Handbook of Microbiological Media, Fourth Edition part 182 ppt

2.5g Preparation of Brain Heart Infusion Broth: Add components to distilled/deionized water and bring volume to 1.0L.. 2.5g Preparation of Brain Heart Infusion Broth: Add components to d

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Transgrow Medium 1805

Use: For the cultivation and maintenance of Pseudomonas species.

TPT 18 Medium (DSMZ Medium 1127) Composition per liter:

Glucose 0.5g

Yeast extract 0.1g

Pancreatic digest of casein 0.1g

MgSO4·7H2O 0.05g

CaCl2·2H2O 0.02g

pH 6.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L Mix thoroughly Autoclave for 15 min

at 15 psi pressure–121°C Adjust pH to 6.0

Use: For the cultivation of Mucilaginibacter gracilis.

TPY Medium Compositionper liter:

Glucose 5.0g

Pancreatic digest of soybean meal 5.0g

Yeast extract 2.5g

K2HPO4 2.0g

Agar 1.5g

Cysteine·HCl 0.5g

MgCl2·6H2O 0.5g

ZnSO4·7H2O 0.25g

CaCl2 0.15g

FeCl3 1.0μg

Tween™ 80 1.0mL

pH 6.5 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized

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

dis-solved Distribute into tubes or flasks Autoclave for 15 min at 15 psi

pressure–121°C

Use: For the isolation and cultivation of Bifidobacterium species.

TPYG Medium Compositionper liter:

Peptone 5.0g

Yeast extract 5.0g

Glucose solution 50.0mL

Glucose Solution:

Compositionper 100.0mL:

Glucose 20.0g

Preparation of Glucose Solution: Add glucose to

distilled/deion-ized water and bring volume to 100.0mL Mix thoroughly Filter

ster-ilize

Preparation of Medium: Add components, except glucose

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

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

Aseptical-ly add 50.0mL of sterile glucose solution Mix thoroughAseptical-ly AsepticalAseptical-ly

distribute into sterile tubes or flasks

Use: For the cultivation of Clostridium felsineum.

Trace Elements Solution HO-LE Compositionper liter:

H3BO3 2.85g MnCl2·4H2O 1.8g Sodium tartrate 1.77g FeSO4·7H2O 1.36g CoCl2·6H2O 0.04g CuCl2·2H2O 0.027g

Na2MoO4·2H2O 0.025g ZnCl2 0.02g

Preparation of Trace Elements Solution HO-LE: Add components

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

Use: For the enrichment of other media requiring added trace metals

Transgrow Medium Compositionper liter:

GC agar base 730.0mL Hemoglobin solution 250.0mL Vitox supplement 10.0mL VCN antibiotic solution 10.0mL

pH 7.3 ± 0.2 at 25°C

GC Agar Base:

Special peptone 15.0g Agar 20.0g NaCl 5.0g

K2HPO4 4.0g Cornstarch 1.0g

KH2PO4 1.0g

pH 7.2 ± 0.2 at 25°C

Preparation of GC Agar Base: Add components of GC medium base and the hemoglobin to distilled/deionized water and bring volume

to 730.0mL Mix thoroughly Gently heat until boiling Autoclave for

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

Hemoglobin Solution:

Hemoglobin 5.0g

Preparation of Hemoglobin Solution: Add hemoglobin to dis-tilled/deionized water and bring volume to 250.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Vitox Supplement:

Glucose 2.0g L-Cysteine·HCl 0.518g L-Glutamine 0.2g L-Cystine 0.022g Adenine sulfate 0.01g Nicotinamide adenine dinucleotide 5.0mg Cocarboxylase 2.0mg Guanine·HCl 0.6mg Fe(NO3)3·6H2O 0.4mg

p-Aminobenzoic acid 0.26mg

Vitamin B12 0.2mg Thiamine·HCl 0.06mg

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

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1806 Transgrow Medium

VCN Antibiotic Solution:

Colistin methane sulfonate 7.5mg

Vancomycin 3.0mg

Nystatin 12,500U

Preparation of VCN Antibiotic Solution: Add components to

distilled/deionized water and bring volume to 10.0mL Mix

thorough-ly Filter sterilize

Preparation of Medium: To 730.0mL of cooled, sterile GC agar

base, aseptically add 250.0mL of sterile hemoglobin solution, 10.0mL

of sterile Vitox supplement, and 10.0mL of VCN antibiotic solution

Mix thoroughly Pour into sterile Petri dishes or distribute into sterile

tubes

Use: For the cultivation and transport of fastidious microorganisms,

especially Neisseria species.

Transgrow Medium Compositionper liter:

GC medium base 730.0mL

Hemoglobin solution 250.0mL

Supplement B 10.0mL

VCNT antibiotic solution 10.0mL

pH 7.3 ± 0.2 at 25°C

GC Medium Base:

Proteose peptone No 3 15.0g

Agar 20.0g

NaCl 5.0g

K2HPO4 4.0g

Glucose 1.5g

Cornstarch 1.0g

KH2PO4 1.0g

pH 7.2 ± 0.2 at 25°C

Preparation of GC Medium Base: Add components to distilled/

deionized water and bring volume to 730.0mL Mix thoroughly Gently

heat until boiling Autoclave for 15 min at 15 psi pressure–121°C Cool

to 45°–50°C

Hemoglobin Solution:

Hemoglobin 10.0g

Preparation of Hemoglobin Solution: Add hemoglobin to

dis-tilled/deionized water and bring volume to 250.0mL Mix thoroughly

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

Supplement B:

Supplement B contains yeast concentrate, glutamine, coenzyme,

co-carboxylase, hematin, and growth factors

Preparation of Supplement B: Add components to

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

ster-ilize

Source: Supplement B is available as a premixed powder from BD

Diagnostic Systems

VCNT Antibiotic Solution:

Colistin methane sulfonate 7.5mg

Trimethoprim lactate 5.0mg

Vancomycin 3.0mg

Nystatin 12,500U

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

thorough-ly Filter sterilize

Preparation of Medium: To 730.0mL of cooled, sterile GC

medi-um base, aseptically add 250.0mL of sterile hemoglobin solution, 10.0mL of sterile supplement B, and 10.0mL of VCNT antibiotic solu-tion Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the cultivation and transport of fastidious microorganisms,

especially Neisseria species.

Transgrow Medium with Trimethoprim Compositionper liter:

Agar 20.0g Hemoglobin 10.0g Pancreatic digest of casein 7.5g Selected meat peptone 7.5g NaCl 5.0g

K2HPO4 4.0g Glucose 1.5g Cornstarch 1.0g

KH2PO4 1.0g Supplement solution 10.0mL VCNT inhibitor 10.0mL

pH 6.7 ± 0.2 at 25°C

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

Supplement Solution:

Compositionper liter:

Glucose 100.0g L-Cysteine·HCl 25.9g L-Glutamine 10.0g L-Cystine 1.1g Adenine 1.0g Nicotinamide adenine dinucleotide 0.25g Vitamin B12 0.1g Thiamine pyrophosphate 0.1g Guanine·HCl 0.03g Fe(NO3)3·6H2O 0.02g

p-Aminobenzoic acid 0.013g

Thiamine·HCl 3.0mg

Source: The supplement solution (IsoVitaleX® enrichment) is avail-able from BD Diagnostic Systems This enrichment may be replaced

by supplement VX from BD Diagnostic Systems

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

VCNT Inhibitor:

Colistin 7.5mg Trimethoprim lactate 5.0mg Vancomycin 3.0mg Nystatin 12,500U

Preparation of VCNT Inhibitor: Add components to distilled/de-ionized water and bring volume to 10.0mL Mix thoroughly Filter ster-ilize

Preparation of Medium: Add components, except supplement so-lution and VCNT inhibitor, to distilled/deionized water and bring

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vol-Trebouxia Agar 1807

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

un-der 5–30% CO2 Aseptically add 10.0mL of sterile supplement

solu-tion and 10.0mL of sterile VCNT inhibitor Mix thoroughly

Aseptically distribute under 5–30% CO2 into sterile screw-capped

tubes

Use: For the transportation and recovery of pathogenic Neisseria

spe-cies.

Transgrow Medium without Trimethoprim

Agar 20.0g

Hemoglobin 10.0g

Selected meat peptone 7.5g

NaCl 5.0g

K2HPO4 4.0g

Glucose 1.5g

Cornstarch 1.0g

KH2PO4 1.0g

Supplement solution 10.0mL

VCN inhibitor 10.0mL

pH 6.7 ± 0.2 at 25°C

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

Di-agnostic Systems

Supplemement Solution:

Glucose 100.0g

L-Cysteine·HCl 25.9g

L-Glutamine 10.0g

L-Cystine 1.1g

Adenine 1.0g

Nicotinamide adenine dinucleotide 0.25g

Vitamin B12 0.1g

Thiamine pyrophosphate 0.1g

Guanine·HCl 0.03g

Fe(NO3)3·6H2O 0.02g

p-Aminobenzoic acid 0.013g

Thiamine·HCl 3.0mg

Source: The supplement solution IsoVitaleX® enrichment is available

from BD Diagnostic Systems This enrichment may be replaced by

supplement VX from BD Diagnostic Systems

Preparation of Supplement Solution: Add components to

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

sterilize

VCN Inhibitor:

Colistin 7.5mg

Vancomycin 3.0mg

Nystatin 12,500U

Preparation of VCN Inhibitor: Add components to

ster-ilize

Preparation of Medium: Add components, except supplement

so-lution and VCN inhibitor, to distilled/deionized water and bring

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

un-der 5–30% CO2 Aseptically add 10.0mL of sterile supplement

solu-tion and 10.0mL of sterile VCN inhibitor Mix thoroughly Aseptically distribute under 5–30% CO2 into sterile screw-capped tubes

Use: For the transportation and recovery of pathogenic Neisseria spe-cies.

Transport Medium Compositionper liter:

Sodium glycerophosphate 10.0g Agar 3.0g Sodium thioglycolate 1.0g CaCl2·2H2O 0.1g Methylene Blue 2.0mg

pH 7.3 ± 0.2 at 25°C

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

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat while stirring and bring to boiling Distribute into screw-capped tubes or vi-als Fill tubes nearly to capacity Leave only enough space so that when

a small swab is introduced the tube does not overflow Autoclave for

10 min at 15 psi pressure–121°C Tighten caps on tubes

Use: For the transportation of swab specimens for the recovery of a

wide variety of microorganisms, including Neisseria gonorrhoeae.

Transport Medium Stuart Compositionper liter:

Sodium glycerophosphate 10.0g Agar 3.0g Sodium thioglycolate 0.9g CaCl2·2H2O 0.1g Methylene Blue 2.0mg

pH 7.3 ± 0.2 at 25°C

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

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Gently heat while stirring and bring to boiling Distribute into screw-capped tubes or vi-als Fill tubes nearly to capacity Leave only enough space so that when

a small swab is introduced the tube does not overflow Autoclave for

10 min at 15 psi pressure–121°C Tighten caps on tubes

Use: For the transportation of swab specimens for the recovery of a

wide variety of microorganisms, including Neisseria gonorrhoeae.

Trebouxia Agar

Bristol's solution 850.0mL Soil extract 140.0mL Glucose 20.0g Agar 15.0g Proteose peptone 10.0g

Bristol's Solution:

NaNO3 solution 10.0g

KH2PO4 solution 7.0g

K2HPO4 solution 3.0g MgSO4·7H2O solution 3.0g CaCl2 solution 1.0g

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1808 Treponema bryantii Medium

NaCl solution 1.0g

FeCl3 solution 0.1mL

NaNO 3 Solution:

NaNO3 10.0g

Preparation of NaNO 3 Solution: Add NaNO3 to

distilled/deion-ized water and bring volume to 400.0mL Mix thoroughly

CaCl 2 Solution:

CaCl2 1.0g

Preparation of CaCl 2 Solution: Add CaCl2 to distilled/deionized

water and bring volume to 400.0mL Mix thoroughly

MgSO 4 ·7H 2 O Solution:

MgSO4·7H2O 3.0g

Preparation of MgSO 4 ·7H 2 O Solution: Add MgSO4·7H2Oto

distilled/deionized water and bring volume to 400.0mL Mix

thorough-ly

K 2 HPO 4 Solution:

K2HPO4 3.0g

Preparation of K 2 HPO 4 Solution: Add K2HPO4 to

distilled/de-ionized water and bring volume to 400.0mL Mix thoroughly

KH 2 PO 4 Solution:

KH2PO4 7.0g

Preparation of KH 2 PO 4 Solution: Add KH2PO4 to

distilled/de-ionized water and bring volume to 400.0mL Mix thoroughly

NaCl Solution:

NaCl 1.0g

Preparation of NaCl Solution: Add NaClto distilled/deionized

FeCl 3 Solution:

FeCl3 1.0g

Preparation of FeCl 3 Solution: Add FeCl3 to distilled/deionized

Preparation of Bristol's Solution: Add 10.0mL of NaNO3

solu-tion, 10.0mL of CaCl2 solution, 10.0mL of MgSO4·7H2O solution,

10.0mL of NaNO3 solution, 10.0mL of K2HPO4 solution, 10.0mL of

KH2PO4 solution, and 10.0mL of NaCl solution to distilled/deionized

water and bring volume to 1.0L Mix thoroughly Add 0.1mL of FeCl3

solution Mix thoroughly

Soil Extract:

African Violet soil 77.0g

Na2CO3 0.2g

Preparation of Soil Extract: Add components to

distilled/deion-ized water and bring volume to 200.0mL Mix thoroughly Autoclave

for 60 min at 15 psi pressure–121°C Filter through Whatman #1 filter

paper

Preparation of Medium: Combine components Mix thoroughly

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 of Brachiomonas submarina and Trebouxia

magna.

Treponema bryantii Medium

L-Cysteine·HCl 1.0g NaCl 0.9g (NH4)2SO4 0.9g

K2HPO4 0.45g

KH2PO4 0.45g MgSO4·7H2O 0.18g CaCl2·2H2O 0.12g Resazurin 1.0mg Rumen fluid, clarified 300.0mL NaHCO3 solution 100.0mL Glucose solution (10% w/v) 20.0mL

pH 7.0 ± 0.2 at 25°C

NaHCO 3 Solution:

NaHCO3 5.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly Filter sterilize Sparge with 100% CO2

Glucose 2.0g

Preparation of Glucose Solution : Add glucose to

distilled/deion-ized water and bring volume to 20.0mL Mix thoroughly Filter sterilize Sparge with 100% CO2

Preparation of Medium: Prepare and dispense medium under 100% CO2 Add components, except rumen fluid, NaHCO3 solution, and glucose solution, to distilled/deionized water and bring volume to 580.0mL Mix thoroughly Adjust pH to 7.0 with KOH Sparge with 100% CO2 Autoclave for 15 min at 15 psi pressure–121°C Cool to room temperature Aseptically add 300.0mL of sterile rumen fluid, 100.0mL of sterile NaHCO3 solution, and 20.0mL of sterile glucose so-lution Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation and maintenance of Treponema bryantii.

Treponema denticola Medium

(DSMZ Medium 909) Composition per 1045.0mL:

Solution A 1.0L Solution B 45.0mL

pH 7.1 ± 0.2 at 25°C

Solution A:

Trypticase™ 10.0g Yeast extract 2.5g Agar 3.0g Glucose 2.0g L-Cysteine·HCl 1.0g Na-thioglycolate 0.5g L-Asparagine 0.25g Brain heart infusion broth 450.0mL

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Treponema Isolation Medium 1809

Preparation of Solution A: Add components to distilled/deionized

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

to boiling Boil for 5 min Cool to room temperature while sparging

with 80% N2 + 20% CO2 Distribute to anaerobe tubes or bottles under

80% N2 + 20% CO2 Autoclave for 15 min at 15 psi pressure–121°C

Cool to room temperature

Brain Heart Infusion Broth:

Pancreatic digest of gelatin 14.5g

Brain heart, solids from infusion 6.0g

Peptic digest of animal tissue 6.0g

NaCl 5.0g

Casein 5.0g

Glucose 3.0g

Na2HPO4 2.5g

Preparation of Brain Heart Infusion Broth: Add components

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

Solution B:

Composition per 65.0mL:

NaHCO3 2.0g

Thiamine pyrophosphate 6.0mg

Volatile fatty acid solution 20.0mL

Rabbit serum 20.0mL

Volatile Fatty Acid Solution:

Composition per 102.0mL:

KOH, 0.1N 100.0mL

Isobutyric acid 0.5mL

D,L-2-methylbutyric acid 0.5mL

Isovaleric acid 0.5mL

Valeric acid 0.5mL

Preparation of Volatile Fatty Acid Solution: Combine

compo-nents and mix thoroughly

Preparation of Solution B: Add components, except rabbit serum,

to distilled/deionized water and bring volume to 45.0mL Mix

thor-oughly Filter sterilize Add 20.0mL rabbit serum

Preparation of Medium: Aseptically and anaerobically add

0.45mL solution B and 10.0mL solution A to individual tubes

Pressur-ize the tubes with H2 to 0.5 bar

Use: For the cultivation of Treponema denticola.

Treponema Isolation Medium

Solution A 450.0mL

Spirolate broth 450.0mL

Rabbit serum,

inactivated at 56°C for 30 min 100.0mL

pH 7.4 ± 0.2 at 25°C

Solution A:

Agar 8.0g

Asparagine 0.25g

Sodium thioglycolate 0.25g

Brain heart infusion broth 450.0mL

Preparation of Solution A: Combine components Mix

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

pressure–121°C Cool to 45°–50°C

Pancreatic digest of gelatin 14.5g Brain heart, solids from infusion 6.0g Peptic digest of animal tissue 6.0g NaCl 5.0g Casein 5.0g Glucose 3.0g

Na2HPO4 2.5g

Spirolate Broth:

Composition per liter:

Pancreatic digest of casein 15.0g Glucose 5.0g Yeast extract 5.0g NaCl 2.5g L-Cysteine·HCl·H2O 1.0g Sodium thioglycolate 0.5g Palmitic acid 0.05g Stearic acid 0.05g Oleic acid 0.05g Linoleic acid 0.05g

Preparation of Spirolate Broth: 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 Cool to 25°C

Preparation of Medium: Combine 450.0mL of sterile solution A, 450.0mL of sterile spirolate broth, and 100.0mL of rabbit serum Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the isolation and cultivation of oral, genital, and fecal treponemes

Treponema Isolation Medium

Beef heart, solids from infusion 20.0g Ionagar No 2 7.2g

K2HPO4 2.0g Arabinose 0.8g Glucose 0.8g Maltose 0.8g Polypeptone™ 0.8g Pyruvate 0.8g Starch, soluble 0.8g Sucrose 0.8g Cysteine·HCl 0.68g (NH4)2SO4 0.6g Serine 0.4g Tryptose 0.4g Yeast extract 0.4g NaCl 0.2g Rumen fluid 500.0mL Rabbit serum-cocarboxylase solution 100.0mL

pH 7.2 ± 0.2 at 25°C

Rabbit Serum-Cocarboxylase Solution:

Rabbit serum, heat inactivated 100.0mL Cocarboxylase solution 1.0mL

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1810 Treponema macrodentium Medium

Preparation of Rabbit Serum-Cocarboxylase Solution: Heat

rabbit serum at 56°C for 1 hr Add 1.0mL of cocarboxylase solution

Mix thoroughly

Cocarboxylase Solution:

Cocarboxylase 0.5g

Preparation of Cocarboxylase Solution: Add cocarboxylase to

1.0mL of distilled/deionized water Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except rumen fluid and

rabbit serum-cocarboxylase solution, to distilled/deionized water and

bring volume to 400.0mL 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 500.0mL of sterile rumen fluid and 100.0mL of sterile

rabbit serum-cocarboxylase solution Mix thoroughly Pour into sterile

Petri dishes or distribute into sterile tubes

Use: For the isolation of oral treponemes

Treponema macrodentium Medium

Glucose 1.0g

Nicotinamide 0.4g

Spermine·4HCl 0.15g

Sodium isobutyrate 0.02g

Carboxylase 5.0mg

PPLO agar 900.0mL

Bovine serum 100.0mL

pH 7.0 ± 0.2 at 25°C

PPLO Agar:

Composition per 900.0mL:

Beef heart, infusion from 50.0g

Agar 14.0g

Peptone 10.0g

NaCl 5.0g

pH 7.8 ± 0.2 at 25°C

Preparation of PPLO Agar: Add components to

Preparation of Medium: Combine components, except bovine

se-rum Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Cool to 45°–50°C Aseptically add sterile bovine serum Mix

thor-oughly Aseptically distribute into sterile tubes or flasks

Use: For the isolation and cultivation of Treponema macrodentium.

Treponema Medium

Ionagar No 2 8.0g

Glucose 5.0g

Yeast extract 5.0g

NaCl 2.5g

Cysteine·HCl 0.75g

Horse serum, inactivated 100.0mL

pH 7.4 ± 0.2 at 25°C

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

distilled/deionized water and bring volume to 900.0mL Mix thoroughly

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

121°C Cool to 45°–50°C Aseptically add 100.0mL of sterile horse

se-rum Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the isolation and cultivation of oral treponemes

Spirolate agar 900.0mL Rabbit serum,

Spirolate Agar:

Pancreatic digest of casein 15.0g Agar 14.0g Glucose 5.0g Yeast extract 5.0g NaCl 2.5g L-Cysteine·HCl·H2O 1.0g Sodium thioglycolate 0.5g Palmitic acid 0.05g Stearic acid 0.05g Oleic acid 0.05g Linoleic acid 0.05g

Preparation of Spirolate Agar: Add components to distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Preparation of Medium: To 900.0mL of cooled, sterile spirolate agar, aseptically add 100.0mL of rabbit serum Mix thoroughly Asep-tically distribute into sterile tubes or flasks

Spirolate agar 675.0mL Brain heart infusion broth 225.0mL Rabbit serum,

pH 7.0–7.2 ± 0.2 at 25°C

Spirolate Agar:

Pancreatic digest of casein 15.0g Ionagar No 2 8.0g Glucose 5.0g Yeast extract 5.0g NaCl 2.5g L-Cysteine·HCl·H2O 1.0g Sodium thioglycolate 0.5g Palmitic acid 0.05g Stearic acid 0.05g Oleic acid 0.05g Linoleic acid 0.05g

Preparation of Spirolate Agar: Add components to distilled/de-ionized water and bring volume to 675.0mL Mix thoroughly Auto-clave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C

Pancreatic digest of gelatin 14.5g Brain heart, solids from infusion 6.0g

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Treponema Medium 1811

NaCl 5.0g

Casein 5.0g

Glucose 3.0g

Na2HPO4 2.5g

Preparation of Medium: Aseptically combine 675.0mL of cooled,

sterile spirolate agar, 225.0mL of cooled, sterile brain heart infusion

broth, and 100.0mL of rabbit serum Mix thoroughly Pour into sterile

Petri dishes or distribute into sterile tubes

Solution A 440.0mL

Rabbit serum,

Mucin solution 20.0mL

pH 7.8 ± 0.2 at 25°C

Solution A:

Ionagar No 2 8.0g

NaCl 5.0g

Casein 5.0g

Glucose 3.0g

Na2HPO4 2.5g

Preparation of Solution A: Add 8.0g of ionagar to 440.0mL of brain

heart infusion broth Mix thoroughly Gently heat and bring to boiling

Glucose 5.0g

Yeast extract 5.0g

NaCl 2.5g

L-Cysteine·HCl·H2O 1.0g

Palmitic acid 0.05g

Stearic acid 0.05g

Oleic acid 0.05g

Linoleic acid 0.05g

Preparation of Spirolate Broth: 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 Cool to 25°

Mucin Solution:

Mucin 0.2g

Preparation of Mucin Solution: Add mucin to distilled/deionized water and bring volume to 20.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Aseptically combine 440.0mL of solu-tion A, 440.0mL of spirolate broth, 100.0mL of rabbit serum, and 20.0mL of mucin solution Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the isolation of intestinal treponemes

Agar 13.0g Glucose 1.4g Cysteine·HCl 0.64g (NH4)2SO4 0.5g Polypeptone™ 0.5g Starch, soluble 0.5g Yeast extract 0.5g Resazurin 1.6mg Salts solution 500.0mL Bovine rumen fluid 280.0mL

pH 7.2–7.5 at 25°C

Salts Solution:

NaHCO3 10.0g NaCl 2.0g

K2HPO4 1.0g

KH2PO4 1.0g CaCl2 0.2g MgSO4 0.2g CoCl 3.4mg MnSO4 3.4mg NaMoO4 3.4mg

Preparation of Salts Solution: Add components to distilled/deion-ized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components, except bovine rumen fluid, to distilled/deionized water and bring volume to 720.0mL 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 bovine ru-men fluid Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the isolation of intestinal treponemes

Cysteine·HCl·H2O 1.0g Glucose 1.0g Nicotinamide 0.4g Spermidine·4HCl 0.15g Sodium isobutyrate 0.02g Thiamine pyrophosphate 5.0mg PPLO broth 900.0mL Rabbit serum, inactivated 100.0mL

pH 7.8 ± 0.2 at 25°C

PPLO Broth:

Beef heart, infusion from solids 50.0g Peptone 10.0g NaCl 5.0g

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1812 Treponema Medium

Preparation of PPLO Broth: Add components to

Preparation of Medium: Combine components, except rabbit

rum Mix thoroughly Filter sterilize Aseptically add sterile rabbit

se-rum Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of oral treponemes For the cultivation of

Treponema denticola, Treponema macrodentium, and Treponema

ora-lis

Rabbit serum 100.0mL

Glucose 5.0g

Yeast extract 5.0g

NaCl 2.5g

Palmitic acid 0.05g

Stearic acid 0.05g

Oleic acid 0.05g

Linoleic acid 0.05g

Preparation of Spirolate Broth: Add components to

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

NaCl 5.0g

Casein 5.0g

Glucose 3.0g

Na2HPO4 2.5g

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

Preparation of Medium: Aseptically combine 675.0mL of cooled,

sterile spirolate broth, 225.0mL of cooled, sterile brain heart infusion

broth, and 100.0mL of rabbit serum Mix thoroughly

Use: For the cultivation of oral treponemes

Heart infusion broth, modified 450.0mL

Rabbit serum, inactivated 100.0mL

pH 7.4 ± 0.2 at 25°C

Heart Infusion Broth, Modified:

Beef heart, solids from infusion 500.0g

Tryptose 10.0g

NaCl 5.0g

Asparagine 2.5g Sodium thioglycolate 2.5g Pancreatic digest of casein 2.5g

Preparation of Heart Infusion Broth, Modified: Add compo-nents 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 Cool to 25°C

Pancreatic digest of casein 15.0g Glucose 5.0g Yeast extract 5.0g NaCl 2.5g L-Cysteine·HCl·H2O 1.0g Sodium thioglycolate 0.5g Palmitic acid 0.05g Stearic acid 0.05g Oleic acid 0.05g Linoleic acid 0.05g

Preparation of Spirolate Broth: Add components to distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Autoclave for

Preparation of Medium: Aseptically combine 450.0mL of cooled, sterile spirolate broth, 450.0mL of cooled, sterile heart infusion broth, modified, and 100.0mL of rabbit serum Mix thoroughly Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of treponemes

Beef heart, solids from infusion 250.0g Sucrose 50.0g Tryptose 5.0g NaCl 2.5g Yeast extract 2.5g Agar 0.5g Sodium thioglycolate 0.38g MgSO4 0.05g Horse serum, inactivated 100.0mL

pH 7.4 ± 0.2 at 25°C

Preparation of Medium: Add components, except horse serum, to distilled/deionized water and bring volume to 400.0mL Mix

thorough-ly Adjust pH to 7.4 Gently heat and bring to boiling Distribute into tubes in 4.0mL volumes Autoclave for 15 min at 15 psi pressure– 121°C Cool to 25°C Prior to inoculation, add 1.0mL sterile horse se-rum to each tube

Use: For the cultivation and maintenance of Treponema pallidum and other Treponema species.

Treponema Medium 1

Thioglycolate agar USP, alternate 900.0mL Normal calf serum 100.0mL

pH 7.1 ± 0.2 at 25°C

Thioglycolate Agar USP, Alternate:

Pancreatic digest of casein 15.0g Ionagar No 2 7.0g

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Treponema saccharophilum Medium 1813

Glucose 5.5g

Yeast extract 5.0g

NaCl 2.5g

L-Cystine 0.5g

Preparation of Thioglycolate Agar USP, Alternate: Add

com-ponents to distilled/deionized water and bring volume to 900.0mL

Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool

to 45°–50°C

Preparation of Medium: Aseptically combine 900.0mL of cooled

sterile thioglycolate agar USP, alternate, and 100.0mL of calf serum

Mix thoroughly Pour into sterile Petri dishes or distribute into sterile

tubes

Ionagar No 2 7.0g

Glucose 5.0g

Yeast extract 5.0g

NaCl 2.5g

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except rabbit serum,

to distilled/deionized water and bring volume to 900.0mL Mix

thoroughly Gently heat and bring to boiling Autoclave for 15 min

at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add sterile

rabbit serum Mix thoroughly Pour into sterile Petri dishes or

dis-tribute into sterile tubes

Spirolate agar 675.0mL

Spirolate Agar:

Ionagar No 2 7.0g

Glucose 5.0g

Yeast extract 5.0g

NaCl 2.5g

Palmitic acid 0.05g

Stearic acid 0.05g

Oleic acid 0.05g

Linoleic acid 0.05g

Preparation of Spirolate Agar: Add components to

distilled/de-ionized water and bring volume to 1.0L Mix thoroughly Gently heat

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

Cool to 25°C

Peptic digest of animal tissue 6.0g NaCl 5.0g Casein 5.0g Glucose 3.0g

Na2HPO4 2.5g

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 25°C

Preparation of Medium: Aseptically combine 675.0mL of cooled, sterile spirolate broth, 225.0mL of cooled, sterile brain heart infusion broth, and 100.0mL of rabbit serum Mix thoroughly

Treponema Medium, Prereduced

Agar 1.6g Glucose 1.4g Cysteine·HCl·H2O 0.64g (NH4)2SO4 0.5g Polypeptone™ 0.5g Starch, soluble 0.5g Yeast extract 0.5g Resazurin 1.6mg Salts solution 500.0mL Bovine rumen fluid 280.0mL

pH 7.2–7.5 at 25°C

Salts Solution:

NaHCO3 10.0g NaCl 2.0g

K2HPO4 1.0g

KH2PO4 1.0g CaCl2 0.2g MgSO4 0.2g CoCl 3.4mg MnSO4 3.4mg NaMoO4 3.4mg

Preparation of Salts Solution: Add components to distilled/deion-ized water and bring volume to 1.0L Mix thoroughly

Preparation of Medium: Add components, except bovine rumen fluid, to distilled/deionized water and bring volume to 720.0mL 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 280.0mL of sterile bovine rumen fluid Mix thoroughly Aseptically and anaerobi-cally distribute into sterile tubes or flasks under 100% N2

Use: For the cultivation of fecal and intestinal treponemes

Treponema saccharophilum Medium

Pancreatic digest of casein 2.0g Yeast extract 2.0g L-Cysteine·HCl 1.0g Resazurin 1.0mg Salt solution A 200.0mL Salt solution B 200.0mL NaHCO3 solution 100.0mL Glucose solution 20.0mL

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1814 Treponema succinifaciens Medium

iso-Butyric acid 0.4mL

n-Butyric acid 0.4mL

DL-2-Methylbutyric acid 0.2mL

iso-Valeric acid 0.2mL

n-Valeric acid 0.2mL

pH 6.7–7.0 at 25°C

Salt Solution A:

MgSO4·7H2O 0.96g

CaCl2·2H2O 0.59g

Preparation of Salt Solution A: Add components to

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

Salt Solution B:

K2HPO4 2.25g

KH2PO4 2.25g

NaCl 4.5g

(NH4)2SO4 4.5g

Preparation of Salt Solution B: Add components to

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

NaHCO 3 Solution:

NaHCO3 5.0g

Preparation of NaHCO 3 Solution: Add NaHCO3 to

sterilize Sparge with 100% CO2

Glucose 2.0g

Preparation of Glucose Solution : Add glucose to

distilled/deion-ized water and bring volume to 20.0mL Mix thoroughly Filter

steril-ize Sparge with 100% CO2

Preparation of Medium: Prepare and dispense medium under

100% CO2 Add components, except NaHCO3 solution and glucose

so-lution, to distilled/deionized water and bring volume to 880.0mL Mix

thoroughly Adjust pH to 7.0 with KOH Gently heat and bring to

boil-ing Continue boiling for 5 min Cool to room temperature while

sparg-ing with 100% CO2 Anaerobically distribute 8.8mL into anaerobic

culture tubes Autoclave for 15 min at 15 psi pressure–121°C Using a

syringe, add 1.0mL of sterile NaHCO3 solution and 0.2 mL of sterile

glucose solution to each tube Check that final pH is 6.7–7.0

Use: For the cultivation and maintenance of Treponema

saccharophi-lum.

Treponema succinifaciens Medium

(DSMZ Medium 275) Compositionper liter:

Solution A 875.0mL

Solution B 50.0mL

Solution D 50.0mL

Solution C 25.0mL

pH 7.0 ± 0.2 at 25°C

Solution A:

NaCl 1.0g

K2HPO4 0.5g

Cysteine-HCl·H2O 0.5g

KH2PO4 0.5g Yeast extract 0.5g Peptone 0.5g (NH4)2SO4 0.5g CaCl2·2H2O 0.1g MgSO4·7H2O 0.1g Resazurin 0.001g Rumen fluid, clarified 300.0mL

Preparation of Solution A: Add components to 575.0mL of

dis-tilled/deionized water Mix thoroughly Adjust pH to 6.2–6.3 with 4N

HCl Heat to boiling point Cool to room temperature under 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Solution B:

Glucose 20.0g

Preparation of Solution B: Add glucose to 100.0mL of distilled/de-ionized water Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Solution C:

KH2PO4 0.45g

Na2HPO4·2H2O 0.58g

Preparation of Solution C: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.4 Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Solution D:

NaHCO3 5.0g

Preparation of Solution D: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Sparge with 80%

N2 + 20% CO2 Filter sterilize

Preparation of Medium: Distribute solution A under 100% N2 into anaerobic tubes Autoclave for 20 min at 15 psi pressure–121°C Cool

to 25°C Aseptically and anaerobically add appropriate amounts of so-lutions B, C, and D to achieve final concentrations

Use: For the cultivation of Treponema succinifaciens.

Tributyrin Agar Compositionper liter:

Agar 15.0g Tributyrin (glyceryl tributyrate) 10.0g Peptone 5.0g Yeast extract 3.0g

pH 7.5 ± 0.2 at 25°C

Source: This medium is available as a prepared medium from Oxoid Unipath

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

Use: For the cultivation and enumeration of lipolytic fungi and

bacte-ria, especially Staphylococcus species, Flavobacterium species,

Clos-tridium species, and Pseudomonas species from butter Lipolytic

bac-teria appear as colonies surrounded by a clear zone

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