novobiocin 0.025g

Nalidixic acid 0.02g

Preparation of Antibiotic Solution: Add components to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly Filter

sterilize

Caution: Potassium tellurite is toxic

Caution: Cycloheximide is toxic Avoid skin contact or aerosol

for-mation and inhalation

Preparation of Medium: Add components, except tellurite

solu-tion and antibiotic solusolu-tion, to distilled/deionized water and bring

vol-ume to 980.0mL Mix thoroughly Gently heat and bring to boiling

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

Aseptically add sterile tellurite solution and antibiotic solution Mix

thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the isolation and cultivation of Rhodococcus

(Corynebacte-rium) equi from animal feces, especially from horses and swine The

addition of polymyxin B inhibits the growth of Pseudomonas

aerugi-nosa which may interfere with the isolation of Rhodococcus equi.

Nannocystis Agar

Agar 15.0g

CaCl2·2H2O 1.0g

pH 7.2 ± 0.2 at 25°C

to boiling Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes After the agar has solidified, overlay the surface

with 0.5mL of a suspension of dead (autoclaved) Escherichia coli cells.

Use: For the cultivation and maintenance of Nannocystis species.

Naphthalene Medium Composition per liter:

NH4NO3 2.5g

Na2HPO4·2H2O 1.0g Naphthalene 0.64g MgSO4·7H2O 0.5g Fe(SO4)3·5H2O 0.01g Co(NO3)2·6H2O 5.0mg CaCl2·2H2O 1.0mg

KH2PO4 0.5mg MnSO4·2H2O 0.1mg (NH4)6Mo7O24·4H2O 0.1mg

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

Use: For the cultivation of Pseudomonas alcaligenes.

Naphthalene Mineral Salts Medium

See: Medium for Hydrocarbon-Degrading Bacteria

Naphthalene Sulfonic Acid Medium Composition per 1004.0mL:

Na2HPO4·2H2O 3.5g

KH2PO4 1.0g

NH4Cl 0.31g MgCl2·6H2O 0.1g Ca(NO3)2·4H2O 0.05g Solution A 100.0mL Solution B 3.0mL Trace elements solution SL-4 1.0mL

pH 7.0 ± 0.2 at 25°C

Solution A:

Glucose 3.0g Glycerol 3.0g Sodium succinate 3.0g

Preparation of Solution A : Add components to distilled/deionized

water and bring volume to 1.0L Mix thoroughly Filter sterilize

Solution B:

Naphthalene sulfonic acid 2.3g

Preparation of Solution B : Add naphthalene sulfonic acid to

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

Trace Elements Solution SL-4:

EDTA 0.5g FeSO4·7H2O 0.2g Trace elements solution SL-6 100.0mL

Preparation of Trace Elements Solution SL-4: Add components

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

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