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R2a agar

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R2A agar is a general-purpose culture medium used for the isolation and enumeration of heterotrophic bacteria from water, food, and other environmental samples. It is designed to support the growth of slow-growing, nutrient-sensitive microorganisms. The medium provides a balanced nutrient composition and a low-nutrient environment to encourage the growth of a wide range of bacterial species.

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Lab products found in correlation

Magnesium sulfate heptahydrate, by Merck Group (2 mentions) Casamino acid, by BD (2 mentions) Kanamycin sulfate, by AppliChem (2 mentions) Yeast extract, by Thermo Fisher Scientific (2 mentions) Starch, by Merck Group (2 mentions) K2hpo4, by AppliChem (2 mentions) Proteose peptone no 3, by BD (2 mentions) D glucose monohydrate, by Merck Group (2 mentions) Rifampicin, by Merck Group (2 mentions) Sodium pyruvate, by Merck Group (2 mentions) Indole 3 acetic acid, by Merck Group (1 mentions) Benzyladenine, by Merck Group (1 mentions) Murashige and skoog basal salt mixture, by Merck Group (1 mentions) Tryptone, by AppliChem (1 mentions) Cycloheximide (chx), by Carl Roth (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Lactose broth, by Merck Group (1 mentions) Ec broth, by Merck Group (1 mentions) Brilliant green, by Merck Group (1 mentions) Sodium hydroxide (naoh), by Merck Group (1 mentions)

20 protocols using r2a agar

1

Detecting Bacterial Endospores in Polymers

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Components of the polymer materials were prepared as shown in Table 1, and diluted in the appropriate solvent (0.1–0.5 g/mL, depending on the solubility of the precursors). The method was optimized for detection of cultivable bacterial endospores by including a heat activation step before pour-plating 1 mL aliquots on soybean casein digest agar or R2A agar (Sigma-Aldrich; Steinheim, Germany). Plates were incubated 5–7 d at 30–35°C, followed by 5–7 d at 55–60°C to detect mesophilic and thermophilic spore-formers, respectively. In total, 45–80 cm3 of each material was investigated. If colonies were detected, they were isolated and visualized microscopically after Gram-staining.
Bauermeister A., Mahnert A., Auerbach A., Böker A., Flier N., Weber C., Probst A.J., Moissl-Eichinger C, & Haberer K. (2014). Quantification of Encapsulated Bioburden in Spacecraft Polymer Materials by Cultivation-Dependent and Molecular Methods. PLoS ONE, 9(4), e94265.
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2

Quantifying Agrobacterium Populations in Growing Media

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Growing medium was analysed within 48 hours of sample collection. Five grams of the fresh growing medium were mixed with 45 ml of 0.85% NaCl39 and homogenized for 2 minutes, using a Stomacher80 blender (Stomacher, Seward, Worthing, UK). This suspension was used for the determination of the total cell and Agrobacterium sp. count on each medium. For the total cell count, the suspension was plated on R2A agar (Sigma Aldrich, Diegem, Belgium) with cycloheximide (200 mg/l). Agrobacteria colonies were selected and identified following Shams, et al.40 . A. rhizogenes was isolated using 2E-Te containing erythritol and 320 mg/l K2TeO3 with cycloheximide. After 5 days of incubation at 28 °C, colony forming units (CFU) were counted for both R2A and 2E-TE medium. The calculation of the CFU was following the procedures outlined by Sutton41 , where the detection limit was equal to 1 CFU at the lowest dilution.
Grunert O., Hernandez-Sanabria E., Vilchez-Vargas R., Jauregui R., Pieper D.H., Perneel M., Van Labeke M.C., Reheul D, & Boon N. (2016). Mineral and organic growing media have distinct community structure, stability and functionality in soilless culture systems. Scientific Reports, 6, 18837.
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3

Quantifying Antibiotic-Resistant Bacteria

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R2A agar (as described in 2.2 with 15 g agar (Sigma-Aldrich, United States)) and E. coli ChromoSelect Agar B (Sigma-Aldrich, United States) plates were inoculated to count heterotrophic bacteria and E. coli, respectively. CIP-resistant bacteria and CIP-resistant E. coli were counted on R2A agar supplemented with 2 mg/L of CIP and on ChromoSelect Agar B plates supplemented with 0.25 mg/L of CIP, respectively. R2A agar plates were incubated for 48 h at 31°C, and ChromoSelect Agar B plates overnight at 45°C. Percentage of resistant heterotrophic bacteria and E. coli were then calculated, and statistical significance assessed using ANOVA and Dunnett’s test from DescTools package at a 95% family-wise confidence level in RStudio (Version 2023.6.0).
Naudin S.A., Ferran A.A., Imazaki P.H., Arpaillange N., Marcuzzo C., Vienne M., Demmou S., Bousquet-Mélou A., Ramon-Portugal F., Lacroix M.Z., Hoede C., Barret M., Dupouy V, & Bibbal D. (2024). Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota. Frontiers in Microbiology, 15, 1377047.
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4

Bacterial Strains Characterization for Colonization

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The two bacterial strains Sphingomonas Leaf257 and Rhizobium Leaf68 were selected from the At-LSPHERE collection29 (link) based on their colonization capacity and previous data collected27 (link),55 (link). Both strains were grown on R-2A-agar (Sigma-Aldrich, Buchs, Switzerland) supplemented with 0.5% v/v methanol (R2A + M) for 3 days at 22 °C prior usage.
Hemmerle L., Maier B.A., Bortfeld-Miller M., Ryback B., Gäbelein C.G., Ackermann M, & Vorholt J.A. (2022). Dynamic character displacement among a pair of bacterial phyllosphere commensals in situ. Nature Communications, 13, 2836.
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5

Naphthalene Biodegradation Protocol

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Naphthalene (99.9% purity), was supplied by MERCK. The physicochemical properties of naphthalene used in this study are listed in Table 1. R2A agar, Murashige and Skoog Basal Salt Mixture (MS salts), Benzyl-adenine and Indole-3-acetic acid (IAA) were purchased from Sigma–Aldrich (USA).

Physico-chemical properties of naphthalene

Molecular weight128.19
FormulaC10H8
Boiling point218C
Melting point80.5C
Solubility (at 20°C)32 mg/l
Specific gravity1.145
Henry’s law constant20 atmm3 water/m3 air
Kow2800
A minimal medium (MM) was composed of (L): KH2PO4 1.0 g, NaCl 5 g (NH4) 2SO4 0.3 g, MgSO4 · 7H2O 0.3 g, CaCl2 20 mg, Sugar (2.5 g/L). MM also contained trace elements as follows: (L): ZnSO4 5.0 g, FeCl3 2.3 g, MnSO4 5.0 g, and (NH4) 6Mo7O24 1.0 g. Solid MM plate was composed of (L MM): 20 g agar. Solid LB plate was composed of (L): 5 g NaCl, 10 g peptone, 5 g yeast extract, 20 g agar. Flasks containing the medium were sterilized by autoclaving at 121°C for 20 min. The pH of the medium was between 6.8 and 7.2. All chemicals used were of analytical grade and all reagents were used as supplied.
Karimi B., Habibi M, & Esvand M. (2015). Biodegradation of naphthalene using Pseudomonas aeruginosa by up flow anoxic–aerobic continuous flow combined bioreactor. Journal of Environmental Health Science and Engineering, 13, 26.
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6

Cultivation Protocols for Diverse Bacterial Isolates

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Unless stated otherwise, At-SPHERE isolates were
cultivated at room temperature on R-2A agar (Sigma-Aldrich) or at 28°C in
R-2A broth (0.5 g yeast extract (Oxoid), 0.5 g Proteose peptone No. 3 (Becton,
Dickinson and Company), 0.5 g Casamino acids (Becton, Dickinson and Company),
0.5 g D-glucose monohydrate (Sigma-Aldrich), 0.5 g starch (from potato, Fluka),
0.3 g sodium pyruvate (Sigma-Aldrich), 0.3 g K2HPO4(AppliChem) and 0.05 g magnesium sulfate heptahydrate (Sigma-Aldrich) dissolved
in 1 L deionized water), both supplemented after sterilization with 0.5% (v/v)
methanol (R-2A+M). Minimal medium was prepared as described previously66 (link) and supplemented with 5 mM
maltose (Fluka). For Leaf374 cultivation, minimal medium agar was supplemented
with vitamins (500 μg L-1 D-pantothenic acid hemi calcium
salt, 100 μg L-1 biotin, 400 μg L-1riboflavin, 400 μg L-1 thiamine HCl, 200 μg
L-1 pyridoxal HCl 150 μg L-1 p-amino benzoic
acid, 200 μg L-1 cobalamin, 50 μg L-1 lipoic
acid, 150 μg L-1 nicotinic acid and 100 μg
L-1 folic acid). For selective growth of Leaf272, R-2A+M was
supplemented with 50 μg mL-1 rifampicin (Sigma-Aldrich), since
this strain has a natural resistance towards this antibiotic.
E. coli BL21 (DE3) gold was cultivated in LB-Lennox at
37°C supplemented with 50 μg mL-1 kanamycin sulfate
(AppliChem).
Schäfer M., Vogel C.M., Bortfeld-Miller M., Mittelviefhaus M, & Vorholt J.A. (2022). Mapping phyllosphere microbiota interactions in planta to establish genotype-phenotype relationships. Nature microbiology, 7(6), 856-867.
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7

Bacterial Strain Selection for Phyllosphere

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Bacterial strains were selected from the At-LSPHERE collection20 (link) based on their ability to
reach high cell-numbers in the phyllosphere. A luxCDABE-tagged,kanamycin and rifampicin resistant Pseudomonas syringae pv.
tomato DC3000 (Pst)58 (link) was provided by Thomas Kroj (UMR BGPI, Montpellier,
France). All At-LSPHERE strains were grown on R-2A-agar
(Sigma-Aldrich) supplemented with 0.5% v/v methanol and allowed to grow for 5d
at 22 °C. Pst was grown on King’s B-agar59 (link) for 1-2 d at 22 °C.
Maier B.A., Kiefer P., Field C.M., Hemmerle L., Bortfeld-Miller M., Emmenegger B., Schäfer M., Pfeilmeier S., Sunagawa S., Vogel C.M, & Vorholt J.A. (2021). A general non-self response as part of plant immunity. Nature plants, 7(5), 696-705.
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8

Bacterial Growth Nutrient Requirements

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In order to assess nutrient requirements, bacteria grown in liquid Grace’s medium with 10% FBS were seeded onto R2A agar (Sigma) or onto agarified Grace’s medium containing inorganic salts, vitamins and carbon sources (sucrose, glucose and fructose), as well as one of two different nitrogen sources: (i) peptones from casein (Serva) and tryptone (AppliChem) 5 g/L each, or (ii) ammonium chloride 1 g/L. Bacteria were incubated in 24-well plates as described above.
Nechitaylo T.Y., Westermann M, & Kaltenpoth M. (2014). Cultivation reveals physiological diversity among defensive ‘Streptomyces philanthi’ symbionts of beewolf digger wasps (Hymenoptera, Crabronidae). BMC Microbiology, 14, 202.
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9

Biolog Phenotypic Assay for Carbon and Nitrogen Utilization

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Biolog Phenotypical MicroArray (Biolog, Hayward, CA, USA) for carbon (PM1, 2a) and nitrogen (PM3b) utilization were performed as described [64 (link)]. After two days incubation at 30 °C on R-2A agar (Sigma), P. phymatum cells were prepared for the inoculation of the Biolog PM plates. The PM plates were then incubated for three days at 30 °C under micro-oxic conditions, using the BD GasPak™ system jar (Becton Dickinson, Franklin Lakes, NJ, USA) by changing the sachet once a day. Afterwards, the OD590 of each well was measured using a plate-reader.
Lardi M., Liu Y., Giudice G., Ahrens C.H., Zamboni N, & Pessi G. (2018). Metabolomics and Transcriptomics Identify Multiple Downstream Targets of Paraburkholderia phymatum σ54 During Symbiosis with Phaseolus vulgaris. International Journal of Molecular Sciences, 19(4), 1049.
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10

Cultivation Protocols for Diverse Bacterial Isolates

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Unless stated otherwise, At-SPHERE isolates were
cultivated at room temperature on R-2A agar (Sigma-Aldrich) or at 28°C in
R-2A broth (0.5 g yeast extract (Oxoid), 0.5 g Proteose peptone No. 3 (Becton,
Dickinson and Company), 0.5 g Casamino acids (Becton, Dickinson and Company),
0.5 g D-glucose monohydrate (Sigma-Aldrich), 0.5 g starch (from potato, Fluka),
0.3 g sodium pyruvate (Sigma-Aldrich), 0.3 g K2HPO4(AppliChem) and 0.05 g magnesium sulfate heptahydrate (Sigma-Aldrich) dissolved
in 1 L deionized water), both supplemented after sterilization with 0.5% (v/v)
methanol (R-2A+M). Minimal medium was prepared as described previously66 (link) and supplemented with 5 mM
maltose (Fluka). For Leaf374 cultivation, minimal medium agar was supplemented
with vitamins (500 μg L-1 D-pantothenic acid hemi calcium
salt, 100 μg L-1 biotin, 400 μg L-1riboflavin, 400 μg L-1 thiamine HCl, 200 μg
L-1 pyridoxal HCl 150 μg L-1 p-amino benzoic
acid, 200 μg L-1 cobalamin, 50 μg L-1 lipoic
acid, 150 μg L-1 nicotinic acid and 100 μg
L-1 folic acid). For selective growth of Leaf272, R-2A+M was
supplemented with 50 μg mL-1 rifampicin (Sigma-Aldrich), since
this strain has a natural resistance towards this antibiotic.
E. coli BL21 (DE3) gold was cultivated in LB-Lennox at
37°C supplemented with 50 μg mL-1 kanamycin sulfate
(AppliChem).
Schäfer M., Vogel C.M., Bortfeld-Miller M., Mittelviefhaus M, & Vorholt J.A. (2022). Mapping phyllosphere microbiota interactions in planta to establish genotype-phenotype relationships. Nature microbiology, 7(6), 856-867.
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