Handbook of Microbiological Media, Fourth Edition part 47 ppsx

4.0g Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L.. Add components, except glucose solution, Na2S·9H2O solution, and Wolfe’s mineral soluti

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Costein’s LDS Test Medium 455

Bovine serum 100.0mL

Nystatin solution 1.15mL

L-Cystine (1% solution) 1.0mL

Egg yolk emulsion 10 eggs

pH 7.4 ± 0.2 at 25°C

Solution A:

Compositionper liter:

Meat extract 9.0g

Proteose peptone No 3 9.0g

NaCl 2.7g

Glucose 1.8g

Na2HPO4·12H2O 1.8g

K2TeO3 (2% solution) 75.0mL

L-Cystine (1% solution) 10.0mL

Caution: Potassium tellurite is toxic

Preparation of Solution A: Add components to distilled/deionized

water and bring volume to 985.0mL Mix thoroughly Filter sterilize

Egg Yolk Emulsion:

Composition:

Chicken egg yolks 9

Whole chicken egg 1

Preparation of Egg Yolk Emulsion: Soak eggs with 1:100

dilu-tion of saturated mercuric chloride soludilu-tion for 1 min Crack eggs and

separate yolks from whites Mix egg yolks with 1 chicken egg Filter

sterilize

Nystatin Solution:

Compositionper 10.0mL:

Nystatin 10,000U

Preparation of Nystatin Solution: Add nystatin to

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

ster-ilize

L -Cystine Solution:

L-Cystine 0.1g

Preparation of L -Cystine Solution: Add L-cystine to

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

ster-ilize

Preparation of Medium: To 985.0mL of sterile solution A,

asepti-cally add the remaining components Mix thoroughly Aseptiasepti-cally

dis-tribute into sterile tubes in 2.0–3.0mL volumes

Use: For the isolation and cultivation of Corynebacterium

diphthe-riae.

Corynebacterium Medium with Blood

(DSMZ Medium 240) Compositionper liter:

Agar 15.0g

Casein peptone, tryptic digest 10.0g

Yeast extract 5.0g

Glucose 5.0g

NaCl 5.0g

Distilled water 1000.0mL

Sheep or horse blood, defibrinated 50.0mL

pH 7.3 ± 0.2 at 25°C

Preparation of Medium: Add components, except sheep or horse

blood, to distilled/deionized water and bring volume to 950.0mL 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 sheep or horse blood Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the cultivation of Streptococcus alactolyticus, Corynebacte-rium spp., Desemzia incerta=BrevibacteCorynebacte-rium incertum, and Moraxella bovis.

Corynebacterium Medium CII

CaCO3 20.0g Agar 15.0g Sucrose 10.0g Yeast extract 4.0g

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 of Clavibacter michiganensis.

Corynebacterium Medium with Salt

(DSMZ Medium 229) Compositionper liter:

NaCl 65.0g Agar 15.0g Casein peptone, tryptic digest 10.0g Yeast extract 5.0g Glucose 5.0g

pH 7.3 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Nesterenkonia halo-bia=Micrococcus halobius.

Costein’s LDS Test Medium Composition per liter:

Meat peptone 4.5g Papaic digest of soybean meal 2.0g Yeast extract 3.0g NaCl 5.0g

D-Glucose 1.0g

L-Lysine monohydrochloride 10.0g

Na2S2O3 0.2g Fe(NH4)2(SO4)2·6H2O 0.2g Bromocresol Purple 0.032 Agar 6.0g

pH 5.6 ± 0.2 at 25°C

Source: This medium is available from HiMedia

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Distribute into tubes Overlay with viscous parrafin Autoclave for 15 min at 15 psi pressure– 121°C Allow tubes to solidify in a vertical position

Use: For the identification of members of Enterobacteriaceae on the basis of lysine decarboxylase and hydrogen sulfide production

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456 Cow Manure Agar

Cow Manure Agar Compositionper liter:

Cow manure 50.0g

Agar 15.0g

Preparation of Medium: Add cow manure to tap water and bring

volume to 1.0L Gently heat and bring to boiling Boil for 1 hr Filter

through cheesecloth Filter through Whatman filter paper Bring volume to

1.0L with tap water Add agar Mix thoroughly Gently heat and bring to

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

pres-sure–121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation of Streptomyces species.

CP Medium Compositionper liter:

Peptone 2.5g

Starch 2.0g

NaNO3 0.38g

Tris(hydroxymethyl)aminomethane buffer 0.25g

K2HPO4 0.038g

MgSO4·7H2O 0.038g

CaCl2·2H2O 0.017g

NaCl 0.013g

TC vitamins minimal eagle, 100X 5.0mL

Solution 1 1.0mL

Solution 2 1.0mL

Solution 3 1.0mL

Solution 4 1.0mL

Vitamin B12 solution 0.2mL

pH 8.7 ± 0.2 at 25°C

TC Vitamins Minimal Eagle 100X:

Inositol 2.0mg

Choline chloride 1.0mg

Folic acid 1.0mg

Nicotinamide 1.0mg

Calcium pantothenate 1.0mg

Pyridoxal 1.0mg

Thiamine·HCl 1.0mg

Riboflavin 0.1mg

Preparation of TC Vitamins Minimal Eagle, 100X: Add

com-ponents to distilled/deionized water and bring volume to 1.0L Mix

thoroughly Filter sterilize

Solution 1:

EDTA 5.0g

KOH 3.1g

Preparation of Solution 1: Add components to distilled/deionized

water and bring volume to 100.0mL Mix thoroughly

Solution 2:

FeSO4·7H2O 4.98g

Preparation of Solution 2: Add FeSO4·7H2O to distilled/deionized

water acidified with 1.0mL of H2SO4 Bring volume to 1.0L Mix

thor-oughly

Solution 3:

H3BO3 1.14g

Preparation of Solution 3: Add H3BO3 to distilled/deionized water and bring volume to 100.0mL Mix thoroughly

Solution 4:

ZnSO4·7H2O 0.88g MnCl2·4H2O 0.144g MoO3 0.071g CoNO3·6H2O 0.049g CuSO4·5H2O 0.016g

Preparation of Solution 4: Add components to distilled/deionized water and bring volume to 100.0mL Mix thoroughly

Vitamin B 12 Solution Compositionper 10.0mL:

Vitamin B12 10.0mg

Preparation of Vitamin B 12 Solution: Add vitamin B12 to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except for vitamin so-lutions, to distilled/deionized water and bring volume to 995.0mL Mix thoroughly Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Aseptically add vitamin solutions Mix thorough-ly

Use: For the cultivation of Lysobacter species

CP Medium for Coprothermobacter proteolyticus

NaHCO3 8.4g Pancreatic digest of casein 2.0g Yeast extract 2.0g MgCl2·6H2O 1.0g

NH4Cl 1.0g CaCl2·2H2O 0.4g

K2HPO4·3H2O 0.4g Resazurin 0.5mg Gelatin solution 100.0mL

Na2S·9H2O solution 10.0mL Wolfe’s mineral solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Gelatin Solution:

Gelatin 3.0g

Preparation of Gelatin Solution: Add gelatin to distilled/deion-ized water and bring volume to 100.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Na 2 S·9H 2 O Solution:

Na2S·9H2O 0.5g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C Before use, neutralize to pH 7.0 with sterile HCl

Wolfe’s Mineral Solution:

MgSO4·7H2O 3.0g Nitrilotriacetic acid 1.5g NaCl 1.0g MnSO4·2H2O 0.5g

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CPC Agar Base with Cellobiose, Colistin, and Polymyxin B 457

CoCl2·6H2O 0.1g

ZnSO4·7H2O 0.1g

CaCl2·2H2O 0.1g

FeSO4·7H2O 0.1g

NiCl2·6H2O 0.025g

KAl(SO4)2·12H2O 0.02g

CuSO4·5H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Na2SeO3·5H2O 0.3mg

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic

acid to 500.0mL of distilled/deionized water Adjust pH to 6.5 with

KOH Add remaining components Add distilled/deionized water to

1.0L Adjust pH to 6.8

Preparation of Medium: Prepare medium anaerobically under

100% CO2 Add components, except gelatin solution, Na2S·9H2O

so-lution, and Wolfe’s mineral soso-lution, to distilled/deionized water and

bring volume to 880.0mL Mix thoroughly Gently heat and bring to

boiling Cool to room temperature while sparging with 100% CO2

Sparge with 100% CO2 for 20 min Adjust pH to 7.0 Autoclave for

15 min at 15 psi pressure–121°C Aseptically and anaerobically add

100.0mL of sterile gelatin solution, 10.0mL of sterile Na2S·9H2O

so-lution, and 10.0mL of sterile Wolfe’s mineral solution to each tube

Mix thoroughly

Use: For the cultivation of Coprothermobacter proteolyticus.

CP Medium for Thermobacteroides leptospartum

NaHCO3 8.4g

Pancreatic digest of casein 2.0g

Yeast extract 2.0g

MgCl2·6H2O 1.0g

NH4Cl 1.0g

CaCl2·2H2O 0.4g

K2HPO4·3H2O 0.4g

Resazurin 0.5mg

Glucose solution 100.0mL

Na2S·9H2O solution 10.0mL

Wolfe’s mineral solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Glucose Solution:

D-Glucose 5.0g

Preparation of Glucose Solution: Add glucose to

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

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

Na2S·9H2O 0.5g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to

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

Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Before use, neutralize to pH 7.0 with sterile HCl

Wolfe’s Mineral Solution:

MgSO4·7H2O 3.0g

Nitrilotriacetic acid 1.5g

NaCl 1.0g

MnSO4·2H2O 0.5g CoCl2·6H2O 0.1g ZnSO4·7H2O 0.1g CaCl2·2H2O 0.1g FeSO4·7H2O 0.1g NiCl2·6H2O 0.025g KAl(SO4)2·12H2O 0.02g CuSO4·5H2O 0.01g

H3BO3 0.01g

Na2MoO4·2H2O 0.01g

Na2SeO3·5H2O 0.3mg

Preparation of Wolfe’s Mineral Solution: Add nitrilotriacetic acid to 500.0mL of distilled/deionized water Adjust pH to 6.5 with KOH Add remaining components Add distilled/deionized water to 1.0L Adjust pH to 6.8

Preparation of Medium: Prepare medium anaerobically under 100%

CO2 Add components, except glucose solution, Na2S·9H2O solution, and Wolfe’s mineral solution, to distilled/deionized water and bring volume to 880.0mL Mix thoroughly Gently heat and bring to boiling Cool to room temperature while sparging with 100% CO2 Sparge with 100% CO2 for

20 min Adjust pH to 7.0 Autoclave for 15 min at 15 psi pressure–121°C Aseptically and anaerobically add 100.0mL of sterile glucose solution, 10.0mL of sterile Na2S·9H2O solution, and 10.0mL of sterile Wolfe’s min-eral solution to each tube Mix thoroughly

Use: For the cultivation of Thermobacteroides leptospartum.

CPC Agar

See: Cellobiose Polymyxin Colistin Agar

CPC Agar Base with Cellobiose, Colistin, and Polymyxin B Compositionper liter:

NaCl 20.0g Agar 15.0g Cellobiose 15.0g Peptic digest of animal tissue 10.0g Beef extract 5.0g Bromthymol Blue 0.04g Cresol Red 0.04g Cellobiose colistin polymyxin B solution 100.0mL

pH 7.6 ± 0.2 at 25°C

Source: This medium, without cellobiose colistin polymyxin B solu-tion, is available as a premixed powder from HiMedia

Cellobiose Colistin Polymyxin B Solution:

Cellobiose 15.0g Colistin 1,360,000U Polymyxin B 100,000U

Preparation of Cellobiose Colistin Polymyxin B Solution:

Add components to distilled/deionized water and bring volume to 100.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except cellobiose co-listin polymyxin B solution, to tap water and bring volume to 1.0L Mix thoroughly Distribute into tubes or flasks Autoclave for 15 min

at 15 psi pressure–121°C Aseptically add 100.0mL of sterile

cellobio-se colistin polymyxin B solution to 900.0 mL of the cooled agar bacellobio-se Mix thoroughly Pour into sterile Petri dishes Use within 7 days

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458 CPC HiVeg Agar Base with Cellobiose, Colistin, and Polymyxin B

Use: For the cultivation and identification of Vibrio species from

foods

CPC HiVeg Agar Base with Cellobiose, Colistin, and Polymyxin B

NaCl 20.0g

Agar 15.0g

Cellobiose 15.0g

Plant peptone 10.0g

Plant extract 5.0g

Bromthymol Blue 0.04g

Cresol Red 0.04g

Cellobiose colistin polymyxin B solution 100.0mL

pH 7.6 ± 0.2 at 25°C

Source: This medium, without cellobiose colistin polymyxin B

solu-tion, is available as a premixed powder from HiMedia

Cellobiose Colistin Polymyxin B Solution:

Cellobiose 15.0g

Colistin 1,360,000U

Polymyxin B 100,000U

Preparation of Cellobiose Colistin Polymyxin B Solution:

Add components to distilled/deionized water and bring volume to

100.0mL Mix thoroughly Filter sterilize

Preparation of Medium: Add components, except cellobiose

co-listin polymyxin B solution, to tap water and bring volume to 1.0L

Mix thoroughly Distribute into tubes or flasks Autoclave for 15 min

at 15 psi pressure–121°C Aseptically add 100.0mL of sterile

cellobio-se colistin polymyxin B solution to 900.0 mL of the cooled agar bacellobio-se

Mix thoroughly Pour into sterile Petri dishes Use within 7 days

Use: For the cultivation and identification of Vibrio species from

foods

CPC Medium Compositionper liter:

Sucrose 30.0g

Peptone 2.0g

Casein hydrolysate 1.0g

K2HPO4·3H2O 1.0g

KCl 0.5g

MgSO4·7H2O 0.5g

FeSO4·7H2O 0.1g

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to tap water and bring

volume to 1.0L Mix thoroughly Distribute into tubes or flasks

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

Use: For the cultivation of Actinoplanes species.

CR Agar

See: Congo Red Agar

Craig’s Medium Composition per liter:

Casein acid hydrolysate 30.0g

Yeast extract 4.0g

K2HPO4 0.5g Glucose solution 20.0mL

pH 7.2 ± 0.2 at 25°C

Source: This medium is available from HiMedia

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 980.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C Cool to 50°C Aseptically add glucose solution Mix thoroughly Aseptically distribute into sterile tubes

Use: For the enrichment and cultivation of Vibrio cholerae during

test-ing of enterotoxigenicity

CRAMP Agar

See: Congo Red Acid Morpholinepropanesulfonic Acid

Pigmentation Agar

CRAMP HiVeg Agar Base Compositionper liter:

Agarose 14.0g Morpholine propane sulfonic acid 8.4g Tricine 1.8g NaCl 2.9g Galactose 2.0g Plant acid hydrolysate 2.0g

Na2S2O3 0.6g

NH4Cl 0.5g

K2HPO4 0.24g MgSO4 0.0986g Congo Red 0.005g

pH 5.3 ± 0.2 at 25°C

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

to boiling Adjust pH to 5.3 Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C Pour into sterile Petri dishes or leave in tubes

Use: For the cultivation of Yersinia species with plasmids.

CREA (Creatine Agar) Compositionper liter:

Sucrose 30.0g Agar 15.0g Creatine·H2O 3.0g

K3P04·7H2O 1.6g Bromcresol Purple 50.0mg Minerals solution 10.0mL Trace minerals solution 1.0mL

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Creatinine/NMH Medium 459

Minerals Solution:

KCl 5.0g

MgSO4·7H2O 5.0g

FeSO4·7H2O 0.1g

Preparation of Minerals Solution: Add components to distilled/

deionized water and bring volume to 100.0mL Mix thoroughly

Trace Minerals Solution:

ZnS04·7H2O 1.0g

CuSO4·5H2O 0.5g

Preparation of Trace Minerals Solution: Add components to

distilled/deionized water and bring volume to 100.0mL Mix

thorough-ly

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 Penicillium species

Creatinine Agar Compositionper liter:

Agar 15.0g

Na2HPO4·12H2O 9.0g

NaCl 5.0g

KH2PO4 1.5g

Creatinine 1.0g

Meat extract 1.0g

Yeast extract 1.0g

MgSO4·7H2O 0.2g

MnCl2·4H2O 20.0mg

CaCl2 1.2mg

Glucose solution 100.0mL

Glucose 5.0g

Preparation of Glucose Solution: Add glucose to

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

ster-ilize Warm to 50°C

Preparation of Medium: Add components, except glucose

solu-tion, 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 50°–55°C Aseptically add 100.0mL of

sterile glucose solution Mix thoroughly Pour into sterile Petri dishes

or distribute into sterile tubes

Use: For the cultivation and maintenance of Pseudomonas species

and other bacteria that can utilize creatinine

Creatinine Medium Compositionper liter:

Creatinine 5.0g

Agar 2.0g

Fumaric acid 2.0g

K2HPO4 2.0g

Yeast extract 1.0g

Salt solution 10.0mL

pH 6.8 ± 0.2 at 25°C

Salt Solution:

MgSO4 12.2g FeSO4·7H2O 2.8g MnSO4·H2O 1.7g CaCl2·2H2O 0.76g NaCl 0.6g

Na2MoO4·2H2O 0.1g ZnSO4·7H2O 0.06g

HCl (0.1N solution) 1.0L

Preparation of Salt Solution: Dissolve salts in 1.0L of 0.1N HCl

solution Mix thoroughly

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L Mix thoroughly Adjust pH to 6.8 with NaOH or KOH Gently heat until boiling Distribute into tubes or flasks Autoclave for 15 min at 15 psi pressure–121°C

Use: For the cultivation and maintenance of Pseudomonas species

Creatinine Medium (LMG 107) Compositionper liter:

Creatinine 5.0g Fumaric acid 2.0g

K2HPO4 2.0g Yeast extract 1.0g Salt solution 10.0mL

pH 6.8 ± 0.2 at 25°C

Salt Solution:

MgSO4·7H2O 25.0g FeSO4·7H2O 2.8g MnSO4·H2O 1.7g CaCl2·2H2O 0.76g NaCl 0.6g

Na2MoO4·2H2O 0.1g ZnSO4·7H2O 60.0mg

HCl (0.1M solution) 1.0L

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

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

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

Use: For the cultivation and maintenance of Flavobacterium filamen-tosum

Creatinine/NMH Medium Compositionper 1100.0mL:

Yeast extract 5.0g NaCl 1.0g NaHCO3 1.0g MgSO4·7H2O 0.5g

Na2S·9H2O 0.5g MnCl2·4H2O 0.06g CaSO4·2H2O 0.05g FeSO4·7H2O 0.01g

Na2SeO3·5H2O 26.0μg Vitamin B12 20.0μg Resazurin 1.0mg

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460 CreDm1 Medium

Phosphate solution 100.0mL

Creatinine solution 100.0mL

Trace elements solution SL-4 10.0mL

Vitamin solution 10.0mL

L-Cysteine·HCl solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Phosphate Solution:

K2HPO4 5.33g

KH2PO4 2.64g

Preparation of Phosphate Solution: Add components to

Creatinine Solution:

Creatinine 5.5g

Preparation of Creatinine Solution: Add creatinine to distilled/

deionized water and bring volume to 100.0mL Mix thoroughly Sparge

with 100% N2 Filter sterilize

Trace Elements Solution SL-4:

EDTA 0.5g

FeSO4·7H2O 0.2g

Trace elements solution SL-6 100.0mL

MnCl2·4H2O 0.5g

H3BO3 0.3g

CoCl2·6H2O 0.2g

ZnSO4·7H2O 0.1g

Na2MoO4·2H2O 0.03g

NiCl2·6H2O 0.02g

CuCl2·2H2O 0.01g

Preparation of Trace Elements Solution SL-6: Add components

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

Preparation of Trace Elements Solution SL-4: Add components

Vitamin Solution:

Pyridoxine·HCl 10.0mg

Calcium DL-pantothenate 5.0mg

Lipoic acid 5.0mg

Nicotinic acid 5.0mg

p-Aminobenzoic acid 5.0mg

Riboflavin 5.0mg

Thiamine·HCl 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

Preparation of Vitamin Solution : Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly

L -Cysteine·HCl Solution:

L-Cysteine·HCl 0.5g

Preparation of L -Cysteine·HCl Solution: Add L-cysteine·HCl to

thorough-ly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure– 121°C

Na2S·9H2O 0.5g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

Preparation of Medium: Add components, except creatinine solu-tion, phosphate solusolu-tion, L-cysteine·HCl solution, and Na2S·9H2O so-lution, to distilled/deionized water and bring volume to 880.0mL Mix thoroughly Adjust pH to 7.0 Autoclave for 15 min at 15 psi pressure– 121°C Aseptically and anaerobically add 100.0mL of sterile phos-phate solution, 10.0mL of sterile L-cysteine·HCl solution, and 10.0mL

of sterile Na2S·9H2O solution Immediately prior to use, aseptically and anaerobically add 100.0mL of sterile creatinine solution Mix thor-oughly Aseptically and anaerobically distribute into tubes or bottles

Use: For the cultivation and maintenance of Clostridium species

CreDm1 Medium Compositionper 1002.0mL:

Solution A 980.0mL Solution D (Vitamin solution) 10.0mL Solution E 10.0mL Solution B (Trace elements solution SL-10) 1.0mL Solution C (Selentite-tungstate solution) 1.0mL

pH 6.7–6.9 at 25°C

Solution A:

KH2PO4 1.4g

NH4Cl 0.5g MgCl2·6H2O 0.2g CaCl2·2H2O 0.15g Yeast extract 50.0mg

Preparation of Solution A : Add components to distilled/deionized

water and bring volume to 980.0mL Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Solution B (Trace Elements Solution SL-10):

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

Na2MoO4·2H2O 36.0mg NiCl2·6H2O 24.0mg

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

Preparation of Solution B (Trace Elements Solution SL-10):

Add FeCl2·4H2O to 10.0mL of HCl solution Mix thoroughly Add dis-tilled/deionized water and bring volume to 1.0L Add remaining com-ponents Mix thoroughly Autoclave for 15 min at 15 psi pressure– 121°C

Solution C (Selenite-Tungstate Solution):

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

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Cryptoanaerobacter Medium 461

Preparation of Solution C (Selenite-Tungstate Solution):

Add components to distilled/deionized water and bring volume to

1.0L Mix thoroughly Autoclave for 15 min at 15 psi pressure–121°C

Solution D (Vitamin Solution):

Pyridoxine·HCl 10.0mg

Calcium DL-pantothenate 5.0mg

Lipoic acid 5.0mg

Riboflavin 5.0mg

Thiamine·HCl 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

Preparation of Solution D (Vitamin Solution): Add components

Filter sterilize

Solution E:

Disodium-DL-malate 1.6g

Preparation of Solution E: Add disodium-DL-malate to distilled/

deionized water and bring volume to 10.0mL Mix thoroughly

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

Preparation of Medium: Aseptically combine 980.0mL of sterile

solution A with 1.0mL of sterile solution B, 1.0mL of sterile solution

C, 10.0mL of sterile solution D, and 10.0mL of sterile solution E, in

that order Mix thoroughly Adjust pH to 6.7–6.9 Aseptically distribute

into sterile tubes or flasks

Use: For the cultivation of Campylobacter species.

Crithidia Medium

Sucrose 15.0g

Pancreatic digest of casein 6.0g

Yeast extract 1.0g

Liver concentrate 0.1g

Hemin solution 5.0mL

pH 7.8 ± 0.2 at 25°C

Hemin Solution:

Hemin 25.0mg

Triethanolamine (TEA) 2.5mL

Preparation of Hemin Solution: Add hemin to 2.5mL

trietha-nolamine Mix thoroughly

water and bring volume to 1.0L Mix thoroughly Adjust pH to 7.8

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

121°C Slight hemin precipitate may occur

Use: For the cultivation of Crithidia acanthocephali, Crithidia deanei,

Crithidia fasciculata, Crithidia harmosa, Crithidia hutneri, Crithidia

luciliae, Crithidia mellificae, Crithidia oncopelti, Crithidia species,

Herpetomonas samuelpessoai, Leptomonas pyrrhocoris, and

Phyto-monas davidi.

CRMOX Agar

Crossley Milk Medium Composition per liter:

Skim milk powder 100.0g Peptone 10.0g Bromcresol Purple 0.1g

pH 5.8 ± 0.2 at 25°C

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

Preparation of Medium: Add components to a very small volume

of distilled/deionized water and mix to a paste Gradually add more dis-tilled/deionized water and bring volume to 1.0L Distribute in 10.0mL volumes into tubes Autoclave for 5 min at 15 psi pressure–121°C

Use: For the routine examination of canned food samples for anaero-bic bacteria

Cryptoanaerobacter Medium

(DSMZ Medium 1022) Composition per liter:

Solution A 650.0mL

Clostridium sporogenes supernatant 350.0mL

pH 7.5–8.0 at 25°C

Solution A:

Yeast extract 5.0g NaHCO3 4.0g Casamino acids 1.0g 4-Hydroxybenzoic acid 0.45g

KH2PO4 0.4g

NH4Cl 0.4g Resazurin 0.5mg Vitamin solution 10.0mL Magnesium chloride solution 10.0mL Calcium chloride solution 10.0mL Trace element solution SL-10 1.0mL Selenite/tungstate solution .1.0mL

Selenite/Tungstate Solution:

NaOH 0.5g

Na2WO4·2H2O 4.0mg

Na2SeO3·5H2O 3.0mg

Preparation of Selenite/Tungstate Solution: Add components

to distilled/deionized water and bring volume to 1.0L Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–121°C

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

Na2MoO4·2H2O 36.0mg NiCl2·6H2O 24.0mg

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

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

to 10.0mL of HCl solution Mix thoroughly Add distilled/deionized water and bring volume to 1.0L Add remaining components Mix

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thor-462 CRYS Medium

oughly Sparge with 100% N2 Autoclave for 15 min at 15 psi pressure–

121°C

Pyridoxine-HCl 10.0mg

Thiamine-HCl·2H2O 5.0mg

Riboflavin 5.0mg

D-Ca-pantothenate 5.0mg

Lipoic acid 5.0mg

Biotin 2.0mg

Folic acid 2.0mg

Vitamin B12 0.1mg

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 1.0L Mix thoroughly Sparge

with 80% H2 + 20% CO2 Filter sterilize

Magnesium Chloride Solution:

MgCl2·6H2O 0.08g

Preparation of Magnesium Chloride Solution: Add

MgCl2·6H2O to distilled/deionized water and bring volume to 1.0L

Mix thoroughly Sparge with 100% N2 Autoclave for 15 min at 15 psi

pressure–121°C

Calcium Chloride Solution:

CaCl2·2H2O 0.06g

Preparation of Calcium Chloride Solution: Add CaCl2·2H2O

Preparation of Solution A: Add components, except bicarbonate,

magnesium chloride and calcium chloride solution, to

distilled/deion-ized water and bring volume to 630.0mL Mix thoroughly Adjust pH

to 7.0–7.5 Gently heat and bring to boiling Boil for 3 min Cool while

sparging with 80% N2 + 20% CO2 Add the solid bicarbonate Adjust

the pH to 7.8 Dispense under 80% N2 + 20% CO2 gas atmosphere into

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

Asep-tically and anoxically add magnesium chloride and calcium chloride

solutions Adjust final pH of the medium topH 7.7 Note: It may be

necessary to add 10–20 mg sodium dithionite per liter (e.g., from 5%

(w/v) solution, freshly prepared under N2 and filter sterilized), if the

so-lution is not completely reduced after inoculation

Clostridium sporogenes Supernatant:

Yeast extract 5.0g

NaHCO3 4.0g

Casamino acids 1.0g

KH2PO4 0.4g

NH4Cl 0.4g

Resazurin 0.5mg

Vitamin solution 10.0mL

Magnesium chloride solution 10.0mL

Calcium chloride solution 10.0mL

Trace element solution SL-10 1.0mL

Selenite-tungstate solution 1.0mL

Clostridium sporogenes Variable

Na 2 S·9H 2 O Solution :

Na2S·9H2O 0.3g

Preparation of Na 2 S·9H 2 O Solution: Add Na2S·9H2O to dis-tilled/deionized water and bring volume to 10.0mL Mix thoroughly Autoclave under 100% N2 for 15 min at 15 psi pressure–121°C Cool

to room temperature

Preparation of Clostridium sporogenes Supernatant: Add components, except bicarbonate, Na2S·9H2O solution, magnesium chloride and calcium chloride solution, to distilled/deionized water and bring volume to 980.0mL Mix thoroughly Adjust pH to 7.0–7.5 Gen-tly heat and bring to boiling Boil for 3 min Cool while sparging with 80% N2 + 20% CO2 Add the solid bicarbonate Adjust the pH to 7.8 Dispense under 80% N2 + 20% CO2 gas atmosphereinto anaerobic bottles Autoclave for 15 min at 15 psi pressure–121°C Aseptically and anoxically add Na2S·9H2O solution, magnesium chloride and cal-cium chloride solutions Adjust final pH to pH 7.0 Inoculate with

Clostridium sp DSM 754 Incubate for 5 to 8 days at 37°C Disrupt

cells of the grown culture by autoclaving for 20 min at 15 psi pressure– 121°C Centrifuge autoclaved culture at 18,000g for 20 min Discard cell pellet Store the supernatant in screw-capped bottles at −20°C Be-fore use sterilize the supernatant by autoclaving under 100% N2 for 15 min at 15 psi pressure–121°C Cool to room temperature under 100%

N2

Preparation of Medium: Aseptically and anoxically combine

650.0mL of solution A with 350.0mL of Clostridum sporogenes

super-natant

Use: For the cultivation of Cryptoanaerobacter spp.

Cryptococcus neoformans Screen Medium

CRYS Medium Compositionper liter:

Stock extract 500.0mL 2× PP medium 500.0mL

Stock Extract:

Cerophyll 5.0g Brown rice 5.0g Yeast extract 5.0g Dried seaweed 5.0g

Preparation of Stock Extract: Add components to distilled/deion-ized water and bring volume to 500.0mL Mix thoroughly Gently heat and bring to boiling Continue boiling for 5 min Filter three times through Whatman #1 filter paper while still hot Cool to room temper-ature Adjust pH to 7.2 Bring volume to 500.0mL with distilled/deion-ized water Autoclave for 15 min at 15 psi pressure–121°C

2 PP Medium:

Proteose peptone 10.0g Pancreatic digest of peptone 10.0g

Ribonucleic acid from Torula yeast 1.0g

Asolectin 0.2g Artificial seawater 167.0mL Vitamin solution 2.0mL

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Crystal Violet Lactose Agar 463

Artificial Seawater:

Aqua-Marin sea salts 6.95g

Source: Aqua-Marin sea salts are available from Aquatrol, Inc.,

Ana-heim, CA

Preparation of Artificial Seawater: Add Aqua-Marin sea salts to

thorough-ly Filter sterilize

Thiamine·HCl 150.0mg

Calcium D-(+)-pantothenate 100.0mg

Folic acid 50.0mg

Nicotinamide 50.0mg

Pyridoxal·HCl 50.0mg

Riboflavin 50.0mg

DL-6-Thioctic acid 1.0mg

Biotin solution 10.0mL

Biotin Solution:

Biotin 0.01mg

Preparation of Biotin Solution: Add biotin to 10.0mL of absolute

ethanol Mix thoroughly

Preparation of Vitamin Solution: Add components to distilled/

deionized water and bring volume to 100.0mL Mix thoroughly Filter

sterilize For long-term storage, preserve under nitrogen at −20°C

Preparation of 2× PP Medium: Add asolectin to 200.0mL of

dis-tilled/deionized water Gently heat to 80°C Mix thoroughly Add other

components, except artificial seawater and vitamin solution, to

Adjust pH to 7.2 Autoclave for 15 min at 15 psi pressure–121°C

Aseptically add 167.0mL of sterile artificial seawater and 2.0mL of

sterile vitamin solution Mix thoroughly

Preparation of Medium: Aseptically combine 500.0mL of sterile

stock extract with 500.0mL of sterile 2× PP medium Mix thoroughly

Aseptically distribute into sterile tubes or flasks

Use: For the cultivation of Pseudocohnilembus marinus.

Crystal Violet Agar Compositionper liter:

Agar 15.0g

Lactose 10.0g

Proteose peptone 5.0g

Beef extract 3.0g

Crystal Violet 3.3mg

pH 6.8 ± 0.1 at 25°C

wa-ter and bring volume to 1.0L Mix thoroughly Gently heat until 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 differentiation of pathogenic staphylococci from

non-pathogenic staphylococci Hemolytic and coagulating strains of

Staph-ylococcus aureus appear as purple or yellow colonies Nonhemolytic

and noncoagulating strains of Staphylococcus species appear as white

colonies

Crystal Violet Azide Esculin Agar Compositionper liter:

Agar 15.0g Glucose 5.0g NaCl 5.0g Proteose peptone 5.0g Pancreatic digest of casein 5.0g Meat extract 3.0g Esculin 1.0g NaN3 1.0g Crystal Violet 0.1g Bovine blood, citrated 100.0mL

pH 7.5 ± 0.2 at 25°C

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

Preparation of Medium: Add components, except citrated bovine blood, 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, cit-rated bovine blood Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the cultivation of Erysipelothrix rhusiopathiae.

Crystal Violet Esculin Agar Compositionper liter:

Agar 15.0g Glucose 5.0g NaCl 5.0g Proteose peptone 5.0g Pancreatic digest of casein 5.0g Meat extract 3.0g Esculin 1.0g Crystal Violet 2.0mg Blood, citrated 100.0mL

pH 7.5 ± 0.2 at 25°C

Preparation of Medium: Add components, except citrated blood,

to distilled/deionized water and bring volume to 900.0mL Mix thor-oughly Gently heat and bring to boiling Autoclave for 15 min at 15 psi pressure–121°C Cool to 45°–50°C Aseptically add citrated blood Mix thoroughly Pour into sterile Petri dishes or distribute into sterile tubes

Use: For the cultivation of Erysipelothrix rhusiopathiae.

Crystal Violet Lactose Agar Compositionper liter:

Agar 15.0g Lactose 10.0g Proteose peptone 5.0g Beef extract 3.0g Crystal Violet 3.3mg

pH 6.8 ± 0.2 at 25°C

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464 Crystal Violet Lactose Broth

Use: For the differentiation of pure cultures of pathogenic and

non-pathogenic staphylococci

Crystal Violet Lactose Broth

Lactose 5.0g

Peptic digest of animal tissue 5.0g

K2HPO4 5.0g

KH2PO4 1.0g

pH 7.4 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from

Hi-Media

Crystal Violet Lactose HiVeg Agar

Agar 15.0g

Lactose 10.0g

Plant peptone No 3 5.0g

Plant extract 3.0g

pH 6.8 ± 0.2 at 25°C

Source: This medium is available as a premixed powder from

Hi-Media

Crystal Violet Pectate Medium

(CVP Medium) Compositionper liter:

Sodium polypectate 9.0g

Agar 2.0g

NaNO3 1.0g

NaOH (1N solution) 4.5mL

CaCl2·H2O (10% solution) 3.0mL

Crystal Violet (0.075% solution) 1.0mL

Sodium lauryl sulfate (10% solution) 0.5mL

pH 7.2 ± 0.2 at 25°C

Preparation of Medium: In a preheated blender, add 500.0mL of

boiling distilled/deionized water and the components, except sodium

polypectate and sodium lauryl sulfate solution Blend at high speed for

15 sec Continue blending at low speed and slowly add 9.0g of sodium

polypectate Pour the incomplete medium into a 2L flask and add

0.5mL of sodium lauryl sulfate solution Mix thoroughly Cap flask

with an aluminum foil seal rather than cotton Autoclave for 25 min at

15 psi pressure–121°C Pour medium quickly into sterile Petri dishes

Allow plates to dry at 25°C for 48 hr before use

Use: For the cultivation of pectinolytic microorganisms, such as

Erwinia species, from foods.

Crystal Violet Pectate Medium

(CVP Medium) Compositionper liter:

Sodium polypectate 18.0g Agar 4.0g NaNO3 2.0g CaCl2·2H2O 0.6g NaOH 0.36g Sodium lauryl sulfate 0.1g Crystal Violet 1.5mg

pH 7.2 ± 0.2 at 25°C

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

Use: For the cultivation of pectinolytic microorganisms, such as

Erwinia species, from foods.

Crystal Violet Tetrazolium Agar Base Compositionper liter:

Agar 15.0g Casein enzymatic hydrolysate 5.0g Yeast extract 2.5g Glucose 1.0g Crystal Violet 1.0mg 2,3,5-Triphenyltetrazolium chloride solution 10.0mL

pH 7.0 ± 0.2 at 25°C

Source: This medium, without triphenyltetrazolium chloride solution,

is available as a premixed powder from HiMedia

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

2,3,5-Triphenyltetrazolium Chloride Solution:

2,3,5-Triphenyltetrazolium chloride 0.1g

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

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

Use: For the detection of Gram-negative psychrotrophic bacteria caus-ing food spoilage

Crystal Violet Tetrazolium HiVeg Agar Base Compositionper liter:

Agar 15.0g Plant hydrolysate 5.0g Yeast extract 2.5g Glucose 1.0g

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