Sixty-seven fish with a size of 3.5–30 cm was obtained. Bacteria were isolated from the kidneys of diseased catfish The bacteria were cultured in glutamate starch phenyl (GSP) medium (Merck, Darmstadt, Germany), a selective medium for Aeromonas and Pseudomonas, at 30°C for 24 h. Aeromonas and Pseudomonas grew in yellow and red colonies, respectively. Furthermore, yellow single colonies were recovered in the tryptic soy broth (TSB) medium (Merck). The isolates were stored in TSB medium (Merck) with 20% glycerol at −80°C for further assay.
Tsb medium
Manufactured by Merck Group
Sourced in Germany, United States, Ireland
TSB medium is a general-purpose liquid growth medium used for the cultivation of various microorganisms, including bacteria and fungi. It provides the necessary nutrients and growth factors to support the proliferation of a wide range of microbial species.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 7900, by Agilent Technologies (1 mentions)
1s verbenone, by Merck Group (1 mentions)
S cis verbenol, by Merck Group (1 mentions)
Multiskan sky microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
μ slide vi0, by Ibidi (1 mentions)
Human fibronectin protein, by R&D Systems (1 mentions)
Iron assay kit, by Merck Group (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Crystal violet solution, by Merck Group (1 mentions)
Synergy h1 plate reader, by Agilent Technologies (1 mentions)
Simmons citrate, by Merck Group (1 mentions)
Urea agar, by Merck Group (1 mentions)
Tsa plate, by Merck Group (1 mentions)
18 protocols using tsb medium
1
Isolation and Identification of Aeromonas and Pseudomonas from Diseased Catfish
2
Genomic DNA Isolation from Bacterial Cultures
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Genomic DNA of both strains was isolated from liquid cultures grown in TSB medium (Sigma-Aldrich) as described by Hopwood et al. [9 ]. DNA was checked by electrophoresis in a 0.8% (w/v) agarose gel. DNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and stored at -20°C.
3
Quantifying Heavy Metal Bioaccumulation
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Renuspore® and L. rhamnosus GG were cultured aerobically in TSB medium (Sigma-Aldrich, Ireland Ltd.) supplemented with 1 ppm of lead or mercury (Sigma-Aldrich, Ireland Ltd.) until they reached a final concentration of 1 × 108 CFU/mL. Cultures were centrifuged at 6,000 RPM for 15 min at room temperature, and the supernatants were saved. TSB medium supplemented with 1 ppm lead or mercury was set as control. Saved supernatants and controls were filter-sterilized and sent to Eurofins Food Testing Ireland Limited for quantification by ICP-MS analysis. ICP-MS (Agilent 7900) consists of plasma, a mass spectrometer with a quadrupole mass filter, and a detector. All liquids were converted to aerosol by pumping into the nebulizer. The saved supernatants and control samples were carried in a gas stream of plasma in the form of an aerosol. The high-temperature electrical discharge of plasma transformed samples into vapors to atomize and ionize the elements. The ionized particles were then separated, and the analyte ions were filtered by the quadrupole mass filter. The filtered mass was finally counted by the detector (González-Antuña et al., 2017 (link)).
4
Staphylococcal Enterotoxin Extraction
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To determine their enterotoxin contents, staphylococcal supernatants were prepared the same way as for the stimulation assays above, except that overnight cultures were grown in TSB medium (Sigma-Aldrich) for 14 h to obtain higher yields of supernatant proteins. Equivalent amounts of bacteria were confirmed by optical density measurements at 600 nm and by CFU counting. Proteins were obtained from the supernatants by means of chloroform/methanol precipitation, using 4 vol. methanol, 1 vol. chloroform and 3 vol. H2Odd per vol. overnight culture. After centrifugation for 45 min at 4.800x g and 12°C, the aqueous phase was removed and 6 vol. methanol were added, followed by a second centrifugation step using the same conditions in order to pellet the proteins. The protein pellets were air-dried and stored at −20°C.
5
Verbenone conversion experiments
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In the species specific verbenone conversion experiments, TSA and TSB medium were purchased from Sigma-Aldrich (Shanghai, China). (S)-cis-verbenol (95% purity), (1S)-(-)-verbenone (94% purity) and heptyl acetate (≥98% purity) were purchased from Sigma-Aldrich (Shanghai, China) for use in all experiments.
6
Streptococcus mutans Culture Protocol
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A pure culture of Streptococcus mutans ATCC® 25175™ was used while gram staining was performed for microscopic confirmation of the purity of the bacteria. The tryptic soy broth (TSB) medium (Sigma, St. Louis, MO, USA) containing bacterial inoculum was incubated at 37 °C for 24 h in a 5% CO2 incubator (ESCO CCL-050B-8, Singapore). The bacterial suspensions were adjusted to a turbidity of 0.5 McFarland standard, equivalent to 1.5 × 108 CFU/mL, using a Multiskan Sky Microplate Spectrophotometer (Thermo Scientific™, Waltham, MA, USA).
7
Isolation and Cultivation of Streptomyces Endophytes
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Streptomyces strains VV/E1 and VV/R4 were isolated from the root system (endophytic) and the rhizosphere of young grapevines, respectively [8 (link)]. Both strains were grown in tryptic soy broth (TSB) medium (Sigma-Aldrich, St. Louis, Missouri, USA) at 220 rpm and 30°C for 3 days.
8
Biomolecule Immobilization Protocol
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Poly(dimethylammonium chloride) (PDDA) and
poly(sodium-4-styrenesulfonate) (PSS) were purchased from Sigma-Aldrich
(DK). Polyammonium chloride (PAX-XL60) was purchased from Kemira Miljo
(DK) and sulfate modified polystyrene colloidal particles in water
were purchased from Invitrogen. S. aureus DMS 20231
was purchased form DSMZ, Germany. Human fibronectin protein was purchased
from R&D systems (USA). All standard DNA oligonucleotides DBCO-PS3d,
DBCO-R3, PS3i-CyB3, and R3-CyB3 sequences were purchased from IDT
(DK). DBCO-NHS A124 was obtained from the click chemistry tool. PLL
P8920, streptavidin 189730, biotin-NHS H1759-5MG, glycin50046, TBE
buffer T4415-1L, and TSB medium were obtained from Sigma-Aldrich (DK).
Biotin-BSA 29130 and SDS-PAGE (EA03552BOX) were obtained from Thermo
Fisher. NHS-dPEG4-biotin (BD1-A0401-045) from quantabiodesign
(USA). μ-Slide VI 0.4 and ibidi μ-slide VI 0.5 glass bottom
channel slide cat. no. 80607 from ibidi (DK). NHS-PEG4-azide (CLK-AZ103-100)
was obtained from Jena bioscience (DE).
poly(sodium-4-styrenesulfonate) (PSS) were purchased from Sigma-Aldrich
(DK). Polyammonium chloride (PAX-XL60) was purchased from Kemira Miljo
(DK) and sulfate modified polystyrene colloidal particles in water
were purchased from Invitrogen. S. aureus DMS 20231
was purchased form DSMZ, Germany. Human fibronectin protein was purchased
from R&D systems (USA). All standard DNA oligonucleotides DBCO-PS3d,
DBCO-R3, PS3i-CyB3, and R3-CyB3 sequences were purchased from IDT
(DK). DBCO-NHS A124 was obtained from the click chemistry tool. PLL
P8920, streptavidin 189730, biotin-NHS H1759-5MG, glycin50046, TBE
buffer T4415-1L, and TSB medium were obtained from Sigma-Aldrich (DK).
Biotin-BSA 29130 and SDS-PAGE (EA03552BOX) were obtained from Thermo
Fisher. NHS-dPEG4-biotin (BD1-A0401-045) from quantabiodesign
(USA). μ-Slide VI 0.4 and ibidi μ-slide VI 0.5 glass bottom
channel slide cat. no. 80607 from ibidi (DK). NHS-PEG4-azide (CLK-AZ103-100)
was obtained from Jena bioscience (DE).
9
Bacterial Mineral Uptake Quantification
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Renuspore® and L. rhamnosus GG were cultured aerobically in TSB medium (Sigma-Aldrich, Ireland Ltd.) supplemented with 500 μM of calcium and 10 ppm of iron or magnesium (Sigma-Aldrich, Ireland Ltd.), until they reached a final concentration of 108 CFU/mL. Cultures were centrifuged at 6,000 RPM for 15 min at room temperature, and the supernatants were saved. TSB medium supplemented with 500 μM of calcium and 10 ppm of iron or magnesium was set as control. Saved supernatants and controls were filter-sterilized and analyzed using an Iron Assay Kit (Sigma-Aldrich, MAK025, Ireland Ltd.), Calcium Assay Kit (Cell Biolabs, MET5121, USA), and Magnesium Microplate Assay Kit (Cohesion biosciences, CAK1107), according to the manufacturer's instructions.
10
Isolation and Culture of Commensal and Pathogenic Bacteria
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Commensal strains were previously isolated from wild A. thaliana plants (Bai et al., 2015) ( http://www.at-sphere.com/ ) ( Table 1 ) . The Pto mutant D36E was previously described (Wei et al., 2015) . Bacterial strains were cultured at 20ºC (commensal strains) or 28ºC ( Pto and D36E) at 200 rpm in liquid 50% TSB medium (Sigma-Aldrich, USA).
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