Handbook of Microbiological Media, Fourth Edition part 30 pdf

Burk’s Medium 285
Burke’s Modified Nitrogen-Free Medium with Benzoate
Composition per liter:
Sodium benzoate 0.72g
MgSO
4
·7H
2
O 0.2g
Na
2
HPO
4
0.189g
NaHCO
3
0.05g
CaSO
4
·2H
2
O 0.02g
KH
2
PO
4
0.011g
SrCl
2
·6H
2

O 0.01g
NaCl 0.01g
Adenine 0.01g
FeSO
4·
7H
2
O 6.0mg
Na
2
MoO
3
0.5mg
pH 7.8 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes
or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of Pseudomonas species and other microor-
ganisms which can utilize benzoate as sole carbon source.
Burkholderia cepacia Agar
Composition per liter:
Agar 12.0g
Sodium pyruvate 7.0g
Peptone 5.0g
KH
2
PO
4
4.4g
Yeast extract 4.0g

Bile salts 1.5g
Na
2
HPO
4
1.4g
(NH
4
)
2
SO
4
1.0g
MgSO
4
0.2
Phenol Red 0.02g
Fe(NH
4
)
2
(SO
4
)
2
·6H
2
O 0.01g
Crystal Violet 0.001g
Selective supplement solution 10.0mL

pH 6.2 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from Oxoid
Unipath.
Selective Supplement Solution:
Composition
per 10.0mL:
Polymyxin B 150,000IU
Ticarcillin 100.0mg
Gentamicin 5.0mg
Preparation of Selective Supplement Solution: Add components
to distilled/deionized water and bring volume to 10.0mL. Mix thor-
oughly. Filter sterilize.
Preparation of Medium: Add components, except selective sup-
plement solution, to distilled/deionized water and bring volume to
990.0mL. Mix thoroughly. Gently heat while stirring and bring to boil-
ing. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°C.
Aseptially add 10.0mL selective supplement solution. Mix thoroughly.
Pour into sterile Petri dishes.
Use: For the selective isolation of Burkholderia cepacia from the
respiratory secretions of patients with cystic fibrosis and for routine
testing of non-sterile inorganic salt solutions containing preservative.
Slow growing B. cepacia can be missed on conventional media such as
blood or MacConkey agar due to overgrowth caused by other faster
growing organisms found in the respiratory tract of CF patients such as
mucoid Klebsiella species, Pseudomonas aeruginosa, and Staphylo-
coccus species. This may lead to the infection being missed or wrongly
diagnosed.
Burkholderia pseudomallei Selective Agar
(BPSA)
Composition per liter:

Agar 15.0g
Pancreatic Digest of Casein 5.0g
Maltose 4.0g
Yeast Extract 2.5g
Glucose 1.0g
Neutral Red 0.1g
Gentamicin solution 10.0mL
Glycerol 10.0mL
Nile Blue solution 1.0mL
pH 7.0 ± 0.2 at 25°C
Gentamicin Solution:
Composition
per 10.0mL:
Gentamicin 20.0mg
Preparation of Gentamicin Solution: Add gentamicin to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Filter
sterilize.
Nile Blue Solution:
Composition
per 10.0mL:
Nile Blue 0.2g
Preparation of Nile Blue Solution: Add Nile blue to 10.0mL of a
1% solution of dimethyl sulfoxide. Mix thoroughly. Filter sterilize.
Preparation of Medium: Add components, except gentamcin solu-
tion, Nile Blue solution, and glycerol, to distilled/deionized water and
bring volume to 979.0mL. Mix thoroughly. Gently heat while stirring
and bring to boiling. Autoclave for 15 min at 15 psi pressure–121°C.
Cool to 45°C. Aseptially add 10.0mL sterile gentamicin solution,
1.0mL sterile Nile blue solution, and 10.0mL filter-sterilized glycerol.
Mix thoroughly for 5 min on a heated magnetic stirrer at 40°C. Pour

into sterile Petri dishes.
Use: For the cultivation of Burkholderia pseudomallei from clinical
specimens collected from non-sterile sites with improved recovery of
the more easily inhibited strains of B. pseudomallei.
Burk’s Medium
Composition per liter:
Sucrose 20.0g
MgSO
4
·7H
2
O 0.2g
K
2
HPO
4
0.8g
KH
2
PO
4
0.25g
CaSO
4
0.13g
FeCl
3
1.45mg
Na
2

MoO
3
0.253mg
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
with inverted Durham tubes. Autoclave for 15 min at 15 psi pressure–
121°C.
Use: For thecultivation of nitrogen fixing bacteria, such as Azotrobacter
spp., from soil.
© 2010 by Taylor and Francis Group, LLC
286 Bushnell-Haas Agar
Bushnell-Haas Agar
Composition per liter:
Agar 15.0g
KH
2
PO
4
1.0g
K
2
HPO
4
1.0g
NH
4
NO
3
1.0g

MgSO
4
·7H
2
O 0.2g
FeCl
3
0.05g
CaCl
2
·2H
2
O 0.02g
pH 7.0 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15
psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes. For
use in cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0% hy-
drocarbon on agar surface or aseptically add sterile hydrocarbon to
cooled agar prior to pouring plates.
Use: For examining fuels for microbial contamination and for study-
ing hydrocarbon utilization by microorganisms. Also for the cultiva-
tion of Nocardia species.
Bushnell-Haas Broth
Composition per liter:
KH
2
PO
4

1.0g
K
2
HPO
4
1.0g
NH
4
NO
3
1.0g
MgSO
4
·7H
2
O 0.2g
FeCl
3
0.05g
CaCl
2
·2H
2
O 0.02g
pH 7.0 ± 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. Distribute into tubes
or flasks. Autoclave for 15 min at 15 psi pressure–121°C. For use in

cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0% hydrocar-
bon on broth surface or add directly to broth.
Use: For examining fuels for microbial contamination and for studying
the hydrocarbon utilization by microorganisms. Also for the cultivation of
Nocardia species.
Bushnell-Haas Medium
Composition per liter:
KH
2
PO
4
1.0g
K
2
HPO
4
1.0g
NH
4
NO
3
1.0g
Cholesterol 0.3g
MgSO
4
·7H
2
O 0.2g
FeCl
3

0.05g
CaCl
2
·2H
2
O 0.02g
pH 7.0 ± 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. Distribute into tubes
or flasks. Autoclave for 15 min at 15 psi pressure–121°C. For use in
cultivating hydrocarbon-utilizing bacteria, layer 0.1–1.0% hydrocar-
bon on broth surface or add directly to broth.
Use: For the cultivation of Nocardia species.
Butanediol Medium
Composition per liter:
NaH
2
PO
4
·H
2
O 2.1g
1,4-Butanediol 1.0g
NaCl 1.0g
NH
4
Cl 1.0g
CaCl

2
·2H
2
O 0.5g
MgSO
4
·7H
2
O 0.5g
K
2
HPO
4
0.3g
Yeast extract 0.2g
Modified Wolfe’s mineral solution 10.0mL
pH 7.0 ± 0.2 at 25°C
Modified Wolfe’s Mineral Solution:
Composition
per liter:
MgSO
4
·7H
2
O 3.0g
Nitrilotriacetic acid 1.5g
NaCl 1.0g
MnSO
4
·H

2
O 0.5g
CaCl
2
0.1g
CoCl
2
·6H
2
O 0.1g
FeSO
4
·7H
2
O 0.1g
ZnSO
4
·7H
2
O 0.1g
AlK(SO
4
)
2
·12H
2
O 0.01g
CuSO
4
·5H

2
O 0.01g
H
3
BO
3
0.01g
Na
2
MoO
4
·2H
2
O 0.01g
Na
2
SeO
3
0.01g
NaWO
4
·2H
2
O 0.01g
NiC1
2
·6H
2
O 0.01g
Preparation of Modified Wolfe’s Mineral Solution: Add

nitrilotriacetic acid to 500.0mL of distilled/deionized water. Adjust pH
to 6.5 with KOH. Add remaining components one at a time. Add dis-
tilled/deionized water to 1.0L. Adjust pH to 6.8.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.0.
Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–
121°C.
Use: For the cultivation of Pseudomonas putida.
Butyrivibrio Species Medium
Composition per 1001.0mL:
Na
2
CO
3
4.0g
Pancreatic digest of casein 2.0g
Yeast extract 2.0g
K
2
HPO
4
.0.3g
Hemin 1.0mg
Resazurin 1.0mg
Rumen fluid, clarified 150.0mL
Minerals solution 75.0mL
Carbohydrate solution 20.0mL
L-Cysteine·HCl·H
2
O solution 10.0mL

Na
2
S·9H
2
O solution 10.0mL
Volatile fatty acid mixture 3.1mL
pH 6.7 ± 0.2 at 25°C
Minerals Solution:
Composition
per liter:
NaCl 12.0g
KH
2
PO
4
6.0g
(NH
4
)
2
SO
4
6.0g
© 2010 by Taylor and Francis Group, LLC
BYE Agar 287
MgSO
4
·7H
2
O 2.5g

CaCl
2
·2H
2
O 1.6g
Preparation of Minerals Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly.
L-Cysteine·HCl·H
2
O Solution:
Composition
per 10.0mL:
L-Cysteine·HCl·H
2
O 0.25g
Preparation of L-Cysteine·HCl·H
2
O Solution: Add L-cys-
teine·HCl·H
2
O to distilled/deionized water and bring volume to 10.0mL.
Mix thoroughly. Sparge with 100% CO
2
. Autoclave for 15 min at 15 psi
pressure–121°C.
Na
2
S·9H
2
O Solution:

Composition
per 10.0mL:
Na
2
S·9H
2
O 0.25g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% CO
2
. Autoclave for 15 min at 15 psi pressure–
121°C.
Carbohydrate Solution:
Composition
per 20.0mL:
Glucose 1.0g
Cellobiose 1.0g
Glycerol 1.0g
Maltose 1.0g
Starch, soluble 1.0g
Preparation of Carbohydrate Solution: Add components to dis-

tilled/deionized water and bring volume to 20.0mL. Mix thoroughly.
Sparge under 100% CO
2
. Autoclave for 15 min at 15 psi pressure–
121°C.
Volatile Fatty Acid Mixture:
Composition
per 7.75mL:
Acetic acid 4.25mL
Propionic acid 1.50mL
Butyric acid 1.0mL
DL-2-Methyl butyric acid 0.25mL
iso-Butyric acid 0.25mL
iso-Valeric acid 0.25mL
n-Valeric acid 0.25mL
Preparation of Volatile Fatty Acid Mixture: Combine compo-
nents. Mix thoroughly.
Preparation of Medium: Prepare and dispense medium under
100% CO
2
. Add components, except carbohydrate solution, Na
2
CO
3
,
L-cysteine·HCl·H
2
O solution, and Na
2
S·9H

2
O solution, to distilled/de-
ionized water and bring volume to 960.0mL Mix thoroughly. Gently
heat and bring to boiling. Continue boiling for 5 min. Cool to room
temperature while sparging with 100% CO
2
. Add Na
2
CO
3
. Continue
sparging with 100% CO
2
until pH reaches 6.8. Distribute into rubber-
stoppered tubes under 100% CO
2
. Autoclave for 15 min at 15 psi pres-
sure–121°C. Aseptically and anaerobically add 20.0mL of sterile car-
bohydrate solution, 10.0mL of sterile
L-cysteine·HCl·H
2
O solution,
and 10.0mL of sterile Na
2
S·9H
2
O solution or, using a syringe, inject
the appropriate amount of sterile carbohydrate solution, sterile
Na
2

S·9H
2
O solution, and sterile L-cysteine·HCl·H
2
O solution into in-
dividual tubes containing medium.
Use: For the cultivation of Butyrivibrio species.
Butzler Medium
See: Campylobacter Selective Medium, Butzler’s
Butzler’s Campylobacter Medium
See: Campylobacter Selective Medium, Butzler’s
BY Agar Medium
(ATCC Medium 2038)
Composition per liter:
Agar 15.0g
Yeast extract 5.0g
Pancreatic digest of casein 5.0g
Beef extract 5.0g
NaCl 2.5g
K
2
HPO
4
0.1g
MgSO
4
·7H
2
O 0.05g
pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15
psi pressure–121°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation and maintenance of Paracoccus thiocyanatus.
BY+ Medium
Composition per liter:
Glucose 5.0g
Peptone 1.0g
Yeast extract 1.0g
Seawater 1.0L
Preparation of Medium: Combine components. Mix thoroughly.
Gently heat and bring to boiling. Distribute into flasks or tubes. Auto-
clave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of Arenariomyces triseptatus, Haliphthoros
milfordensis, Haliphthoros philippinensis, Halosphaeria salina,
Japonochytrium species, Lignincola laevis, Lindra thalassiae,
Schizochytrium aggregatum, Thraustochytrium species, and Torpe-
dospora radiata.
BYE Agar
Composition per liter:
Pancreatic digest of casein 16.0g
Agar 13.5g
Brain heart, solids from infusion 8.0g
Peptic digest of animal tissue 5.0g
NaCl 5.0g
Glucose 2.0g
Na
2
HPO

4
2.5g
Yeast extract 2.0g
Blood, human or animal, sterile 150.0mL
pH 7.8–8.0 ± 0.2 at 25°C
Preparation of Medium: Add components, except blood, to dis-
tilled/deionized water and bring volume to 850.0mL. Mix thoroughly.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Aseptically add 150.0mL of sterile blood. Outdated, citrated, or hepa-
rinized blood (blood from a blood bank is acceptable). Pour into sterile
Petri dishes.
© 2010 by Taylor and Francis Group, LLC
288 BYE HiVeg Agar with Blood
Use: For the isolation and cultivation of Mycoplasma species and L-
forms of bacteria. For the detection of Mycoplasma species in tissue cul-
ture and cell lines.
BYE HiVeg Agar with Blood
Composition per liter:
Agar 13.0g
Plant infusion 10.0g
Plant peptone No. 3 10.0g
Plant special infusion 7.5g
NaCl 5.0g
Na
2
HPO
4
2.5g
Glucose 2.0g
Yeast extract 2.0g

Horse or human blood, sterile 150.0mL
pH 7.9 ± 0.2 at 25°C
Source: This medium, without blood, is available as a premixed pow-
der from HiMedia.
Preparation of Medium: Add components, except blood, to dis-
tilled/deionized water and bring volume to 850.0mL. Mix thoroughly.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Aseptically add 150.0mL of sterile blood. Outdated, citrated, or hepa-
rinized blood (blood from a blood bank is acceptable). Pour into sterile
Petri dishes.
Use: For the isolation and cultivation of Mycoplasma species and L-
forms of bacteria. For the detection of Mycoplasma species in tissue cul-
ture and cell lines.
BYE HiVeg Broth with Blood
Composition per liter:
Plant infusion 10.0g
Plant peptone No. 3 10.0g
Plant special infusion 7.5g
NaCl 5.0g
Na
2
HPO
4
2.5g
Glucose 2.0g
Yeast extract 2.0g
Horse or human blood, sterile 150.0mL
pH 7.9 ± 0.2 at 25°C
Source: This medium, without blood, is available as a premixed pow-
der from HiMedia.

Preparation of Medium: Add components, except blood, to dis-
tilled/deionized water and bring volume to 850.0mL. Mix thoroughly.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Aseptically add 150.0mL of sterile blood. Outdated, citrated, or hepa-
rinized blood (blood from a blood bank is acceptable).
Use: For the cultivation of Mycoplasma species and L-forms of bacteria.
BYEB
(Buffered Yeast Extract Broth)
Composition per liter:
ACES buffer (2-[(2-amino-2-oxoethyl)-
amino]-ethane sulfonic acid) 10.0g
Yeast extract 10.0g
α-Ketoglutarate 1.0g
L-Cysteine·HCl·H
2
O 0.4g
Fe
4
(P
2
O
7
)
3
·9H
2
O 0.25g
pH 6.9 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.9. Fil-

ter sterilize. Aseptically distribute into sterile tubes or flasks.
Use: For the cultivation of Legionella pneumophila.
C/10 Agar
Composition per liter:
Agar 15.0g
Pancreatic digest of casein 3.0g
CaCl
2
·2H
2
O 1.36g
pH 7.2 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Adjust pH t o 7.2. 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 Cytophaga flevensis, Flexibacter filiformis,
Myxococcus
C/10 Medium Reichenbach
Composition per liter:
Agar 15.0g
Pancreatic digest of casein 3.0g
CaCl
2
1.0g
pH 7.2 ± 0.2 at 25°C
Preparation of Medium: Add components, except agar, to distilled/
deionized water and bring volume to 1.0L. Adjust pH to 7.2. Add agar.
Mix thoroughly. Gently heat 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 Flexibacter filiformis.
fulvus, and Myxococcus xanthus.
C 3G Spiroplasma Medium
Composition per liter:
Sucrose 100.0g
Phenol Red 10.0mg
PPLO broth without Crystal Violet 500.0mL
Horse serum 150.0mL
Fresh yeast extract solution 50.0mL
CMRL-1066 medium 5.0mL
pH 7.5 ± 0.2 at 25°C
Source: PPLO broth without Crystal Violet is available as a premixed
powder from BD Diagnostic Systems.
PPLO Broth without Crystal Violet:
Composition
per 500.0mL:
Beef heart, infusion from 11.52g
Peptone 2.32g
NaCl 1.15g
Preparation of PPLO Broth without Crystal Violet: Add
components to distilled/deionized water and bring volume to 500.0mL.
Mix thoroughly.
Fresh Yeast Extract Solution:
Composition
per 100.0mL:
Baker’s yeast, live, pressed, starch-free 25.0g
© 2010 by Taylor and Francis Group, LLC
C 3N Spiroplasma Medium 289

Preparation of Fresh Yeast Extract Solution: Add the live Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8. Filter sterilize.
CMRL-1066 Medium:
Composition
per liter:
NaCl 6.8g
NaHCO
3
2.2g
D-Glucose 1.0g
KCl 0.4g
L-Cysteine·HCl·H
2
O 0.26g
CaCl
2
, anhydrous 0.2g
MgSO
4
·7H
2
O 0.2g
NaH
2
PO
4
·H
2

O 0.14g
L-Glutamine 0.1g
Sodium acetate·3H
2
O 0.083g
L-Glutamic acid 0.075g
L-Arginine·HCl 0.07g
L-Lysine·HCl 0.07g
L-Leucine 0.06g
Glycine 0.05g
Ascorbic acid 0.05g
L-Proline 0.04g
L-Tyrosine 0.04g
L-Aspartic acid 0.03g
L-Threonine 0.03g
L-Alanine 0.025g
L-Phenylalanine 0.025g
L-Serine 0.025g
L-Valine 0.025g
L-Cystine 0.02g
L-Histidine·HCl·H
2
O 0.02g
L-Isoleucine 0.02g
Phenol Red 0.02g
L-Methionine 0.015g
Deoxyadenosine 0.01g
Deoxycytidine 0.01g
Deoxyguanosine 0.01g
Glutathione, reduced 0.01g

Thymidine 0.01g
Hydroxy-
L-proline 0.01g
L-Tryptophan 0.01g
Nicotinamide adenine dinucleotide 7.0mg
Tween™ 80 5.0mg
Sodium glucoronate·H
2
O 4.2mg
Coenzyme A 2.5mg
Cocarboxylase 1.0mg
Flavin adenine dinucleotide 1.0mg
Nicotinamide adenine
dinucleotide phosphate 1.0mg
Uridine triphosphate 1.0mg
Choline chloride 0.5mg
Cholesterol 0.2mg
5-Methyldeoxycytidine 0.1mg
Inositol 0.05mg
p-Aminobenzoic acid 0.05mg
Niacin 0.025mg
Niacinamide 0.025mg
Pyridoxine 0.025mg
Pyridoxal·HCl 0.025mg
Biotin 0.01mg
D-Calcium pantothenate 0.01mg
Folic acid 0.01mg
Riboflavin 0.01mg
Thiamine·HCl 0.01mg
Source: CMRL-1066 medium is available as a premixed powder

from BD Diagnostics.
Preparation of CMRL-1066 Medium: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad-
just pH to 7.2. Filter sterilize.
Preparation of Medium: Add components—except horse serum,
fresh yeast extract, and CMRL medium—to distilled/deionized water
and bring volume to 795.0mL. Adjust pH to 7.5. Autoclave for 15 min
at 15 psi pressure–121°C. Aseptically add 150.0mL of sterile horse se-
rum, 50.0mL of sterile fresh yeast extract solution, and 5.0mL of sterile
CMRL medium. Distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Spiroplasma species.
C 3N Spiroplasma Medium
Composition per 100.0mL:
Sucrose 12.0g
Phenol Red 10.0mg
PPLO broth without Crystal Violet 50.0mL
Horse serum 20.0mL
Fresh yeast extract solution 5.0mL
CMRL-1066 medium 0.5mL
pH 7.5 ± 0.2 at 25°C
PPLO Broth without Crystal Violet:
Composition
per 500.0mL:
Beef heart, infusion from 11.52g
Peptone 2.32g
NaCl 1.15g
Source: PPLO broth without Crystal Violet is available as a premixed
powder from BD Diagnostic Systems.
Preparation of PPLO Broth without Crystal Violet: Add
components to distilled/deionized water and bring volume to 500.0mL.

Mix thoroughly.
Fresh Yeast Extract Solution:
Composition
per 100.0mL:
Baker’s yeast, live, pressed, starch-free 25.0g
Preparation of Fresh Yeast Extract Solution: Add the live Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8. Filter sterilize.
CMRL-1066 Medium:
Composition
per liter:
NaCl 6.8g
NaHCO
3
2.2g
D-Glucose 1.0g
KCl 0.4g
L-Cysteine·HCl·H
2
O 0.26g
CaCl
2
, anhydrous 0.2g
MgSO
4
·7H
2
O 0.2g
NaH

2
PO
4
·H
2
O 0.14g
L-Glutamine 0.1g
Sodium acetate·3H
2
O 0.083g
L-Glutamic acid 0.075g
L-Arginine·HCl 0.07g
L-Lysine·HCl 0.07g
© 2010 by Taylor and Francis Group, LLC
290 CAE Agar Base with Triphenyltetrazolium Chloride
L-Leucine 0.06g
Glycine 0.05g
Ascorbic acid 0.05g
L-Proline 0.04g
L-Tyrosine 0.04g
L-Aspartic acid 0.03g
L-Threonine 0.03g
L-Alanine 0.025g
L-Phenylalanine 0.025g
L-Serine 0.025g
L-Valine 0.025g
L-Cystine 0.02g
L-Histidine·HCl·H
2
O 0.02g

L-Isoleucine 0.02g
Phenol Red 0.02g
L-Methionine 0.015g
Deoxyadenosine 0.01g
Deoxycytidine 0.01g
Deoxyguanosine 0.01g
Glutathione, reduced 0.01g
Thymidine 0.01g
Hydroxy-
L-proline 0.01g
L-Tryptophan 0.01g
Nicotinamide adenine dinucleotide 7.0mg
Tween™ 80 5.0mg
Sodium glucoronate·H
2
O 4.2mg
Coenzyme A 2.5mg
Cocarboxylase 1.0mg
Flavin adenine dinucleotide 1.0mg
Nicotinamide adenine
dinucleotide phosphate 1.0mg
Uridine triphosphate 1.0mg
Choline chloride 0.5mg
Cholesterol 0.2mg
5-Methyldeoxycytidine 0.1mg
Inositol 0.05mg
p-Aminobenzoic acid 0.05mg
Niacin 0.025mg
Niacinamide 0.025mg
Pyridoxine 0.025mg

Pyridoxal·HCl 0.025mg
Biotin 0.01mg
D-Calcium pantothenate 0.01mg
Folic acid 0.01mg
Riboflavin 0.01mg
Thiamine·HCl 0.01mg
Source: CMRL-1066 medium is available as a premixed powder
from BD Diagnostics.
Preparation of CMRL-1066 Medium: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad-
just pH to 7.2. Filter sterilize.
Preparation of Medium: Add components—except horse serum,
fresh yeast extract, and CMRL medium—to distilled/deionized water
and bring volume to 75.0mL. Adjust pH to 7.5. Autoclave for 15 min
at 15 psi pressure–121°C. Aseptically add 20.0mL of sterile horse se-
rum, 5.0mL of sterile yeast extract, and 0.5mL of sterile CMRL medi-
um. Distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Spiroplasma kunkelii.
CA
See: Carrot Decoction Agar
CA YE Broth
See: Casamino Acids Yeast Extract Salts Broth, Gorbach
Cadmium Fluoride Acriflavin Tellurite Medium
See: CFAT Medium
CAE Agar Base with Triphenyltetrazolium Chloride
(Citrate Azide Enterococcus HiVeg Agar Base)
Composition per liter:
Agar 15.0g
Casein enzymatic hydrolysate 15.0g
Sodium citrate 15.0g

KH
2
PO
4
5.0g
Yeast extract 5.0g
Na
2
CO
3
2.0g
Polysorbate 80 1.0g
NaN
3
0.4g
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:
Composition
per 10.0mL:
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 isolation, cultivation, and enumeration of entercocci in
water, sewage, and feces by the membrane filter method. For the direct
plating of specimens for the detection and enumeration of fecal strep-
tococci.
CAE HiVeg Agar Base
with Triphenyltetrazolium Chloride
(Citrate Azide Enterococcus HiVeg Agar Base)
Composition per liter:
Agar 15.0g
Plant hydrolysate 15.0g
Sodium citrate 15.0g
KH
2
PO
4
5.0g
Yeast extract 5.0g
Na
2
CO
3
2.0g
Polysorbate 80 1.0g
NaN

3
0.4g
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.
© 2010 by Taylor and Francis Group, LLC
CAL Broth 291
Caution: Sodium azide is toxic. Azides also react with metals and
disposal must be highly diluted.
2,3,5-Triphenyltetrazolium Chloride Solution:
Composition
per 10.0mL:
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 isolation, cultivation, and enumeration of entercocci in
water, sewage, and feces by the membrane filter method. For the direct
plating of specimens for the detection and enumeration of fecal strep-
tococci.
Caffeic Acid Ferric Citrate Test Medium

(CAFC Test Medium)
(Caffeic Acid Agar)
Composition per liter:
Agar 20.0g
(NH
4
)
2
SO
4
5.0g
Glucose 5.0g
Yeast extract 2.0g
K
2
HPO
4
0.8g
MgSO
4
·3H
2
O 0.7g
Caffeic acid·
1/2H
2
O 0.18g
Chloramphenicol 0.05g
Ferric citrate solution 4.0mL
pH 6.5 ± 0.2 at 25°C

Ferric Citrate Solution:
Composition per 20.0mL:
Ferric citrate 100.0mg
Preparation of Ferric Citrate Solution: Add ferric citrate to
20.0mL of distilled/deionized water. Mix thoroughly.
Preparation of Medium: Add components, except chlorampheni-
col, to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Heat to boiling. Autoclave for 15 min at 15 psi pressure–
121°C. Cool to 45°–50°C. Aseptically add 0.05g of chloramphenicol.
Mix thoroughly. Pour into sterile Petri dishes.
Use: For the isolation and presumptive identification of Cryptococcus
neoformans. Cryptococcus neoformans appears as dark brown colo-
nies. All other Cryptococcus species appear as light brown or nonpig-
mented colonies.
Caffeine Medium
Composition per liter:
Agar 15.0g
Solution A 400.0mL
Solution B 400.0mL
Solution C 200.0mL
pH 5.0 ± 0.2 at 25°C
Solution A:
Composition
per 400.0mL:
Na
2
HPO
4
7.8g
KH

2
PO
4
3.0g
Caffeine 1.0g
NaCl 0.58g
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 400.0mL. Mix thoroughly. Adjust pH to 5.0.
Solution B:
Composition
per 400.0mL:
MgSO
4
·7H
2
O 0.12g
CaCl
2
·2H
2
O 11.0mg
Preparation of Solution B: Add components to distilled/deionized
water and bring volume to 400.0mL. Mix thoroughly.
Solution C:
Composition
per 200.0mL:
FeCl
3
16.0mg
Preparation of Solution C: Add FeCl

3
to distilled/deionized water
and bring volume to 200.0mL. Mix thoroughly.
Preparation of Medium: To 400.0mL of solution A, add 400.0mL
of solution B and 200.0mL of solution C. Adjust pH to 5.0. Add agar.
Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min
at 15 psi pressure–121°C. Pour into sterile Petri dishes or distribute
into sterile tubes.
Use: For the cultivation of Pseudomonas species.
CAGV Medium
See: Casamino Acid Glucose Medium
CAL Agar
(Cellobiose Arginine Lysine Agar)
(Yersinia Isolation Agar)
Composition per liter:
Agar 20.0g
L-Arginine·HCl 6.5g
L-Lysine·HCl 6.5g
NaCl 5.0g
Cellobiose 3.5g
Yeast extract 3.0g
Sodium deoxycholate 1.5g
Neutral Red 0.03g
pH 7.3 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Heat to boiling. Do
not autoclave. Pour into sterile Petri dishes.
Use: For the isolation and characterization of Yersinia enterocolitica
from fecal specimens and enumeration of Yersinia enterocolitica from
water and other liquid specimens.

CAL Broth
(Cellobiose Arginine Lysine Broth)
Composition per liter:
L-Arginine·HCl 6.5g
L-Lysine·HCl 6.5g
NaCl 5.0g
Cellobiose 3.5g
Yeast extract 3.0g
© 2010 by Taylor and Francis Group, LLC
292 CAL HiVeg Agar
Sodium deoxycholate 1.5g
Neutral Red 0.03g
pH 7.3 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Heat to boiling. Do
not autoclave. Distribute into sterile tubes in 6.0–8.0mL volumes.
Use: For the isolation and characterization of Yersinia enterocolitica
from fecal specimens and enumeration of Yersinia enterocolitica from
water and other liquid specimens.
CAL HiVeg Agar
(Cellobiose Arginine Lysine HiVeg Agar)
Composition per liter:
Agar 20.0g
L-Arginine 6.5g
L-Lysine hydrochloride 6.5g
NaCl 5.0g
Cellobiose 3.5g
Yeast extract 3.0g
Synthetic detergent No. III 1.5g
Neutral Red 0.03g

pH 7.1 ± 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. Heat to boiling. Do
not autoclave. Pour into sterile Petri dishes.
Use: For the isolation and characterization of Yersinia enterocolitica
from fecal specimens and enumeration of Y. enterocolitica from water.
CAL HiVeg Broth
(Cellobiose Arginine Lysine HiVeg Broth)
Composition per liter:
L-Arginine 6.5g
L-Lysine hydrochloride 6.5g
NaCl 5.0g
Cellobiose 3.5g
Yeast extract 3.0g
Synthetic detergent No. III 1.5g
Neutral Red 0.03g
pH 7.1 ± 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. Heat to boiling. Do
not autoclave. Distribute into sterile tubes.
Use: For the isolation and characterization of Yersinia enterocolitica
from fecal specimens and enumeration of Yersinia enterocolitica from
water and other liquid specimens.
Calcium Caseinate Agar
Composition per liter:
Agar 13.0g

Peptic digest of animal tissue 4.0g
Calcium caseinate 3.5g
Meat extract 2.0g
Casein enzymic hydrolysate 2.0g
CaCl
2
·2H
2
O 0.2g
Tri-potassium citrate 0.35g
Na
2
HPO
4
0.105g
KH
2
PO
4
0.035g
NaCl 5.0g
pH 7.0 ± 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
or flasks. Gently heat and bring to boiling. Boil for 10 min. Autoclave
for 15 min at 15 psi pressure–121°C. Mix thoroughly while pouring
into Petri dishes.
Use: For the detection and enumeration of proteolytic microorganisms
in foodstuffs and other materials.

Calcium Caseinate Agar with Skim Milk
Composition per liter:
Agar 13.0g
Skim Milk 10.0g
Peptic digest of animal tissue 4.0g
Calcium caseinate 3.5g
Meat extract 2.0g
Casein enzymic hydrolysate 2.0g
CaCl
2
·2H
2
O 0.2g
Tri-potassium citrate 0.35g
Na
2
HPO
4
0.105g
KH
2
PO
4
0.035g
NaCl 5.0g
pH 7.0 ± 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
or flasks. Gently heat and bring to boiling. Boil for 10 min. Autoclave

for 15 min at 15 psi pressure–121°C. Mix thoroughly while pouring
into Petri dishes.
Use: For the detection and enumeration of proteolytic microorganisms
in foodstuffs and other materials.
Caldicellulosiruptor Medium
Composition per liter:
Pancreatic digest of casein 2.0g
K
2
HPO
4
1.5g
Cellobiose 1.0g
Yeast extract 1.0g
NaCl 0.9g
NH
4
Cl 0.9g
KH
2
PO
4
0.75g
L-Cysteine·HCl 0.75g
MgCl
2
·6H
2
O 0.4g
FeCl

3
·6H
2
O 2.5mg
Resazurin 0.5mg
Trace elements solution SL-10 1.0mL
pH 7.2 ± 0.2 at 25°C
Trace Elements Solution SL-10:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
CoCl
2
·6H
2
O 190.0mg
MnCl
2
·4H
2
O 100.0mg
© 2010 by Taylor and Francis Group, LLC
Caldisphaera Medium 293
ZnCl
2
70.0mg

Na
2
MoO
4
·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10: Add
FeCl
2
·4H
2
O to 10.0mL of HCl solution. Mix thoroughly. Add distilled/
deionized water and bring volume to 1.0L. Add remaining compo-

nents. Mix thoroughly. Sparge with 100% N
2
. Autoclave for 15 min at
15 psi pressure–121°C.
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Add components to distilled/deionized water and bring vol-
ume to 1.0L. Mix thoroughly. Sparge with 100% N
2
. Anaerobically
distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–
121°C.
Use: For the cultivation of Caldicellulosiruptor saccharolyticus.
Caldicellulosiruptor Medium
Composition per 1001.0mL:
Pancreatic digest of casein 2.0g
K
2
HPO
4
1.5g
Cellulose 1.0g
Cellobiose 1.0g
Yeast extract 1.0g
NaCl 0.9g
NH
4
Cl 0.9g
KH

2
PO
4
0.75g
MgCl
2
·6H
2
O 0.4g
FeCl
3
·6H
2
O 2.5mg
L-Cysteine·HCl 0.75g
Resazurin 0.5mg
pH 7.2 ± 0.2 at 25°C
Trace Elements Solution SL-10:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
CoCl
2
·6H
2
O 190.0mg

MnCl
2
·4H
2
O 100.0mg
ZnCl
2
70.0mg
Na
2
MoO
4
·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg

HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10: Add FeCl
2
·4H
2
O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–
121°C.
Preparation of Medium: Prepare and dispense medium under
100% N
2
. Add components to distilled/deionized water and bring vol-
ume to 1.0L. Mix thoroughly. Sparge with 100% N
2
. Anaerobically
distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–
121°C.
Use: For the cultivation of Caldicellulosiruptor saccharolyticus.
Caldisphaera Medium
(DSMZ Medium 991)
Composition per liter:
Sulfur, powder 10.0g
MnCl
2
·4H
2

O 1.8g
(NH
4
)
2
SO
4
1.3g
KH
2
PO
4
0.28g
MgSO
4
·7H
2
O 0.25g
CaCl
2
·2H
2
O 0.07g
FeCl
3
·6H
2
O 0.02g
Na
2

B
4
O
7
·10H
2
O 4.5mg
Resazurin 1.0mg
ZnSO
4
·7H
2
O 0.22mg
CuCl
2
·2H
2
O 0.05mg
Na
2
MoO
4
·4H
2
O 0.03mg
VOSO
4
·2H
2
O 0.03mg

Na
3
-citrate·2H
2
O 0.03mg
CoSO
4
0.01mg
Vitamin solution 10.0mL
Na
2
S·9H
2
O solution 10.0mL
Yeast extract solution 5.0mL
pH 4.3 ± 0.2 at 25°C
Yeast Extract Solution:
Composition
per 5.0mL:
Yeast extract 0.5g
Preparation of Yeast Extract Solution: Add yeast extract to dis-
tilled/deionized water and bring volume to 5.0mL. Mix thoroughly.
Sparge under 100% N
2
gas for 3 min. Autoclave for 15 min at 15 psi
pressure–121°C. Store under N
2
gas.
Na
2

S·9H
2
O Solution:
Composition per 10.0mL:
Na
2
S·9H
2
O 0.5g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Autoclave under 100% N
2
for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
Vitamin Solution:
Composition
per liter:
Pyridoxine-HCl 10.0mg
Thiamine-HCl·2H
2
O 5.0mg

Riboflavin 5.0mg
Nicotinic acid 5.0mg
D-Ca-pantothenate 5.0mg
p-Aminobenzoic acid 5.0mg
Lipoic acid 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge
with 80% H
2
+ 20% CO
2
. Filter sterilize.
Preparation of Medium: Add components, except vitamin solution,
yeast extract solution, sulfur, and Na
2
S·9H
2
O solution, to distilled/deion-
ized water and bring volume to 975.0mL. Mix thoroughly. Gently heat
and bring to boiling. Boil for 3 min. Cool to room temperature under
80% N
2
+ 20% CO
2
. Adjust pH to 3.5 with 10N H

2
SO
4
. Dispense under
same gas atmosphere in suitable culture vessels (e.g., 20.0mL of the
medium in 120 mL serum bottles). Autoclave for 15 min at 15 psi pres-
sure–121°C. Steam sulfur for 3 hr on each of 3 successive days. Asep-
© 2010 by Taylor and Francis Group, LLC
294 Calditerrivibrio Medium
tically mix the sterilized sulfur with the medium and add vitamins and
yeast extract from sterile, anaerobic stock solutions. Prior to inocula-
tion change atmosphere to 80% H
2
+ 20% CO
2
. Aseptically and anox-
ically add Na
2
S·9H
2
O. Adjust pH to 4.0–4.5 if necessary. After
inoculation pressurize vials to 1 bar overpressure with 80% H
2
+ 20%
CO
2
gas mixture.
Use: For the cultivation of Caldisphaera spp.
Calditerrivibrio Medium
(DSMZ Medium 1112)

Composition per liter:
NaNO
3
0.85g
Na-acetate 0.82g
NH
4
Cl 0.54g
MgCl
2
·6H
2
O 0.2g
CaCl
2
·2H
2
O 0.15g
KH
2
PO
4
0.14g
Resazurin 0.5mg
NaHCO
3
solution 10.0mL
Vitamin solution 10.0mL
Na
2

S·9H
2
O solution 10.0mL
Trace element solution SL-10 1.0mL
Selenite/tungstate solution 1.0mL
pH 7.0 ± 0.2 at 25°C
NaHCO
3
Solution:
Composition per 10.0mL:
NaHCO
3
2.5g
Preparation of NaHCO
3
Solution: Add components to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Sparge
with 20% CO
2
+ 80% H
2
. Autoclave for 15 min at 15 psi pressure–
121°C. Cool to room temperature.
Na
2
S·9H
2
O Solution:
Composition per 10.0mL:
Na

2
S·9H
2
O 0.5g
Preparation of Na
2
S·9H
2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Autoclave under 100% N
2
for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
Selenite/Tungstate Solution:
Composition
per liter:
NaOH 0.5g
Na
2
WO
4
·2H
2
O 4.0mg
Na

2
SeO
3
·5H
2
O 3.0mg
Preparation of Selenite/Tungstate Solution: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–121°C.
Vitamin Solution:
Composition
per liter:
Pyridoxine-HCl 10.0mg
Thiamine-HCl·2H
2
O 5.0mg
Riboflavin 5.0mg
Nicotinic acid 5.0mg
D-Ca-pantothenate 5.0mg
p-Aminobenzoic acid 5.0mg
Lipoic acid 5.0mg
Biotin 2.0mg
Folic acid 2.0mg
Vitamin B
12
0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Sparge

with 80% H
2
+ 20% CO
2
. Filter sterilize.
Trace Elements Solution SL-10:
Composition
per liter:
FeCl
2
·4H
2
O 1.5g
CoCl
2
·6H
2
O 190.0mg
MnCl
2
·4H
2
O 100.0mg
ZnCl
2
70.0mg
Na
2
MoO
4

·2H
2
O 36.0mg
NiCl
2
·6H
2
O 24.0mg
H
3
BO
3
6.0mg
CuCl
2
·2H
2
O 2.0mg
HCl (25% solution) 10.0mL
Preparation of Trace Elements Solution SL-10: Add FeCl
2
·4H
2
O
to 10.0mL of HCl solution. Mix thoroughly. Add distilled/deionized
water and bring volume to 1.0L. Add remaining components. Mix thor-
oughly. Sparge with 100% N
2
. Autoclave for 15 min at 15 psi pressure–
121°C.

Preparation of Medium: Add components, except bicarbonate so-
lution, sulfide solution, and vitamin solution, to distilled/deionized wa-
ter and bring volume to 970.0mL. Mix thoroughly. Gently heat and
bring to boiling. Boils for several minutes. Cool to room temperature
while sparging with 80% N
2
+ 20% CO
2
. Distribute into screw-capped
tubes or bottles under an atmosphere of 80% N
2
+ 20% CO
2
. Autoclave
for 15 min at 15 psi pressure–121°C. Add the bicarbonate solution, sul-
fide solution, and vitamin solution. Adjust the final pH to 7.0.
Use: For the cultivation of Calditerrivibrio spp.
Caldivirga Medium
(DSMZ Medium 883)
Composition per liter:
(NH
4
)
2
SO
4
1.3g
Sulfur, powdered 10.0g
Na
2

S·9H
2
O 0.5 g
KH
2
PO
4
0.28g
MgSO
4
·7H
2
O 0.25g
CaCl
2
·2H
2
O 0.07g
FeCl
3
·6H
2
O 0.02g
Na
2
B
4
O
7
·10H

2
O 4.5mg
MnCl
2
·4H
2
O 1.8mg
Resazurin 0.5mg
ZnSO
4
·7H
2
O 0.22mg
CuCl
2
·2H
2
O 0.05mg
Na
2
MoO
4
·2H
2
O 0.03mg
VOSO
4
·2H
2
O 0.03mg

CoSO
4
0.01mg
Na
2
S·9H
2
O solution 7.5mL
Yeast extract solution 5.0mL
Na
3
-citrate·2H
2
O 3.0mL
Vitamin solution 1.0mL
pH 4.0± 0.2 at 25°C
Na
2
S·9H
2
O Solution:
Composition per 10.0mL:
Na
2
S·9H
2
O 0.3g
Preparation of Na
2
S·9H

2
O Solution: Add Na
2
S·9H
2
O to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Autoclave under 100% N
2
for 15 min at 15 psi pressure–121°C. Cool
to room temperature.
© 2010 by Taylor and Francis Group, LLC