Handbook of Microbiological Media, Fourth Edition part 38 potx

Preparation of Medium: To 1.0L of chopped meat extract filtrate, add the remaining components, except the L-cysteine·HCl·H2O and chopped meat solids.. Preparation of Medium: To 1.0L of c

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Chopped Meat Broth 365

Preparation of Medium: Add components to distilled/deionized

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

to boiling Continue boiling for 2–3 min Allow precipitate to settle out

Distribute supernatant into 125.0mL flasks in 10.0mL volumes Add

standard solution or test solutions to each flask Adjust the volume of

each flask to 20.0mL with distilled/deionized water Autoclave for 10

min at 15 psi pressure–121°C

Use: For the microbiological assay of choline using Neurospora

crassa as the test microorganism.

Choline Medium

Compositionper liter:

NaCl 30.0g

Choline chloride 5.0g

K2HPO4 1.0g

MgSO4 0.5g

FeSO4 0.01g

pH 7.4 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.4

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

121°C

Use: For the cultivation of Achromobacter holinophagum.

Chondromyces VYZ Agar

Agar 15.0g

Fresh baker’s yeast cake 5.0g

CaCl2 1.0g

pH 7.2 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.2

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

121°C

Use: For the cultivation of Chondromyces species.

Chopped Liver Broth

Fresh beef liver 500.0g

Peptone 10.0g

K2HPO4 1.0g

Soluble starch 1.0g

pH 7.0 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from

Hi-Media

Preparation of Medium: Grind fresh beef liver Add to 1.0L of

dis-tilled/deionized water Gently heat and bring to boiling Continue

boil-ing for 60 min Cool to 25°C Adjust pH to 7.0 Gently heat and brboil-ing

to boiling Continue boiling for 10 min Filter through cheesecloth

Save chopped liver particles To filtrate, add remaining components

Bring volume to 1.0L with distilled/deionized water Adjust pH to 7.0

Filter through Whatman #1 filter paper Add chopped liver particles to

test tubes to a depth of 1.2–2.5 cm Add 10.0mL of broth to each tube

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

Use: For the isolation and cultivation of Clostridium botulinum,

Clostridium perfringens, and other anaerobic bacteria from foods.

Chopped Liver HiVeg Broth

Plant infusion 100.0g Plant peptone 10.0g

K2HPO4 1.0g Starch, soluble 1.0g

pH 7.0 ± 0.2 at 25°C

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

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

to boiling Distribute into tubes or flasks Autoclave for 20 min at 15 psi pressure–121°C

Use: For the isolation and cultivation of Clostridium botulinum, Clostridium perfringens, and other anaerobic bacteria from foods.

Chopped Meat Agar

Ground meat, fat free 500.0g Pancreatic digest of casein 30.0g Agar 15.0g

K2HPO4 5.0g Yeast extract 5.0g L-Cysteine·HCl 0.5g Resazurin 1.0mg

NaOH (1N solution) 25.0mL

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Use lean beef or horse meat Remove fat and connective tissue Grind finely Add ground meat and 25.0mL of NaOH solution to distilled/deionized water and bring volume to 1025.0mL Gently heat and bring to boiling Continue boiling for 15 min without stirring Cool to room temperature Remove fat from sur-face Filter and retain both meat particles and filtrate Adjust volume of filtrate to 1.0L with distilled/deionized water Add pancreatic digest of casein, agar, K2HPO4, yeast extract, and resazurin Gently heat and bring to boiling Boil for 1–2 min Add L-cysteine·HCl Mix

thorough-ly Distribute 7.0mL into tubes that contain meat particles (1 part meat particles to 5 parts fluid) Autoclave for 30 min at 15 psi pressure– 121°C

Use: For the cultivation of various anaerobes

Chopped Meat Broth

Ground meat, fat free 500.0g Pancreatic digest of casein 30.0g

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Use lean beef or horse meat Remove fat and connective tissue Grind finely Add ground meat and 25.0mL of NaOH solution to distilled/deionized water and bring volume to 1025.0mL Gently heat and bring to boiling Continue boiling for 15 min without stirring Cool to room temperature Remove fat from sur-face Filter and retain both meat particles and filtrate Adjust volume of filtrate to 1.0L with distilled/deionized water Add pancreatic digest of casein, K2HPO4, yeast extract, and resazurin Gently heat and bring to

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366 Chopped Meat Broth with Carbohydrates

boiling Boil for 1–2 min Add L-cysteine·HCl Mix thoroughly

Dis-tribute 7.0mL into tubes that contain meat particles (1 part meat

parti-cles to 5 parts fluid) Autoclave for 30 min at 15 psi pressure–121°C

Use: For the cultivation of various anaerobes

Chopped Meat Broth with Carbohydrates

(DSMZ Medium 110)

Ground meat, fat free 500.0g

Pancreatic digest of casein 30.0g

K2HPO4 5.0g

Yeast extract 5.0g

Glucose 4.0g

Cellobiose 1.0g

Maltose 1.0g

Starch, soluble 1.0g

L-Cysteine·HCl 0.5g

Resazurin 1.0mg

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Use lean beef or horse meat Remove fat

and connective tissue Grind finely Add ground meat and 25.0mL of

NaOH solution to distilled/deionized water and bring volume to

1025.0mL Gently heat and bring to boiling Continue boiling for 15

min without stirring Cool to room temperature Remove fat from

sur-face Filter and retain both meat particles and filtrate Adjust volume of

filtrate to 1.0L with distilled/deionized water Add pancreatic digest of

casein, K2HPO4, yeast extract, and resazurin Gently heat and bring to

boiling Boil for 1–2 min Add glucose, cellobiose, maltose, and

solu-ble starch Add L-cysteine·HCl Mix thoroughly Distribute 7.0mL into

tubes that contain meat particles (1 part meat particles to 5 parts fluid)

Use: For the cultivation of numerous anaerobes, including

Actinomy-ces suis, Anaerobiospirillum succiniciproducens, Bacteroides

distaso-nis, Bacteroides eggerthii, Bacteroides fragilis, Bacteroides

helco-genes, Bacteroides macacae, Bacteroides pyohelco-genes, Bacteroides

splanchnicus, Bacteroides suis, Centipeda periodontii, numerous

Clostridium species, Eubacterium brachy, Eubacterium eligens,

Eubacterium hallii, Eubacterium limosum, Eubacterium plautii,

Eubacterium ruminantium, Eubacterium saburreum, Eubacterium

sir-aeum, Eubacterium species, Eubacterium tarantellus, Eubacterium

tenue, Eubacterium ventriosum, Megamonas hypermegas,

Peptococ-cus niger, PeptostreptococPeptococ-cus productus, Prevotella buccae,

votella buccalis, Prevotella denticola, Prevotella intermedia,

Pre-votella oralis, and Selenomonas sputigena.

Chopped Meat Broth with Formate and Fumarate

(LMG Medium 69)

K2HPO4 5.0g

Yeast extract 5.0g

Resazurin 1.0mg

Formate-fumarate solution 7.7mL

pH 7.0 ± 0.2 at 25°C

Formate-Fumarate Solution:

Compositionper 100.0mL:

Sodium formate 6.0g Fumaric acid 6.0g

Preparation of Formate-Fumarate Solution: Add components

to distilled/deionized water and bring volume to 100.0mL Adjust pH

to 7.0 Filter sterilize

thorough-ly Distribute 6.5mL into tubes that contain meat particles (1 part meat particles to 5 parts fluid) Autoclave for 30 min at 15 psi pressure– 121°C Cool to 25°C Aseptically add 50 µL of sterile formate/fumar-ate solution to each tube prior to inoculation

Use: For the cultivation of various Clostridium spp.

(LMG Medium 70)

Ground meat, fat free 500.0g Pancreatic digest of casein 30.0g

Vitamin K1 solution 7.7mL

pH 7.0 ± 0.2 at 25°C

Vitamin K 1 Solution:

Ethanol (20% solution) 50.0mL Vitamin K1 0.7gL

Preparation of Vitamin K 1 Solution: Mix components Filter sterilize Store solution protected from light at 5°C Discard after one month

thorough-ly Distribute 6.5mL into tubes that contain meat particles (1 part meat particles to 5 parts fluid) Autoclave for 30 min at 15 psi pressure– 121°C Cool to 25°C Aseptically add 50 µL of sterile vitamin K1 solu-tion to each tube prior to inoculasolu-tion

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Chopped Meat Carbohydrate Medium with Tween™ 80 367

Chopped Meat Carbohydrate Medium

Peptone 30.0g

K2HPO4 5.0g

Yeast extract 5.0g

Cellobiose 1.0g

Maltose 1.0g

Starch 1.0g

L-Cysteine·HCl·H2O 0.5g

Chopped meat extract filtrate 1.0L

Chopped meat extract solids 200.0mL

Resazurin (0.025% solution) 4.0mL

pH 7.0 ± 0.2 at 25°C

Chopped Meat Extract:

Beef or horse meat 500.0g

Preparation of Chopped Meat Extract: Use lean beef or horse

meat Remove fat and connective tissue Grind Add meat and NaOH

to distilled/deionized water and bring volume to 1.0L Gently heat and

bring to boiling while stirring Cool to 25°C Remove fat from surface

Filter Reserve ground meat particles and filtrate Add

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

Preparation of Medium: To 1.0L of chopped meat extract filtrate,

add the remaining components, except the L-cysteine·HCl·H2O and

chopped meat solids Mix thoroughly Gently heat to boiling Cool to

room temperature Add the L-cysteine·HCl·H2O Adjust pH to 7.0

Dis-tribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by

volume) into tubes under O2-free 97% N2 + 3% H2 Cap with rubber

stoppers and place tubes in a press Autoclave for 15 min at 15 psi

pres-sure–121°C with fast exhaust

Use: For the cultivation of anaerobic bacteria, including Clostridium

species, Eubacterium species, and Gemmiger formicilis.

Chopped Meat Carbohydrate

Medium with Rumen Fluid

(ATCC Medium 1016)

Peptone 30.0g

K2HPO4 5.0g

Yeast extract 5.0g

Cellobiose 1.0g

Maltose 1.0g

Starch 1.0g

Rumen fluid 150.0mL

pH 7.0 ± 0.2 at 25°C

Filter Reserve ground meat particles and filtrate Add distilled/deion-ized water to filtrate and bring volume to 1.0L

Preparation of Medium: To 1.0L of chopped meat extract filtrate, add the remaining components, except L-cysteine·HCl·H2O and chopped meat solids Mix thoroughly Gently heat to boiling Cool to room temperature Add the L-cysteine·HCl·H2O Adjust pH to 7.0 Dis-tribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by volume) into tubes under O2-free 97% N2 + 3% H2 Cap with rubber stoppers and place tubes in a press Autoclave for 15 min at 15 psi pres-sure–121°C with fast exhaust

Use: For the cultivation of anaerobic bacteria, including Butyrivibrio crossotus, Eubacterium species, and Ruminococcus species.

Chopped Meat Carbohydrate Medium with Rumen Fluid

Peptone 30.0g

K2HPO4 5.0g Yeast extract 5.0g Glucose 4.0g Cellobiose 1.0g Maltose 1.0g Starch 1.0g L-Cysteine·HCl·H2O 0.5g Chopped meat extract filtrate 1.0L Chopped meat extract solids 200.0mL Rumen fluid 150.0mL Resazurin (0.025% solution) 4.0mL

pH 7.0 ± 0.2 at 25°C

Preparation of Chopped Meat Extract: Use lean beef or horse meat Remove fat and connective tissue Grind Add meat and NaOH

to distilled/deionized water and bring volume to 1.0L Gently heat and bring to boiling while stirring Cool to 25°C Remove fat from surface Filter Reserve ground meat particles and filtrate Add distilled/deion-ized water to filtrate and bring volume to 1.0L

Use: For the cultivation of anaerobic bacteria, including Fusobacte-rium prausnitzii, EubacteFusobacte-rium species, and Prevotella ruminicola.

Chopped Meat Carbohydrate Medium with Tween™ 80

Peptone 30.0g

K2HPO4 5.0g Yeast extract 5.0g Cellobiose 1.0g Maltose 1.0g Starch 1.0g

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368 Chopped Meat Glucose Agar

Tween™ 80 1.0g

pH 7.0 ± 0.2 at 25°C

add the remaining components, except L-cysteine·HCl·H2O and

Use: For the cultivation of Coprococcus species and

Peptostreptococ-cus micros.

Chopped Meat Glucose Agar

(LMG Medium 68)

Agar 15.0g

Glucose 10.0g

K2HPO4 5.0g

Yeast extract 5.0g

Resazurin 1.0mg

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Use lean beef or horse meat Remove fat

and connective tissue Grind finely Add ground meat and 25.0mL of

NaOH solution to distilled/deionized water and bring volume to

1025.0mL Gently heat and bring to boiling Continue boiling for 15

min without stirring Cool to room temperature Remove fat from

sur-face Filter and retain both meat particles and filtrate Adjust volume of

filtrate to 1.0L with distilled/deionized water Add pancreatic digest of

casein, agar, K2HPO4, yeast extract, and resazurin Gently heat and

bring to boiling Boil for 1–2 min Add L-cysteine·HCl Mix

thorough-ly Distribute 7.0mL into tubes that contain meat particles (1 part meat

particles to 5 parts fluid) Autoclave for 30 min at 15 psi pressure–

121°C

Chopped Meat Glucose Broth

(LMG Medium 68)

Glucose 10.0g

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Use lean beef or horse meat Remove fat and connective tissue Grind finely Add ground meat and 25.0mL of NaOH solution to distilled/deionized water and bring volume to 1025mL Gently heat and bring to boiling Continue boiling for 15 min without stirring Cool to room temperature Remove fat from surface Filter and retain both meat particles and filtrate Adjust volume of fil-trate to 1.0L with distilled/deionized water Add pancreatic digest of casein, agar, K2HPO4, yeast extract, and resazurin Gently heat and bring to boiling Boil for 1–2 min Add L-cysteine·HCl Mix

thorough-ly Distribute 7.0mL into tubes that contain meat particles (1 part meat particles to 5 parts fluid) Autoclave for 30 min at 15 psi pressure– 121°C

Chopped Meat Glucose Medium

Peptone 30.0g

K2HPO4 5.0g Yeast extract 5.0g Glucose 5.0g L-Cysteine·HCl·H2O 0.5g Chopped meat extract filtrate 1.0L Chopped meat extract solids 200.0mL Resazurin (0.025% solution) 4.0mL

pH 7.0 ± 0.2 at 25°C

Use: For the cultivation of Clostridium species and Selenomonas noxia.

Chopped Meat Glucose Medium with NaCl

NaCl 30.0g Peptone 30.0g

K2HPO4 5.0g Yeast extract 5.0g

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Chopped Meat Medium with Formate and Fumarate 369

Glucose 5.0g

pH 7.0 ± 0.2 at 25°C

Use: For the cultivation and maintenance of anaerobic halophilic

bac-teria

Chopped Meat Medium

Peptone 30.0g

K2HPO4 5.0g

Yeast extract 5.0g

pH 7.0 ± 0.2 at 25°C

Use: For the cultivation and maintenance of a variety of anaerobic

bacteria, including Bacteroides species, Bifidobacterium species,

Cap-nocytophaga species, Clostridium species, Eubacterium species, Fuso-bacterium species, Peptostreptococcus species, Prevotella species, Propionibacterium species, Ruminococcus species, and others.

Chopped Meat Medium with 10% Fetal Calf Serum

Peptone 30.0g

K2HPO4 5.0g Yeast extract 5.0g L-Cysteine·HCl·H2O 0.5g Chopped meat extract filtrate 1.0L Chopped meat extract solids 200.0mL Fetal calf serum 100.0mL Resazurin (0.025% solution) 4.0mL

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: To 1.0L of chopped meat extract filtrate, add the remaining components, except the L-cysteine·HCl·H2O and chopped meat solids Mix thoroughly Gently heat to boiling Cool to room temperature Add the L-cysteine·HCl·H2O Adjust pH to 7.0 Dis-tribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by volume) into tubes under O2-free 97% N2 + 3% H2 Cap with rubber stoppers and place tubes in a press Autoclave for 15 min at 15 psi pres-sure–121°C with fast exhaust

Use: For the cultivation and maintenance of Actinomyces hordeovul-neris.

Chopped Meat Medium with Formate and Fumarate

Peptone 30.0g

K2HPO4 5.0g Yeast extract 5.0g L-Cysteine·HCl·H2O 0.5g Chopped meat extract filtrate 1.0L Chopped meat extract solids 200.0mL Resazurin (0.025% solution) 4.0mL Formate-fumarate solution 0.05mL

pH 7.0 ± 0.2 at 25°C

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370 Chopped Meat Medium with 1% Glucose

Sodium formate 6.0g

Fumaric acid 6.0g

add the remaining components, except the L-cysteine·HCl·H2O,

formate-fumarate solution, and chopped meat solids Mix thoroughly Gently heat

to boiling Cool to room temperature Add the L-cysteine·HCl·H2O

Ad-just pH to 7.0 Distribute 1 part chopped meat solids (by volume) and 5

parts of liquid (by volume) into tubes under O2-free 97% N2 + 3% H2

Cap with rubber stoppers and place tubes in a press Autoclave for 15

min at 15 psi pressure–121°C with fast exhaust Prior to inoculation, add

0.05mL of formate-fumarate solution to each tube containing

approxi-mately 6.5mL of chopped meat medium

Use: For the cultivation and maintenance of Bacteroides ureolyticus

and Wolinella species.

Chopped Meat Medium with 1% Glucose

Peptone 30.0g

Glucose 10.0g

K2HPO4 5.0g

Yeast extract 5.0g

pH 7.0 ± 0.2 at 25°C

room temperature Add the L-cysteine Adjust pH to 7.0 Distribute 1

part chopped meat solids (by volume) and 5 parts of liquid (by volume)

into tubes under O2-free 97% N2 + 3% H2 Cap with rubber stoppers

and place tubes in a press Autoclave for 15 min at 15 psi pressure–

121°C with fast exhaust

Use: For the cultivation and maintenance of anaerobic bacteria,

including Bacteroides disiens, Coprococcus eutastus, Eubacterium

formicigenerans, Prevotella disiens, Ruminococcus torques, and

Streptococcus hansenii.

Chopped Meat Medium with Menadione

Peptone 30.0g

K2HPO4 5.0g

Yeast extract 5.0g L-Cysteine·HCl·H2O 0.5g Chopped meat extract filtrate 1.0L Chopped meat extract solids 200.0mL Resazurin (0.025% solution) 4.0mL Menadione solution 0.25mL

pH 7.0 ± 0.2 at 25°C

Menadione Solution:

Menadione (vitamin K3) 50.0μg Ethanol (20% solution) 25.0mL

Preparation of Menadione Solution: Dissolve menadione in eth-anol Filter sterilize

Preparation of Medium: To 1.0L of chopped meat extract filtrate, add the remaining components, except the L-cysteine·HCl·H2O, me-nadione solution, and chopped meat solids Mix thoroughly Gently heat to boiling Cool to room temperature Add the L-cysteine·HCl·H2O Adjust pH to 7.0 Distribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by volume) into tubes under

O2-free 97% N2 + 3% H2 Cap with rubber stoppers and place tubes

in a press Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust Prior to inoculation, add 0.25mL of menadione solution to each tube containing approximately 5.0mL of chopped meat medium

Use: For the cultivation and maintenance of Bacteroides gingivalis, Bacteroides macacae, and Porphyromonas gingivalis.

Chopped Meat Medium, Modified (DSMZ Medium 797)

Chopped meat extract solids 200.0g Trypticase™ 30.0g Agar 20.0g

K2HPO4 5.0g Yeast extract 5.0g L-Cysteine·HCl·H2O 0.5g Chopped meat extract filtrate 1.0L Hemin solution 10.0mL Resazurin (0.025% solution) 4.0mL Vitamin K1 solution 0.2mL

pH 7.0 ± 0.2 at 25°C

to distilled/deionized water and bring volume to 1.0L Gently heat and bring to boiling while stirring Cool to 25°C Remove fat from surface

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Chopped Meat Medium, Modified with Arginine 371

Filter Reserve both ground meat particles and filtrate Add distilled/

deionized water to filtrate and bring volume to 1.0L

Hemin Solution:

Composition per 100.0mL:

Hemin 0.05g

Preparation of Hemin Solution: Add components to

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

Ethanol (95% solution) 30.0mL

Vitamin K1 0.15mL

Preparation of Vitamin K 1 Solution: Mix components Store

so-lution protected from light at 5°C Discard after 1 month

add the remaining components, except the L-cysteine·HCl·H2O, hemin

solution, vitamin K1 solution, and chopped meat solids Mix

thor-oughly Gently heat to boiling Boil for 5 min Cool to 25°C while

sparging with 80% N2 + 10% H2 + 10% CO2 Add the

L-cysteine·HCl·H2O, hemin solution, and vitamin K1 solution Adjust pH

to 7.0 Distribute 1 part chopped meat solids (by volume) and 5 parts

of liquid (by volume) into tubes under 80% N2 + 10% H2 + 10% CO2

Use: For the cultivation of Dialister pneumosintes.

Chopped Meat Medium, Modified

Peptone 30.0g

Agar 20.0g

K2HPO4 5.0g

Yeast extract 5.0g

Hemin solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Hemin Solution:

Hemin 0.05g

Ethanol (95% solution) 30.0mL Vitamin K1 0.15mL

Preparation of Vitamin K 1 Solution: Mix components Store so-lution protected from light at 5°C Discard after 1 month

Preparation of Medium: To 1.0L of chopped meat extract filtrate, add the remaining components, except the L-cysteine·HCl·H2O, hemin solution, vitamin K1 solution, and chopped meat solids Mix

thorough-ly Gently heat to boiling Cool to room temperature Add the L-cysteine·HCl·H2O, hemin solution, and vitamin K1 solution Adjust pH

of liquid (by volume) into tubes under O2-free 97% N2 + 3% H2 Cap with rubber stoppers and place tubes in a press Autoclave for 15 min

at 15 psi pressure–121°C with fast exhaust

Use: For the cultivation and maintenance of a variety of anaerobic

bacteria, including Actinomyces species, Bacteroides species, Clostrid-ium species, EubacterClostrid-ium species, FusobacterClostrid-ium species, Peptostrep-tococcus species, Porphyromonas species, Prevotella species, Propi-onibacterium species, Selenomonas species, and others.

Chopped Meat Medium, Modified with Arginine

Pancreatic digest of casein 30.0g Peptone 30.0g Agar 20.0g Arginine 5.0g

K2HPO4 5.0g Yeast extract 5.0g L-Cysteine·HCl·H2O 0.5g Chopped meat extract filtrate 1.0L Chopped meat extract solids 200.0mL Hemin solution 10.0mL Resazurin (0.025% solution) 4.0mL Vitamin K1 solution 0.2mL

pH 7.0 ± 0.2 at 25°C

Tween™ 80 25.0mL

Hemin Solution:

Hemin 0.05g

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

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372 Chopped Meat Medium, Modified with Formate and Fumarate

Preparation of Vitamin K 1 Solution: Mix components Store

so-lution protected from light at 5°C Discard after 1 month

add the remaining components, except the L-cysteine·HCl·H2O, hemin

solution, vitamin K1 solution, and chopped meat solids Mix

thorough-ly Gently heat to boiling Cool to room temperature Add the

L-cysteine·HCl·H2O, hemin solution, and vitamin K1 solution Adjust pH

of liquid (by volume) into tubes under O2-free 97% N2 + 3% H2 Cap

with rubber stoppers and place tubes in a press Autoclave for 15 min

at 15 psi pressure–121°C with fast exhaust

Use: For the cultivation and maintenance of Eubacterium lentum.

Chopped Meat Medium, Modified with Formate and Fumarate

Peptone 30.0g

Agar 20.0g

K2HPO4 5.0g

Yeast extract 5.0g

Hemin solution 10.0mL

Formate-fumarate solution 0.25mL

pH 7.0 ± 0.2 at 25°C

Hemin Solution:

Hemin 0.05g

Sodium formate 6.0g

Fumaric acid 6.0g

Ethanol (95% solution) 30.0mL

Vitamin K1 0.15mL

Preparation of Vitamin K 1 Solution: Mix components Store so-lution protected from light at 5°C Discard after one month

Preparation of Medium: To 1.0L of chopped meat extract filtrate, add the remaining components, except the L-cysteine·HCl·H2O, hemin solution, vitamin K1 solution, formate-fumarate solution, and chopped meat solids Mix thoroughly Gently heat to boiling Cool to room tem-perature Add the L-cysteine·HCl·H2O, hemin solution, and vitamin K1 solution Adjust pH to 7.0 Distribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by volume) into tubes under O2-free 97% N2 + 3% H2 Cap with rubber stoppers and place tubes in a press Autoclave for 15 min at 15 psi pressure–121°C with fast exhaust Prior

to inoculation, add 0.25mL of formate-fumarate solution to each tube containing approximately 5.0mL of chopped meat medium, modified

Use: For the cultivation and maintenance of Bacteroides gracilis, Bacteroides ureolyticus, Campylobacter mucosalis, and Wolinella suc-cinogenes.

Chopped Meat Medium, Modified with Tween™ 80

Pancreatic digest of casein 30.0g Peptone 30.0g Agar 20.0g

K2HPO4 5.0g Yeast extract 5.0g L-Cysteine·HCl·H2O 0.5g Chopped meat extract filtrate 1.0L Chopped meat extract solids 200.0mL Hemin solution 10.0mL Resazurin (0.025% solution) 4.0mL Vitamin K1 solution 0.2mL

pH 7.0 ± 0.2 at 25°C

Tween™ 80 25.0mL

Hemin Solution:

Hemin 0.05g

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

Preparation of Vitamin K 1 Solution: Mix components Store so-lution protected from light at 5°C Discard after 1 month

Preparation of Medium: To 1.0L of chopped meat extract filtrate, add the remaining components, except the L-cysteine·HCl·H2O, hemin solution, vitamin K1 solution, and chopped meat solids Mix thoroughly Gently heat to boiling Cool to room temperature Add the

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L-Chopped Meat Medium with 0.025% Tween™ 80 373

cysteine·HCl·H2O, hemin solution, and vitamin K1 solution Adjust pH

to 7.0 Distribute 1 part chopped meat solids (by volume) and 5 parts of

liquid (by volume) into tubes under O2-free 97% N2 + 3% H2 Cap with

rubber stoppers and place tubes in a press Autoclave for 15 min at 15 psi

pressure–121°C with fast exhaust

Use: For the cultivation and maintenance of Lactobacillus species and

Eubacterium biforme.

Chopped Meat Medium with

10% Reduced Filtered Rumen Fluid

Peptone 30.0g

K2HPO4 5.0g

Yeast extract 5.0g

Rumen fluid, reduced and filtered 100.0mL

pH 7.0 ± 0.2 at 25°C

pres-sure–121°C

Use: For the cultivation and maintenance of Eubacterium hallii.

Chopped Meat Medium for Treponema spp.

(DSMZ Medium 78a)

K2HPO4 5.0g

Yeast extract 5.0g

Resazurin 1.0mg

Amino acid solution 50.0mL

Vitamin solution 5.0mL

pH 7.0 ± 0.2 at 25°C

Amino Acid Solution:

L-Glutamine 0.7g

L-Histidine 0.6g

L-Serine 0.5g

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

Vitamin Solution:

Vitamin B12 50.0mg Pantothenic acid 50.0mg Riboflavin 50.0mg α-Lipoic acid 50.0mg

p-Aminobenzoic acid 50.0mg

Thiamine-HCl·2H2O 50.0mg Nicotinic acid 25.0mg Nicotine amide 25.0mg Biotin 20.0mg Folic acid 20.0mg Pyridoxamine-HCl 10.0mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 1.0L Mix thoroughly Filter ster-ilize

Preparation of Medium: Use lean beef or horse meat Remove fat and connective tissue Grind finely Add ground meat and 25.0mL of NaOH solution to distilled/deionized water and bring volume to 1025.0mL Gently heat and bring to boiling Continue boiling for 15 min without stirring Cool to room temperature Remove fat from sur-face Filter and retain both meat particles and filtrate Adjust volume of filtrate to 1.0L with distilled/deionized water Add pancreatic digest of casein, K2HPO4, yeast extract, and resazurin Gently heat and bring to boiling Boil for 1–2 min Add L-cysteine·HCl Mix thoroughly Auto-clave for 30 min at 15 psi pressure–121°C Aseptically add amino acid and vitamin solutions Mix thoroughly Aseptically distribute 7.0mL amounts into tubes that contain meat particles (1 part meat particles to

5 parts fluid)

Use: For the cultivation of Treponema brennaborense.

Chopped Meat Medium with 0.025% Tween™ 80

Peptone 30.0g

K2HPO4 5.0g Yeast extract 5.0g L-Cysteine·HCl·H2O 0.5g Tween™ 80 0.25g Chopped meat extract filtrate 1.0L Chopped meat extract solids 200.0mL Resazurin (0.025% solution) 4.0mL

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: To 1.0L of chopped meat extract filtrate, add the remaining components, except the L-cysteine·HCl·H2O and chopped meat solids Mix thoroughly Gently heat to boiling Cool to room temperature Add the L-cysteine·HCl·H2O Adjust pH to 7.0

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Dis-374 Chopped Meat Medium with 1% Tween™ 80

tribute 1 part chopped meat solids (by volume) and 5 parts of liquid (by

Use: For the cultivation and maintenance of Eubacterium and

Lacto-bacillus species.

Chopped Meat Medium with 1% Tween™ 80

Peptone 30.0g

Tween™ 80 10.0g

K2HPO4 5.0g

Yeast extract 5.0g

pH 7.0 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Eubacterium biforme and

Lactobacillus species.

Christensen Agar

Agar 15.0g

NaCl 5.0g

Sodium citrate 3.0g

KH2PO4 1.0g

Phenol Red 12.0mg

pH 6.9 ± 0.2 at 25°C

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

to boiling Dispense into tubes or flasks Autoclave for 15 min at 15 psi

pressure–121°C Pour into sterile Petri dishes or leave in tubes Allow

tubes to cool in a slanted position

Use: For the differentiation of enteric pathogens, especially members

of the Enterobacteriaceae, and coliforms based on their ability to utilize

citrate as a carbon source Bacteria that can utilize citrate turn the

medium pink-red

Christensen Agar

Agar 15.0g NaCl 5.0g Sodium citrate 3.0g

KH2PO4 1.0g Yeast extract 0.5g Glucose 0.2g L-Cysteine·HCl·H2O 0.1g Phenol Red 12.0mg

pH 6.9 ± 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 and bring

to boiling Dispense into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes Allow tubes to cool in a slanted position

of the Enterobacteriaceae, and coliforms based on their ability to utilize citrate as a carbon source Bacteria that can utilize citrate turn the medium pink-red

Christensen Citrate Agar (BAM M39)

KH2PO4 1.0g Yeast extract 0.5g Ferric ammonium citrate 0.4g L-Cysteine·HCl·H2O 0.1g

Na2S2O5 0.08g Phenol Red 12.0mg

pH 6.9 ± 0.2 at 25°C

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

of the Enterobacteriaceae, and coliforms based on their ability to utilize citrate as a carbon source Bacteria that can utilize citrate turn the medium pink-red

Christensen Citrate Agar, Modified

(Citrate Agar)

KH2PO4 1.0g Yeast extract 0.5g Glucose 0.2g L-Cysteine·HCl·H2O 0.1g Phenol Red 0.02g

pH 6.7 ± 0.2 at 25°C

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