Handbook of Microbiological Media, Fourth Edition part 150 potx

Regan-Lowe Semisolid Transport Medium 1485
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Distribute into screw-
capped tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the transport and isolation of bacteria from dental plaque,
especially Streptococcus mutans, Streptococcus sanguis, and Lactoba-
cillus species.
Reduced Transport Fluid
Composition per liter:
(NH
4
)
2
SO
4
9.0g
NaCl 9.0g
K
2
HPO
4
4.5g
KH
2
PO
4
4.5g
Na
2
CO
3

4.0g
EDTA (ethylenediamine tetraacetic acid) 3.8g
Dithiothreitol 2.0g
MgSO
4
·7H
2
O 1.8g
pH 8.0 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Asep-
tically distribute into sterile tubes with rubber stoppers.
Use: For the transport and isolation of bacteria from dental plaque,
especially Streptococcus mutans and Streptococcus sanguis. Also used
for the cultivation of a variety of Gram-positive bacteria from the oral
cavity, especially streptococci, actinomycetes, lactobacilli, clostridia,
Bacteroides species, Fusobacterium species, and Veillonella species.
Reduced Transport Fluid
Composition per liter:
Stock mineral salt solution No. 1 75.0mL
Stock mineral salt solution No. 2 75.0mL
Dithiothreitol (1% solution) 20.0mL
Ethylenediamine tetraacetic acid
(1M solution) 10.0mL
Na
2
CO
3
(8% solution) 5.0mL
Resazurin (0.1% solution) 1.0mL

pH 8.0 ± 0.2 at 25°C
Stock Mineral Salt Solution No. 1:
Composition
per 100.0mL:
K
2
HPO
4
0.6g
Preparation of Stock Mineral Salt Solution No. 1: Add
K
2
HPO
4
to distilled/deionized water and bring volume to 100.0mL.
Mix thoroughly.
Stock Mineral Salt Solution No. 2:
Composition
per 100.0mL:
NaCl 1.2g
(NH
4
)
2
SO
4
1.2g
K
2
HPO

4
0.6g
MgSO
4
·7H
2
O 0.25g
Preparation of Stock Mineral Salt Solution No. 2: Add com-
ponents to distilled/deionized water and bring volume to 100.0mL.
Mix thoroughly.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Filter sterilize. Asep-
tically distribute into sterile tubes with rubber stoppers.
Use: For the transport and isolation of bacteria from dental plaque,
especially Streptococcus mutans and Streptococcus sanguis. Also used
for the cultivation of a variety of Gram-positive bacteria from the oral
cavity, especially streptococci, actinomycetes, lactobacilli, clostrida,
Bacteroides, Fusobacteria, and Veillonela.
Regan-Lowe Charcoal Agar
(Regan-Lowe Medium)
Composition per liter:
Agar 12.0g
Beef extract 10.0g
Pancreatic digest of gelatin 10.0g
Soluble starch 10.0g
NaCl 5.0g
Charcoal 4.0g
Niacin 0.01g
Horse blood, defibrinated 100.0mL
Cephalexin solution 10.0mL

pH 7.4 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from BD Di-
agnostic Systems.
Cephalexin Solution:
Composition
per 10.0mL:
Cephalexin 0.04g
Preparation of Cephalexin Solution: Add cephalexin to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Filter sterilize.
Preparation of Medium: Add components, except horse blood and
cephalexin solution, to distilled/deionized water and bring volume to
890.0mL. 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 horse blood and sterile cephalexin solution. Mix thoroughly.
Pour into sterile Petri dishes or distribute into sterile tubes. Swirl me-
dium while dispensing to keep charcoal in suspension.
Use: For the selective isolation and cultivation of Bordetella pertussis
and Bordetella parapertussis from clinical specimens.
Regan-Lowe Semisolid Transport Medium
Composition per liter:
Agar 6.0g
Beef extract 5.0g
Pancreatic digest of gelatin 5.0g
Soluble starch 5.0g
NaCl 2.5g
Charcoal 2.0g
Niacin 0.01g
Horse blood, defibrinated 100.0mL
Cephalexin solution 10.0mL

pH 7.4 ± 0.2 at 25°C
Cephalexin Solution:
Composition
per 10.0mL:
Cephalexin 0.04g
Preparation of Cephalexin Solution: Add cephalexin to dis-
tilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Filter sterilize.
Preparation of Medium: Add components, except horse blood and
cephalexin solution, to distilled/deionized water and bring volume to
890.0mL. 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 ster-
ile horse blood and sterile cephalexin solution. Mix thoroughly. Aseptical-
© 2010 by Taylor and Francis Group, LLC
1486 Reinforced AE Medium
ly distribute into small, sterile, screw-capped tubes. Fill tubes half-full.
Swirl medium while dispensing to keep charcoal in suspension.
Use: For the transport of Bordetella pertussis and Bordetella paraper-
tussis isolated from clinical specimens.
Reinforced AE Medium
(RAE Medium)
(LMG Medium 239)
Composition per liter:
Base medium 500.0mL
Growth medium 500.0mL
pH 5.0 ± 0.2 at 25°C
Base Medium:
Composition
per liter:
Agar 10.0g

Preparation of Base Medium: Add agar to distilled/deionized wa-
ter and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to
boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour as a base
layer into sterile Petri dishes.
Growth Medium:
Composition
per liter:
Glucose 40.0g
Agar 20.0g
Yeast extract 10.0g
Peptone 10.0g
Na
2
HPO
4
·2H
2
O 3.38g
Citric acid·2H
2
O 1.5g
Ethanol 20.0mL
Acetic acid 10.0mL
Preparation of Growth Medium: Add components, except etha-
nol and acetic acid, to distilled/deionized water and bring volume to
970.0mL. 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 20.0mL filter sterilized ethanol and 10.0mL filter sterilized acetic
acid. Mix thoroughly.
Preparation of Growth Medium: This medium is used as a dou-

ble layer. Pour as a layer of base medium into sterile Petri dishes. Al-
low to solidify. Pour a thin layer of growth medium over the solid base
medium. Allow to solidify.
Use: For the isolation and cultivation of Gluconacetobacter spp. and
Acetobacter pomorum.
Reinforced Clostridial Agar
Composition per liter:
Agar 13.5g
Beef extract 10.0g
Pancreatic digest of casein 10.0g
NaCl 5.0g
Glucose 5.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Soluble starch 1.0g
L-Cysteine·HCl·H
2
O 0.5g
pH 6.8 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from BD Di-
agnostic Systems and Oxoid Unipath.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 10
psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation and enumeration of Clostridium species, Bifi-
dobacterium species, other anaerobes (e.g., lactobacilli), and faculta-
tive organisms from clinical specimens and foods.
Reinforced Clostridial Agar with Tween™
(LMG Medium 146)

Composition per liter:
Agar 13.5g
Beef extract 10.0g
Pancreatic digest of casein 10.0g
NaCl 5.0g
Glucose 5.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Tween™ 80 1.0g
Soluble starch 1.0g
L-Cysteine·HCl·H
2
O 0.5g
pH 6.8 ± 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 10
psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation and maintenance of Bifidobacterium meryci-
cum.
Reinforced Clostridial HiVeg Agar
Composition per liter:
Agar 13.5g
Plant extract 10.0g
Plant hydrolysate 10.0g
Glucose 5.0g
NaCl 5.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Starch, soluble 1.0g

L-Cysteine·HCl 0.5g
pH 6.8 ± 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 15 min at 10
psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation and enumeration of clostridia and other anaer-
obes.
Reinforced Clostridial HiVeg Broth
Composition per liter:
Plant extract 10.0g
Plant hydrolysate 10.0g
Glucose 5.0g
NaCl 5.0g
Sodium acetate 3.0g
Yeast extract 3.0g
© 2010 by Taylor and Francis Group, LLC
Reinforced Clostridial Medium with Uric Acid 1487
Starch, soluble 1.0g
Agar 0.5g
L-Cysteine·HCl 0.5g
pH 6.8 ± 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 15 min at 10
psi pressure–115°C.

Use: For the cultivation and enumeration of clostridia and other anaer-
obes.
Reinforced Clostridial Medium
Composition per liter:
Tryptose 10.0g
Beef extract 10.0g
Glucose 5.0g
NaCl 5.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Soluble starch 1.0g
L-Cysteine·HCl·H
2
O 0.5g
Agar 0.5g
pH 6.8 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from BD Di-
agnostic Systems and Oxoid Unipath.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 10
psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the nonselective cultivation and enumeration of Clostridium
species, other anaerobes such as lactobacilli, and facultative organisms
from clinical specimens and foods.
Reinforced Clostridial Medium with Casamino Acids
Composition per liter:
Casamino acids 15.0g
Agar 13.5g
Beef extract 10.0g

Pancreatic digest of casein 10.0g
NaCl 5.0g
Glucose 5.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Soluble starch 1.0g
L-Cysteine·HCl·H
2
O 0.5g
pH 6.8 ± 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 Clostridium aminophilum.
Reinforced Clostridial Medium with Glycerol
Composition per liter:
Agar 13.5g
Beef extract 10.0g
Pancreatic digest of casein 10.0g
NaCl 5.0g
Glucose 5.0g
Glycerol 5.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Soluble starch 1.0g
L-Cysteine·HCl·H
2
O 0.5g
pH 6.8 ± 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 10
psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation of Anaerovibrio glycerini.
Reinforced Clostridial Medium, Modified
(ATCC Medium 2107)
Composition
per liter:
Tryptose 10.0g
Beef extract 10.0g
Glucose 5.0g
NaCl 5.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Soluble starch 1.0g
L-Cysteine·HCl·H
2
O 0.5g
pH 6.8 ± 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 10
psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation of Clostridium saccharobutylicum, Clostrid-
ium frigidicarnis, and Mitsuokella jalaludinii.
Reinforced Clostridial Medium with Sodium Lactate
Composition per liter:
Tryptose 10.0g
Beef extract 10.0g
Glucose 5.0g

NaCl 5.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Soluble starch 1.0g
L-Cysteine·HCl·H
2
O 0.5g
Agar 0.5g
Sodium lactate (60% solution) 15.0mL
pH 6.8 ± 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 10
psi pressure–115°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the nonselective cultivation and enumeration of Clostridium
species, other anaerobes such as lactobacilli, and facultative organisms
from clinical specimens and foods.
Reinforced Clostridial Medium with Uric Acid
Composition per liter:
Agar 13.5g
Beef extract 10.0g
Pancreatic digest of casein 10.0g
NaCl 5.0g
© 2010 by Taylor and Francis Group, LLC
1488 Renibacterium KDM2 Medium
Glucose 5.0g
Uric acid 3.0g
Yeast extract 3.0g
Sodium acetate 3.0g
Soluble starch 1.0g

L-Cysteine·HCl·H
2
O 0.5g
pH 6.8 ± 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 Clostridium acidurici.
Renibacterium KDM2 Medium
Composition per liter:
Agar 15.0g
Peptone 10.0g
L-Cysteine·HCl·H
2
O 1.0g
Yeast extract 0.5g
Fetal calf serum 200.0mL
pH 6.5 ± 0.2 at 25°C
Preparation of Medium: Add components, except fetal calf serum
and agar, to distilled/deionized water and bring volume to 800.0mL. Mix
thoroughly. Adjust pH to 6.5 with NaOH. Add agar. Gently heat while
stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure–
121°C. Cool to 45°–50°C. Aseptically add fetal calf serum. Mix thor-
oughly. Pour into sterile Petri dishes or distribute into sterile tubes.
Use: For the cultivation and maintenance of Renibacterium salmoni-
narum.
Reuters Sorbic Acid Agar Base
Composition per liter:
D-Glucose 20.0g
Agar 16.0g

Casein enzymic hydrolysate 10.0g
Meat extract 10.0g
Yeast extract 5.0g
Sodium acetate 5.0g
Sodium citrate 3.0g
Tween 80 1.0g
MgSO
4
·7H
2
O 0.2g
MnSO
4
·2H
2
O 0.05g
Selective supplement solution 10.0mL
pH 5.0 ± 0.2 at 25°C
Source: This medium is available from HiMedia.
Selective Supplement Solution:
Composition
per 10.0mL:
Sorbic acid 0.4g
Preparation of Selective Supplement Solution: Add sorbic
acid to distilled/deionized water and bring volume to 10.0mL. Mix
thoroughly. 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 and bring to boiling. Mix to dis-
solve components completely. Cool to 50°C. Aseptically add selective

supplement solution. Mix thoroughly. Sterilize for 30 min at 0 psi pres-
sure–100°C. Pour into Petri dishes or aseptically distribute into sterile
tubes.
Use: For the isolation and differentiation of lactobacilli from food-
stuffs, feces, etc.
RF Medium
Composition per liter:
Yeast extract 0.05g
Peptone 0.05g
(NH
4
)
2
SO
4
0.05g
L-Cysteine·HCl·H
2
O 0.05g
Salt solution 50.0mL
Rumen fluid, clarified 30.0mL
Resazurin (1% solution) 0.1mL
pH 7.4 ± 0.2 at 25°C
Salt Solution:
Composition
per liter:
NaHCO
3
10.0g
NaCl 2.0g

K
2
HPO
4
1.0g
KH
2
PO
4
1.0g
CaCl
2
, anhydrous 0.2g
MgSO
4
0.2g
Preparation of Salts Solution: Add CaCl
2
and MgSO
4
to 300.0mL
of distilled/deionized water. Mix thoroughly until dissolved. Bring vol-
ume to 800.0mL with distilled/deionized water. Add remaining com-
ponents while stirring. Bring volume to 1.0L. Mix thoroughly. Store at
4°C.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.2–6.3
with 4N HCl. Gently heat and bring to boiling under 100% N
2
. Anaer-

obically distribute into tubes in 7.0mL volumes. Cap with rubber stop-
pers. Place tubes in a press. Autoclave for 20 min at 15 psi pressure–
121°C with fast exhaust. The pH of the medium should be 7.4 after au-
toclaving.
Use: For the cultivation and maintenance of Treponema bryantii.
RFC Agar
See: Rumen Fluid Cellobiose Agar
RGCA Medium
(Rumen Fluid Glucose Cellobiose Agar)
Composition per 300.3mL:
Rumen fluid 120.0mL
Solution IV 65.0mL
Mineral solution I 45.0mL
Mineral solution II 45.0mL
Na
2
CO
3
solution 20.0mL
L-Cysteine·HCl·H
2
O solution 5.0mL
Solution III 0.3mL
pH 6.6 ± 0.2 at 25°C
Mineral Solution I:
Composition
per 100.0mL:
K
2
HPO

4
0.3g
Preparation of Mineral Solution I: Add K
2
HPO
4
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly.
Mineral Solution II:
Composition
per 100.0mL:
(NH
4
)
2
SO
4
0.6g
NaCl 0.6g
© 2010 by Taylor and Francis Group, LLC
Rhizobium BIII Defined Agar 1489
KH
2
PO
4
0.3g
MgSO
4
0.06g
CaCl

2
0.06g
Preparation of Mineral Solution II: Add components to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Solution III:
Composition
per 10.0mL:
Resazurin 0.01g
Preparation of Solution III: Add resazurin to 10.0mL of distilled/
deionized water. Mix thoroughly.
Solution IV:
Composition per 65.0mL:
Agar 4.5g
Glucose 0.6g
Cellobiose 0.6g
Preparation of Solution IV: Add components to distilled/deion-
ized water and bring volume to 65.0mL. Mix thoroughly.
L-Cysteine·HCl·H
2
O Solution:
Composition
per 100.0mL:
L-Cysteine·HCl·H
2
O 3.0g
Preparation of L-Cysteine·HCl·H
2
O Solution: Add L-cyste-
ine·HCl·H
2

O to distilled/deionized water and bring volume to
100.0mL. Mix thoroughly. Filter sterilize.
Na
2
CO
3
Solution:
Composition
per 100.0mL:
Na
2
CO
3
6.0g
Preparation of Na
2
CO
3
Solution: Add Na
2
CO
3
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Filter
sterilize.
Rumen Fluid:
Composition
per 120.0mL:
Rumen fluid 120.0mL
Preparation of Rumen Fluid: Filter rumen contents, obtained

from a cow on an alfalfa-hay concentrate ration, through two layers of
cheesecloth to remove larger particles. Store under CO
2
in quart milk
bottles in the refrigerator. Much of the particulate matter settles out.
Use the supernatant fluid.
Preparation of Medium: Combine 45.0mL of mineral solution I,
45.0mL of mineral solution II, 0.3mL of solution III, and 65.0mL of so-
lution IV in a 500.0mL flask. Gently heat and bring to boiling. Add
120.0mL of rumen fluid. Gently heat and bring to boiling under 100%
CO
2
. Cap with a rubber stopper and wire the stopper secure. Autoclave
for 20 min at 15 psi pressure–121°C. Cool to 45°–50°C. Remove stop-
per and gas with 100% CO
2
to eliminate O
2
. Aseptically add 5.0mL of
sterile
L-cysteine·HCl·H
2
O solution and 20.0mL of sterile Na
2
CO
3
so-
lution. Mix thoroughly. Aseptically and anaerobically distribute into
tubes under 100% CO
2

in 6.0mL volumes. Cap with rubber stoppers.
Use: For the cultivation and maintenance of Ruminococcus albus,
Ruminococcus flavifaciens, and Succinimonas amylolytica.
Rhamnose Salts Medium
Composition per liter:
Rhamnose 10.0g
Yeast extract 3.0g
K
2
HPO
4
2.9g
KH
2
PO
4
2.1g
NH
4
·Cl 2.0g
MgSO
4
·7H
2
O 0.4g
NaCl 30.0mg
CaCl
2
3.0mg
FeSO

4
·7H
2
O 1.0mg
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. Distribute into tubes
or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of Rhodococcus chlorophenolicus.
Rhizobium Agar
(LMG 201)
Composition per liter:
Agar 20.0g
Mannitol 10.0g
Yeast extract 1.0g
Sodium glutamate 0.5g
KH
2
PO
4
0.5g
MgSO
4
·7H
2
O 0.1g
CaCl
2
·2H
2

O 40.0mg
FeCl
3
4.0mg
pH 6.8 ± 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 6.8.
Gently heat and bring to boiling. Distribute into tubes or flasks. Auto-
clave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes
or leave in tubes.
Use: For the cultivation and maintenance of Rhizobium fredii, Rhizo-
bium galegae, Rhizobium leguminosarum, Rhizobium loti, Rhizobium
meliloti, and Rhizobium tropici.
Rhizobium BIII Defined Agar
Composition per liter:
Agar 13.0g
Mannitol 10.0g
Sodium glutamate 1.1g
K
2
HPO
4
0.23g
MgSO
4
·7H
2
O 0.1g
Trace elements stock 1.0mL
Vitamin stock 1.0mL

pH 7.0 ± 0.2 at 25°C
Trace Elements Stock:
Composition
per liter:
Nitrilotriacetic acid 7.0g
CaCl
2
·2H
2
O 6.62g
H
3
BO
3
0.145g
FeSO
4
·7H
2
O 0.125g
Na
2
MoO
4
0.125g
ZnSO
4
·7H
2
O 0.108g

CoSO
4
·7H
2
O 0.07g
CuSO
4
·5H
2
O 5.0mg
MnCl
2
·4H
2
O 4.3mg
Preparation of Trace Elements Stock: Add components to
500.0mL of distilled/deionized water in the order: CaCl
2
·2H
2
O, H
3
BO
3
,
FeSO
4
·7H
2
O, CoSO

4
·7H
2
O, CuSO
4
·5H
2
O, MnCl
2
·4H
2
O, ZnSO
4
·7H
2
O, and
Na
2
MoO
4
. Adjust pH to 5.0. Add nitrilotriacetic acid. Bring volume to
1.0L with distilled/deionized water.
© 2010 by Taylor and Francis Group, LLC
1490 Rhizobium BIII Defined Broth
Vitamin Stock:
Composition
per liter:
Inositol 0.12g
p-Aminobenzoic acid 0.02g
Biotin 0.02g

Calcium pantothenate 0.02g
Nicotinic acid 0.02g
Pyridoxine·HCl 0.02g
Riboflavin 0.02g
Thiamine·HCl 0.02g
Sodium phosphate buffer (50.0mM solution, pH 7.0) 1.0L
Preparation of Vitamin Stock: Combine components. Mix thor-
oughly. Filter sterilize. Store at 4°C in the dark.
Preparation of Medium: Add components, except vitamin stock, to
distilled/deionized water and bring volume to 999.0mL. Mix thoroughly.
Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pres-
sure–121°C. Cool to 45°–50°C. Aseptically add 1.0mL of sterile vitamin
stock. Mix thoroughly. Pour into sterile Petri dishes or distribute into
sterile tubes.
Use: For the isolation and cultivation of Rhizobium species from root
nodules.
Rhizobium BIII Defined Broth
Composition per liter:
Mannitol 10.0g
Sodium glutamate 1.1g
K
2
HPO
4
0.23g
MgSO
4
·7H
2
O 0.1g

Trace elements stock 1.0mL
Vitamin stock 1.0mL
pH 7.0 ± 0.2 at 25°C
Trace Elements Stock:
Composition
per liter:
Nitrilotriacetic acid 7.0g
CaCl
2
·2H
2
O 6.62g
H
3
BO
3
0.145g
FeSO
4
·7H
2
O 0.125g
Na
2
MoO
4
0.125g
ZnSO
4
·7H

2
O 0.108g
CoSO
4
·7H
2
O 0.07g
CuSO
4
·5H
2
O 5.0mg
MnCl
2
·4H
2
O 4.3mg
Preparation of Trace Elements Stock: Add components, except
nitrilotriacetic acid, to 500.0mL of distilled/deionized water in the or-
der listed. Adjust pH to 5.0. Add nitrilotriacetic acid. Bring volume to
1.0L with distilled/deionized water.
Vitamin Stock:
Composition
per liter:
Inositol 0.12g
p-Aminobenzoic acid 0.02g
Biotin 0.02g
Calcium pantothenate 0.02g
Nicotinic acid 0.02g
Pyridoxine·HCl 0.02g

Riboflavin 0.02g
Thiamine·HCl 0.02g
Sodium phosphate buffer (50.0mM solution, pH 7.0) 1.0L
Preparation of Vitamin Stock: Combine components. Mix thor-
oughly. Filter sterilize. Store at 4°C in the dark.
Preparation of Medium: Add components, except vitamin stock,
to distilled/deionized water and bring volume to 999.0mL. Mix thor-
oughly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C.
Aseptically add 1.0mL of sterile vitamin stock. Mix thoroughly. Asep-
tically distribute into sterile tubes or flasks.
Use: For the isolation and cultivation of Rhizobium species.
Rhizobium japonicum Agar
Composition per liter:
Agar 15.0g
Mannitol 10.0g
Yeast extract 1.0g
Soil extract 200.0mL
Soil Extract:
Composition
per liter:
African Violet soil 77.0g
Na
2
CO
3
0.2g
Preparation of Soil Extract: Add components to 1.0L of tap water.
Autoclave for 15 min at 15 psi pressure–121°C. Filter through What-
man filter paper. Bring volume to 1.0L with tap water.
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 Bradyrhizobium japoni-
cum.
Rhizobium Medium 1
Composition per liter:
Agar 15.0g
Yeast extract 10.0g
K
2
HPO
4
0.5g
MgSO
4
·7H
2
O 0.2g
NaCl 0.2g
FeCl
3
·6H
2
O 0.002g
pH 7.2 ± 0.2 at 25°C
Preparation of Medium: Add components, except agar, to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad-
just pH to 7.2. Add agar. 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 members of the Rhizobiaceae.
Rhizobium Medium 2
Composition per liter:
Agar 15.0g
Glycerol 4.6g
CaSO
4
1.3g
K
2
HPO
4
1.0g
L-Arabinose 1.0g
Yeast extract 1.0g
KNO
3
0.7g
MgSO
4
·7H
2
O 0.36g
FeCl
3
·6H
2
O 4.0mg
pH 7.2 ± 0.2 at 25°C

Preparation of Medium: Add components, except agar, to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad-
just pH to 7.2. Add agar. Gently heat and bring to boiling. Distribute
© 2010 by Taylor and Francis Group, LLC
Rhizomonas Medium 1491
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 members of the Rhizobiaceae.
Rhizobium X Medium
Composition per liter:
Agar 15.0g
Mannitol 10.0g
Yeast extract 1.0g
Soil extract 200.0mL
pH 7.2 ± 0.2 at 25°C
Soil Extract:
Composition
per 200.0mL:
African Violet soil 77.0g
Na
2
CO
3
0.2g
Preparation of Soil Extract: Add components to tap water and
bring volume to 200.0mL. Autoclave for 60 min at 15 psi pressure–
121°C. Filter through Whatman #1 filter paper.
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 Bradyrhizobium japoni-
cum, Rhizobium species, and Sinorhizobium xinjiangensis.
Rhizobium X Medium with Thiram
Composition per liter:
Agar 15.0g
Mannitol 10.0g
Yeast extract 1.0g
Soil extract 200.0mL
Thiram solution 10.0mL
pH 7.2 ± 0.2 at 25°C
Thiram Solution:
Composition
per 10.0mL:
Thiram 1.0mg
Ethanol, absolute 10.0mL
Preparation of Thiram Solution: Add thiram to 10.0mL of abso-
lute ethanol. Mix thoroughly. Filter sterilize.
Soil Extract:
Composition
per 200.0mL:
African Violet soil 77.0g
Na
2
CO
3
0.2g
Preparation of Soil Extract: Add components to tap water and
bring volume to 200.0mL.
Preparation of Medium: Add components, except thiram solution,

to distilled/deionized water and bring volume to 990.0mL. Mix thor-
oughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi
pressure–121°C. Cool to 50°C. Aseptically add 10.0mL of sterile thi-
ram solution. Pour into sterile Petri dishes or distribute into sterile
tubes.
Use: For the cultivation and maintenance of Bradyrhizobium japoni-
cum, Rhizobium species, and Sinorhizobium xinjiangensis.
Rhizoctonia Isolation Medium
Composition per liter:
Agar 20.0g
K
2
HPO
4
1.0g
KCl 0.5g
MgSO
4
·7H
2
O 0.5g
NaNO
2
0.2g
FeSO
4
·7H
2
O 0.01g
Dexon

®
solution 10.0mL
Antibiotic solution 10.0mL
Gallic acid solution 10.0mL
Antibiotic Solution:
Composition
per 10.0mL:
Chloramphenicol 0.05g
Streptomycin 0.05g
Preparation of Antibiotic Solution: Add components to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Filter
sterilize.
Dexon
®
Solution:
Composition
per 10.0mL:
Dexon
®
(Chemagro
®
) wettable powder 0.09g
Preparation of Dexon
®
Solution: Add Dexon
®
to distilled/deion-
ized water and bring volume to 10.0mL. Mix thoroughly. Filter steril-
ize.
Gallic Acid Solution:

Composition
per 10.0mL:
Gallic acid 0.4g
Preparation of Gallic Acid Solution: Add gallic acid to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Filter
sterilize.
Preparation of Medium: Add components—except Dexon
®
solu-
tion, antibiotic solution, and gallic acid solution—to distilled/deion-
ized water and bring volume to 970.0mL. 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 Dexon
®
solution, sterile an-
tibiotic solution, and sterile gallic acid solution. Mix thoroughly. Pour
into sterile Petri dishes or distribute into sterile tubes.
Use: For the isolation and cultivation of Rhizoctonia species.
Rhizomonas Medium
Composition per liter:
Noble agar 11.0g
Pancreatic digest of casein 5.0g
Glucose 2.5g
K
2
HPO
4
1.0g
MgSO
4

·7H
2
O 0.5g
KNO
3
0.5g
Ca(NO
3
)
2
·4H
2
O 0.06g
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 to 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 and maintenance of Rhizomonas suberifa-
ciens.
© 2010 by Taylor and Francis Group, LLC
1492 Rhizomonas suberifaciens Medium
Rhizomonas suberifaciens Medium
Composition per liter:
Pancreatic digest of casein 5.0g
K
2
HPO
4

·3H
2
O 1.3g
Noble agar 1.1g
KNO
3
0.5g
MgSO
4
·7H
2
O 0.5g
Ca(NO
3
)
2
·4H
2
O 60.0mg
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 to 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 Rhizomonas suberifaciens.
Rhodobacter adriaticus Medium
Composition per 1001.0mL:
NaCl 25.0g
NaHCO

3
3.0g
K
2
HPO
4
.1.0g
NH
4
Cl 1.0g
MgCl
2
·6H
2
O 0.5g
Sodium ascorbate 0.5g
CaCl
2
·2H
2
O 0.1g
Trace elements solution SLA 1.0mL
Vitamin solution 1.0mL
pH 7.0 ± 0.2 at 25°C
Trace Elements Solution SLA:
Composition
per liter:
CuCl
2
·2H

2
O 10.0g
FeCl
2
·4H
2
O 1.8g
H
3
BO
3
0.5g
CoCl
2
·6H
2
O 0.25g
ZnCl
2
0.1g
MnCl
2
·4H
2
O 70.0mg
Na
2
MoO
4
·2H

2
O 30.0mg
Na
2
SeO
3
·5H
2
O 10.0mg
NiCl
2
·6H
2
O 10.0mg
Preparation of Trace Elements Solution SLA: Add compo-
nents to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Bring pH to 2.0–3.0.
Vitamin Solution:
Composition
per liter:
Nicotinamide 35.0mg
Thiamine·HCl 30.0mg
p-Aminobenzoic acid 20.0mg
Pyridoxal·HCl 10.0mg
Calcium
DL-pantothenate 10.0mg
Biotin 10.0mg
Vitamin B
12
5.0mg

Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 1.0L. Mix thoroughly. Filter ster-
ilize.
Preparation of Medium: Add components, except vitamin solu-
tion, to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically
add 1.0mL of sterile vitamin solution. Mix thoroughly. Aseptically dis-
tribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Rhodobacter adraiticus.
Rhodobacter changlensis Medium
(DSMZ Medium 1197)
Composition per 1001.0mL:
Yeast extract 0.4g
Sodium pyruvate 3.0g
NH
4
Cl 0.6g
MgCl
2
·6H
2
O 0.5g
KH
2
PO
4
0.5g
NaCl 0.4g
NH
4

Cl 0.6g
CaC
l
2
·2H
2
O 0.05g
Trace elements solution SL-7 1.0mL
Vitamin solution 1.0mL
pH 7.2 ± 0.2 at 25°C
Trace Elements Solution SL-7:
Composition
per liter:
CoCl
2
·6H
2
O 200.0mg
MnCl
2
·4H
2
O 100.0mg
ZnCl
2
70.0mg
H
3
BO
3

60.0mg
Na
2
MoO
4
·2H
2
O 40.0mg
CuCl
2
·2H
2
O 20.0mg
NiCl
2
·6H
2
O 20.0mg
HCl (25%) 1.0mL
Preparation of Trace Elements Solution SL-7: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Vitamin Solution:
Composition
per 100.0mL:
Vitamin B
12
20.0mg
Preparation of Vitamin Solution: Add vitamin B
12
to distilled/

deionized water and bring volume to 100.0mL. Mix thoroughly. Filter
sterilize.
Preparation of Medium: Add components, except vitamin solu-
tion, to distilled/deionized water and bring volume to 1.0L. Mix thor-
oughly. Adjust pH to 7.2. Gently heat and bring to boiling. Autoclave
for 15 min at 15 psi pressure–121°C. Cool to room temperature. Asep-
tically add vitamin solution. Mix thoroughly. Aseptically distribute
into culture vessels.
Use: For the cultivation of Rhodobacter changlensis.
Rhodobacter Medium
(LMG Medium 80)
Composition per liter:
Yeast extract 1.0g
Disodium succinate 1.0g
KH
2
PO
4
0.5g
MgSO
4
·7H2O 0.4g
NaCl 0.4g
NH
4
Cl 0.4g
CaCl
2
·2H
2

O 50.0mg
Ferric citrate solution 5.0mL
Trace elements solution 1.0mL
Ethanol 0.5mL
pH 5.8 ± 0.2 at 25°C
© 2010 by Taylor and Francis Group, LLC
Rhodobacter veldkampii Medium 1493
Ferric Citrate Solution :
Composition
per 100.0mL:
Ferric citrate 0.1g
Preparation of Ferric Citrate Solution: Add ferric citrate to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Trace Elements Solution:
Composition
per liter:
H
3
BO
3
0.3g
CoCl
2
·6H
2
O 0.2g
ZnSO
4
·7H
2

O 0.1g
Na
2
MoO
4
·2H
2
O 30.0mg
MnCl
2
·4H
2
O 30.0mg
NiCl
2
·6H
2
O 20.0mg
CuCl
2
·2H
2
O 10.0mg
Preparation of Trace Elements Solution: Add components to
distilled/deionized 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 40 mL me-
dium into 50 mL screw-capped bottles. Flush each bottle for 1 to 2 min
with nitrogen gas and then close immediately with rubber septa and
screw caps. Autoclave for 15 min at 15 psi pressure–121°C. Sterile sy-

ringes are used to inoculate and remove the samples. Incubate in light
using a tungsten lamp.
Use: For the cultivation of Rhodobacter capsulatus, Rhodobacter spha-
eroides, and Rhodospirillum rubrum.
Rhodobacter veldkampii Medium
(DSMZ Medium 867)
Composition per 2780.0mL:
Solution 1 1540.0mL
Solution 3 1000.0mL
Solution 4 120.0mL
Solution 5 120.0mL
pH 4.0 ± 0.1 at 25°C
Solution 1:
Composition
per 2500.0mL:
CaCl
2
2.0g
Preparation of Solution 1: Add CaCl
2
to distilled/deionized water
and bring volume to 2.5L. Mix thoroughly.
Solution 3:
Composition
per liter:
NaHCO
3
4.5g
Solution 2 100.0mL
Preparation of Solution 3: Add NaHCO

3
to distilled/deionized
water and bring volume to 900.0mL. Mix thoroughly. Sparge with gas-
eous CO
2
for at least 30 min. Add 100.0mL solution 2. Immediately fil-
ter sterilize using CO
2
pressure to push the liquid through (no suction).
Solution 2:
Composition
per 100.0mL:
Sodium ascorbate 2.4g
KH
2
PO
4
1.0g
KCl 1.0g
NH
4
Cl 0.8g
MgCl
2
·6H
2
O 0.8g
Heavy metal solution 50.0mL
Vitamin solution 15.0mL
Vitamin B

12
solution 3.0mL
Preparation of Solution 2: Add components to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly.
Heavy Metal Solution:
Composition
per liter:
EDTA 1.50g
FeSO
4
·7H
2
O 0.2g
ZnSO
4
·7H
2
O 0.1g
MnCl
2
·7H
2
O 0.02g
Modified Hoagland trace elements solution 6.0mL
Preparation of Heavy Metal Solution: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Modified Hoagland Trace Elements Solution:
Composition
per 3.6L:
H

3
BO
3
11.0g
MnCl
2
·4H
2
O 7.0g
ZnCl
2
1.0g
CuCl
2
1.0g
NiCl
2
1.0g
CoCl
2
1.0g
AlCl
3
1.0g
KI 1.0g
KBr 0.5g
LiCl 0.5g
SnCl
2
·2H

2
O 0.5g
BaCl
2
0.5g
Na
2
MoO
4
0.5g
Na
2
SeO
3
0.5g
NaVO
3
·H
2
O 0.1g
Preparation of Modified Hoagland Trace Elements Solu-
tion:
Add components sequentially to distilled/deionized water and
bring final volume to 3.6L. Mix thoroughly after adding each compo-
nent until dissolved. Adjust pH to just below 7.0. Adjust the final pH
to 3–4. The flaky yellow precipitate which is formed after mixing
transforms after standing for one or a few days into a very fine white
precipitate. Mix thoroughly before use.
Vitamin B
12

Solution:
Composition
per 3.0mL:
Vitamin B
12
(cyanocobalamine) 2.0mg
Preparation of Vitamin B
12
Solution: Add vitamin B
12
to dis-
tilled/deionized water and bring volume to 3.0mL. Mix thoroughly.
Vitamin Solution:
Composition
per 100.0mL:
Pyridoxamine·2HCl 5.0mg
Nicotinic acid 2.0mg
Thiamine 1.0mg
Pantothenic acid 0.5mg
Biotin 0.2mg
p-Aminobenzoic acid 0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 100.0mL. Mix thoroughly. Sparge
with 100% CO
2
for 30 min.
Solution 4:
Composition
per 200.0mL:
Na

2
S·9H
2
O 3.0g
Preparation of Solution 4: Add Na
2
S·9H
2
O to distilled/deionized
water in a flask with a magnetic stirrer and bring volume to 200.0mL.
Mix thoroughly. Autoclave under 100% N
2
for 15 min at 15 psi pres-
sure–121°C. Cool to room temperature. Partially neutralize the steril-
© 2010 by Taylor and Francis Group, LLC
1494 Rhodobacter veldkampii Medium
ized solution by adding, on a magnetic stirrer, drop by drop, 2.0mL
sterile 2M H
2
SO
4
.
Solution 5:
Composition
per 200.0mL:
Na-acetate 6.0g
Preparation of Solution 5: Add Na-acetate to distilled/deionized
water and bring volume to 200.0mL. Mix thoroughly. Sparge with
100% N
2

for 5 min. Autoclave under 100% N
2
for 15 min at 15 psi
pressure–121°C. Cool to room temperature.
Preparation of Medium: Distribute solution 1 in 77.0mL amounts
into 20 127mL screw-capped Boston round bottles. Autoclave for 15
min at 15 psi pressure–121°C. Cool to room temperature. Aseptically
add 50.0mL sterile solution 3 to each of the 20 127mL bottles contain-
ing 77.0mL of sterile solution 1 so that the solution completely fills the
bottle. Mix thoroughly. Remove 6.0mL of the medium from the com-
pletely filled bottles. Add 6.0mL of neutralized solution 4 so that the
bottles are again completely filled. Mix thoroughly. Remove 6.0mL of
the medium from the completely filled bottles. Add 6.0mL of solution
5 so that the bottles are again completely filled. Mix thoroughly. Adjust
pH to 4.0. Allow the bottles to stand overnight to develop a hazy, white
precipitate before inoculating. Mix the solution thoroughly before use.
To inoculate, remove 6.0mL of completed medium and replace it with
an equal volume of inoculum. Grow cultures under tungsten light.
Use: For the cultivation of Rhodobacter veldkampii.
Rhodobacter veldkampii Medium
Composition per 127.0mL:
Solution 1 76.2mL
Solution 2 + Solution 3 44.8mL
Solution 4 6.0mL
Solution 1:
Composition
per 2.5L:
CaCl
2
2.0g

Preparation of Solution 1: Add CaCl
2
to distilled/deionized water
and bring volume to 2.5L. Distribute in 80.0mL volumes into 127.0mL
screw-capped bottles. Autoclave for 15 min at 15 psi pressure–121°C.
Solution 2:
Composition
per 100.0mL:
Sodium ascorbate 2.4g
Sodium acetate 1.0g
KC1 1.0g
KH
2
PO
4
1.0g
MgCl
2
·6H
2
O 0.8g
NH
4
Cl 0.8g
Heavy metal solution 50.0mL
Vitamin solution 15.0mL
Vitamin B
12
solution 3.0mL
Preparation of Solution 2: Add components to distilled/deionized

water and bring volume to 100.0mL. Mix thoroughly.
Heavy Metal Solution:
Composition
per liter:
Ethylenediamine tetraacetate (EDTA) 1.5g
FeSO
4
·7H
2
O 0.2g
ZnSO
4
·7H
2
O 0.1g
MnCl
2
·4H
2
O 0.02g
Modified Hoagland trace elements solution 6.0mL
Preparation of Heavy Metal Solution: Dissolve EDTA in 800.0mL
of distilled/deionized water. Add remaining components. Bring volume
to 1.0L with distilled/deionized water. Mix thoroughly.
Modified Hoagland Trace Elements Solution:
Composition
per 3.6L:
H
3
BO

3
11.0g
MnCl
2
·4H
2
O 7.0g
AlCl
3
1.0g
CoCl
2
1.0g
CuCl
2
1.0g
KI 1.0g
NiCl
2
1.0g
ZnCl
2
1.0g
BaCl
2
0.5g
KBr 0.5g
LiCl 0.5g
Na
2

MoO
4
0.5g
SeCl
4
0.5g
SnCl
2
·2H
2
O 0.5g
NaVO
3
·H
2
O 0.1g
Preparation of Modified Hoagland Trace Elements Solu-
tion:
Prepare each component as a separate solution. Dissolve each
salt in approximately 100.0mL of distilled/deionized water. Adjust the
pH of each solution to below 7.0. Combine all the salt solutions and
bring the volume to 3.6L with distilled/deionized water. Adjust the pH
to 3–4. A yellow precipitate may form after mixing. After a few days,
it will turn into a fine white precipitate. Mix the solution thoroughly be-
fore using.
Vitamin Solution:
Composition per 100.0mL:
Pyridoxamine·2HCl 5.0mg
Nicotinic acid 2.0mg
Thiamine 1.0mg

Pantothenic acid 0.5mg
Biotin 0.2mg
p-Aminobenzoic acid 0.1mg
Preparation of Vitamin Solution: Add components to distilled/
deionized water and bring volume to 100.0mL. Mix thoroughly.
Vitamin B
12
Solution:
Composition
per 100.0mL:
Vitamin B
12
(cyanocobalamin) 2.0mg
Preparation of Vitamin B
12
Solution: Add vitamin B
12
to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Solution 3:
Composition
per 900.0mL:
NaHCO
3
4.5g
Preparation of Solution 3: Add NaHCO
3
to distilled/deionized
water and bring volume to 900.0mL. Mix thoroughly. Bubble 100%
CO

2
through the solution for 30 min. After CO
2
saturation of solution
3, add solution 2 and immediately filter the mixture through a Seitz fil-
ter (or a Millipore) using positive CO
2
pressure to push the liquid
through.
Solution 4:
Composition per 200.0mL:
Na
2
S·9H
2
O 3.0g
Preparation of Solution 4: Add Na
2
S·9H
2
O to distilled/deionized
water and bring volume to 200.0mL. Add a magnetic stir bar to the flask.
Autoclave for 15 min at 15 psi pressure–121°C. On a magnetic stirrer,
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