Handbook of Microbiological Media, Fourth Edition part 150 potx

Preparation of Medium: Add components, except horse blood and cephalexin solution, to distilled/deionized water and bring volume to 890.0mL.. Preparation of Medium: Add components, excep

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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:

(NH4)2SO4 9.0g

NaCl 9.0g

K2HPO4 4.5g

KH2PO4 4.5g

Na2CO3 4.0g

EDTA (ethylenediamine tetraacetic acid) 3.8g

Dithiothreitol 2.0g

MgSO4·7H2O 1.8g

pH 8.0 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Filter sterilize

Asep-tically distribute into sterile tubes with rubber stoppers

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

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

Na2CO3 (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:

K2HPO4 0.6g

Preparation of Stock Mineral Salt Solution No 1: Add

K2HPO4 to distilled/deionized water and bring volume to 100.0mL

Mix thoroughly

Stock Mineral Salt Solution No 2:

NaCl 1.2g

(NH4)2SO4 1.2g

K2HPO4 0.6g

MgSO4·7H2O 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

water and bring volume to 1.0L Mix thoroughly Filter sterilize

Asep-tically distribute into sterile tubes with rubber stoppers

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)

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:

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

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:

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 sterster-ile cephalexin solution Mix thoroughly

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Aseptical-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)

Base medium 500.0mL

Growth medium 500.0mL

pH 5.0 ± 0.2 at 25°C

Base Medium:

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:

Glucose 40.0g

Agar 20.0g

Yeast extract 10.0g

Peptone 10.0g

Na2HPO4·2H2O 3.38g

Citric acid·2H2O 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

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·H2O 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)

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·H2O 0.5g

pH 6.8 ± 0.2 at 25°C

Use: For the cultivation and maintenance of Bifidobacterium

meryci-cum.

Reinforced Clostridial HiVeg Agar

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

Use: For the cultivation and enumeration of clostridia and other anaer-obes

Reinforced Clostridial HiVeg Broth

Plant extract 10.0g Plant hydrolysate 10.0g Glucose 5.0g NaCl 5.0g Sodium acetate 3.0g Yeast extract 3.0g

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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

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

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

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

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

Casamino acids 15.0g

Agar 13.5g

Beef extract 10.0g

NaCl 5.0g

Glucose 5.0g

Yeast extract 3.0g

Sodium acetate 3.0g

Soluble starch 1.0g

pH 6.8 ± 0.2 at 25°C

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

Agar 13.5g

Beef extract 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·H2O 0.5g

pH 6.8 ± 0.2 at 25°C

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·H2O 0.5g

pH 6.8 ± 0.2 at 25°C

Use: For the cultivation of Clostridium saccharobutylicum,

Clostrid-ium frigidicarnis, and Mitsuokella jalaludinii.

Reinforced Clostridial Medium with Sodium Lactate

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·H2O 0.5g Agar 0.5g Sodium lactate (60% solution) 15.0mL

pH 6.8 ± 0.2 at 25°C

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

Agar 13.5g Beef extract 10.0g Pancreatic digest of casein 10.0g NaCl 5.0g

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1488 Renibacterium KDM2 Medium

Glucose 5.0g

Uric acid 3.0g

Yeast extract 3.0g

Sodium acetate 3.0g

Soluble starch 1.0g

pH 6.8 ± 0.2 at 25°C

water and bring volume to 1.0L Mix thoroughly Distribute into tubes

Use: For the cultivation of Clostridium acidurici.

Renibacterium KDM2 Medium

Agar 15.0g

Peptone 10.0g

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

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

MgSO4·7H2O 0.2g

MnSO4·2H2O 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:

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

Yeast extract 0.05g Peptone 0.05g (NH4)2SO4 0.05g L-Cysteine·HCl·H2O 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:

NaHCO3 10.0g NaCl 2.0g

K2HPO4 1.0g

KH2PO4 1.0g CaCl2, anhydrous 0.2g MgSO4 0.2g

Preparation of Salts Solution: Add CaCl2 and MgSO4 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% N2 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

Na2CO3 solution 20.0mL L-Cysteine·HCl·H2O solution 5.0mL Solution III 0.3mL

pH 6.6 ± 0.2 at 25°C

Mineral Solution I:

K2HPO4 0.3g

Preparation of Mineral Solution I: Add K2HPO4 to distilled/de-ionized water and bring volume to 100.0mL Mix thoroughly

Mineral Solution II:

(NH4)2SO4 0.6g NaCl 0.6g

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Rhizobium BIII Defined Agar 1489

KH2PO4 0.3g

MgSO4 0.06g

CaCl2 0.06g

Preparation of Mineral Solution II: Add components to

dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly

Solution III:

Resazurin 0.01g

Preparation of Solution III: Add resazurin to 10.0mL of distilled/

deionized water Mix thoroughly

Solution IV:

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:

Preparation of L -Cysteine·HCl·H 2 O Solution: Add

L-cyste-ine·HCl·H2O to distilled/deionized water and bring volume to

100.0mL Mix thoroughly Filter sterilize

Na 2 CO 3 Solution:

Na2CO3 6.0g

Preparation of Na 2 CO 3 Solution: Add Na2CO3 to

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

sterilize

Rumen Fluid:

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 CO2 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%

CO2 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% CO2 to eliminate O2 Aseptically add 5.0mL of

sterile L-cysteine·HCl·H2O solution and 20.0mL of sterile Na2CO3

so-lution Mix thoroughly Aseptically and anaerobically distribute into

tubes under 100% CO2 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

Rhamnose 10.0g

Yeast extract 3.0g

K2HPO4 2.9g

KH2PO4 2.1g

NH4·Cl 2.0g

MgSO4·7H2O 0.4g NaCl 30.0mg CaCl2 3.0mg FeSO4·7H2O 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

Use: For the cultivation of Rhodococcus chlorophenolicus.

Rhizobium Agar

(LMG 201)

Agar 20.0g Mannitol 10.0g Yeast extract 1.0g Sodium glutamate 0.5g

KH2PO4 0.5g MgSO4·7H2O 0.1g CaCl2·2H2O 40.0mg FeCl3 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, RhizoRhizo-bium leguminosarum, RhizoRhizo-bium loti, RhizoRhizo-bium meliloti, and Rhizobium tropici.

Rhizobium BIII Defined Agar

Agar 13.0g Mannitol 10.0g Sodium glutamate 1.1g

K2HPO4 0.23g MgSO4·7H2O 0.1g Trace elements stock 1.0mL Vitamin stock 1.0mL

pH 7.0 ± 0.2 at 25°C

Trace Elements Stock:

Nitrilotriacetic acid 7.0g CaCl2·2H2O 6.62g

H3BO3 0.145g FeSO4·7H2O 0.125g

Na2MoO4 0.125g ZnSO4·7H2O 0.108g CoSO4·7H2O 0.07g CuSO4·5H2O 5.0mg MnCl2·4H2O 4.3mg

Preparation of Trace Elements Stock: Add components to 500.0mL of distilled/deionized water in the order: CaCl2·2H2O, H3BO3, FeSO4·7H2O, CoSO4·7H2O, CuSO4·5H2O, MnCl2·4H2O, ZnSO4·7H2O, and

Na2MoO4 Adjust pH to 5.0 Add nitrilotriacetic acid Bring volume to 1.0L with distilled/deionized water

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1490 Rhizobium BIII Defined Broth

Vitamin Stock:

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

Mannitol 10.0g

Sodium glutamate 1.1g

K2HPO4 0.23g

MgSO4·7H2O 0.1g

Trace elements stock 1.0mL

Vitamin stock 1.0mL

pH 7.0 ± 0.2 at 25°C

Trace Elements Stock:

Nitrilotriacetic acid 7.0g

CaCl2·2H2O 6.62g

H3BO3 0.145g

FeSO4·7H2O 0.125g

Na2MoO4 0.125g

ZnSO4·7H2O 0.108g

CoSO4·7H2O 0.07g

CuSO4·5H2O 5.0mg

MnCl2·4H2O 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:

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

Agar 15.0g Mannitol 10.0g Yeast extract 1.0g Soil extract 200.0mL

Soil Extract:

African Violet soil 77.0g

Na2CO3 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

Use: For the cultivation and maintenance ofBradyrhizobium japoni-cum.

Rhizobium Medium 1

Agar 15.0g Yeast extract 10.0g

K2HPO4 0.5g MgSO4·7H2O 0.2g NaCl 0.2g FeCl3·6H2O 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

Agar 15.0g Glycerol 4.6g CaSO4 1.3g

K2HPO4 1.0g L-Arabinose 1.0g Yeast extract 1.0g KNO3 0.7g MgSO4·7H2O 0.36g FeCl3·6H2O 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

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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:

Na2CO3 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

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:

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:

Na2CO3 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

Agar 20.0g

K2HPO4 1.0g KCl 0.5g MgSO4·7H2O 0.5g NaNO2 0.2g FeSO4·7H2O 0.01g Dexon® solution 10.0mL Antibiotic solution 10.0mL Gallic acid solution 10.0mL

Antibiotic Solution:

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:

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:

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 solusolu-tion, 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

Noble agar 11.0g Pancreatic digest of casein 5.0g Glucose 2.5g

K2HPO4 1.0g MgSO4·7H2O 0.5g KNO3 0.5g Ca(NO3)2·4H2O 0.06g

pH 7.2 ± 0.2 at 25°C

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.

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1492 Rhizomonas suberifaciens Medium

Rhizomonas suberifaciens Medium

K2HPO4·3H2O 1.3g

Noble agar 1.1g

KNO3 0.5g

MgSO4·7H2O 0.5g

Ca(NO3)2·4H2O 60.0mg

pH 7.2 ± 0.2 at 25°C

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

NaCl 25.0g

NaHCO3 3.0g

K2HPO4 1.0g

NH4Cl 1.0g

MgCl2·6H2O 0.5g

Sodium ascorbate 0.5g

CaCl2·2H2O 0.1g

Trace elements solution SLA 1.0mL

Vitamin solution 1.0mL

pH 7.0 ± 0.2 at 25°C

Trace Elements Solution SLA:

CuCl2·2H2O 10.0g

FeCl2·4H2O 1.8g

H3BO3 0.5g

CoCl2·6H2O 0.25g

ZnCl2 0.1g

MnCl2·4H2O 70.0mg

Na2MoO4·2H2O 30.0mg

Na2SeO3·5H2O 10.0mg

NiCl2·6H2O 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:

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 B12 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)

Yeast extract 0.4g Sodium pyruvate 3.0g

NH4Cl 0.6g MgCl2·6H2O 0.5g

KH2PO4 0.5g NaCl 0.4g

NH4Cl 0.6g CaCl2·2H2O 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:

CoCl2·6H2O 200.0mg MnCl2·4H2O 100.0mg ZnCl2 70.0mg

H3BO3 60.0mg

Na2MoO4·2H2O 40.0mg CuCl2·2H2O 20.0mg NiCl2·6H2O 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 B12 20.0mg

Preparation of Vitamin Solution: Add vitamin B12 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 AsepAsep-tically distribute into culture vessels

Use: For the cultivation of Rhodobacter changlensis.

Rhodobacter Medium

(LMG Medium 80)

Yeast extract 1.0g Disodium succinate 1.0g

KH2PO4 0.5g MgSO4·7H2O 0.4g NaCl 0.4g

NH4Cl 0.4g CaCl2·2H2O 50.0mg Ferric citrate solution 5.0mL Trace elements solution 1.0mL Ethanol 0.5mL

pH 5.8 ± 0.2 at 25°C

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Rhodobacter veldkampii Medium 1493

Ferric Citrate Solution :

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:

H3BO3 0.3g

CoCl2·6H2O 0.2g

ZnSO4·7H2O 0.1g

Na2MoO4·2H2O 30.0mg

MnCl2·4H2O 30.0mg

NiCl2·6H2O 20.0mg

CuCl2·2H2O 10.0mg

Preparation of Trace Elements Solution: Add components to

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

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)

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:

CaCl2 2.0g

Preparation of Solution 1: Add CaCl2 to distilled/deionized water

and bring volume to 2.5L Mix thoroughly

Solution 3:

NaHCO3 4.5g

Solution 2 100.0mL

Preparation of Solution 3: Add NaHCO3 to distilled/deionized

water and bring volume to 900.0mL Mix thoroughly Sparge with

gas-eous CO2 for at least 30 min Add 100.0mL solution 2 Immediately

fil-ter sfil-terilize using CO2 pressure to push the liquid through (no suction)

Solution 2:

KH2PO4 1.0g

KCl 1.0g

NH4Cl 0.8g

MgCl2·6H2O 0.8g

Heavy metal solution 50.0mL

Vitamin B12 solution 3.0mL

Preparation of Solution 2: Add components to distilled/deionized water and bring volume to 100.0mL Mix thoroughly

Heavy Metal Solution:

EDTA 1.50g FeSO4·7H2O 0.2g ZnSO4·7H2O 0.1g MnCl2·7H2O 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:

H3BO3 11.0g MnCl2·4H2O 7.0g ZnCl2 1.0g CuCl2 1.0g NiCl2 1.0g CoCl2 1.0g AlCl3 1.0g

KI 1.0g KBr 0.5g LiCl 0.5g SnCl2·2H2O 0.5g BaCl2 0.5g

Na2MoO4 0.5g

Na2SeO3 0.5g NaVO3·H2O 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:

Vitamin B12 (cyanocobalamine) 2.0mg

Preparation of Vitamin B 12 Solution: Add vitamin B12 to dis-tilled/deionized water and bring volume to 3.0mL Mix thoroughly

Pyridoxamine·2HCl 5.0mg Nicotinic acid 2.0mg Thiamine 1.0mg Pantothenic acid 0.5mg Biotin 0.2mg

Preparation of Vitamin Solution: Add components to distilled/ deionized water and bring volume to 100.0mL Mix thoroughly Sparge with 100% CO2 for 30 min

Solution 4:

Na2S·9H2O 3.0g

Preparation of Solution 4: Add Na2S·9H2O to distilled/deionized water in a flask with a magnetic stirrer and bring volume to 200.0mL Mix thoroughly Autoclave under 100% N2 for 15 min at 15 psi pres-sure–121°C Cool to room temperature Partially neutralize the

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steril-1494 Rhodobacter veldkampii Medium

ized solution by adding, on a magnetic stirrer, drop by drop, 2.0mL

sterile 2M H2SO4

Solution 5:

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% N2 for 5 min Autoclave under 100% N2 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

Solution 1 76.2mL

Solution 2 + Solution 3 44.8mL

Solution 4 6.0mL

Solution 1:

CaCl2 2.0g

Preparation of Solution 1: Add CaCl2 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:

Sodium acetate 1.0g

KC1 1.0g

KH2PO4 1.0g

MgCl2·6H2O 0.8g

NH4Cl 0.8g

Heavy metal solution 50.0mL

Vitamin B12 solution 3.0mL

Preparation of Solution 2: Add components to distilled/deionized

water and bring volume to 100.0mL Mix thoroughly

Heavy Metal Solution:

Ethylenediamine tetraacetate (EDTA) 1.5g

FeSO4·7H2O 0.2g

ZnSO4·7H2O 0.1g

MnCl2·4H2O 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:

H3BO3 11.0g MnCl2·4H2O 7.0g AlCl3 1.0g CoCl2 1.0g CuCl2 1.0g

KI 1.0g NiCl2 1.0g ZnCl2 1.0g BaCl2 0.5g KBr 0.5g LiCl 0.5g

Na2MoO4 0.5g SeCl4 0.5g SnCl2·2H2O 0.5g NaVO3·H2O 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

Pyridoxamine·2HCl 5.0mg Nicotinic acid 2.0mg Thiamine 1.0mg Pantothenic acid 0.5mg Biotin 0.2mg

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

Vitamin B 12 Solution:

Vitamin B12 (cyanocobalamin) 2.0mg

Preparation of Vitamin B 12 Solution: Add vitamin B12 to dis-tilled/deionized water and bring volume to 100.0mL Mix thoroughly

Solution 3:

NaHCO3 4.5g

Preparation of Solution 3: Add NaHCO3 to distilled/deionized water and bring volume to 900.0mL Mix thoroughly Bubble 100%

CO2 through the solution for 30 min After CO2 saturation of solution

3, add solution 2 and immediately filter the mixture through a Seitz fil-ter (or a Millipore) using positive CO2 pressure to push the liquid through

Solution 4:

Na2S·9H2O 3.0g

Preparation of Solution 4: Add Na2S·9H2O 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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