The wild-type PAK strain of P. aeruginosa and the PAK strain labelled with the green fluorescent protein (PAK-GFP) were a kind gift from Dr. Stephen Lory, Ph.D., Professor, Department of Microbiology and Molecular Genetics at Harvard Medical School, Boston Massachusetts. For each experiment, frozen bacteria were inoculated into Luria-Bertani broth (Invitrogen, Carlsbad, CA), incubated for 6h at 37°C on a rotating platform, and then diluted 1:100 in fresh Luria-Bertani broth. After 16–18 h of incubation at 37°C, the stationary phase bacteria were pelleted, washed three times in phosphate-buffered saline (PBS), and suspended in PBS to a concentration adjusted by optical density at 600 nm, as 1 × 109 colony forming units/ml (CFU/ml) for in vitro experiments or 2 × 108 CFU/ml for instillation in mice (50µl preparation per mouse).
Luria bertani broth
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, China, Australia, Canada, Italy
Luria-Bertani (LB) broth is a widely used nutrient-rich growth medium for the cultivation of bacteria, particularly Escherichia coli (E. coli). It provides the essential nutrients and conditions required for bacterial growth and proliferation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Glycerol, by Merck Group (4 mentions)
Lb agar, by Thermo Fisher Scientific (4 mentions)
Mrs broth, by Thermo Fisher Scientific (4 mentions)
Ampicillin, by Merck Group (4 mentions)
Kanamycin, by Merck Group (3 mentions)
Macconkey agar, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Formic acid, by Thermo Fisher Scientific (3 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Genesys 10 uv, by Thermo Fisher Scientific (2 mentions)
Hydrochloric acid (hcl), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
D glucose, by Merck Group (2 mentions)
Triethanolamine, by Merck Group (2 mentions)
Bhi broth, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Lb broth, by Thermo Fisher Scientific (2 mentions)
122 protocols using luria bertani broth
1
Cultivation of P. aeruginosa Strains
2
Antimicrobial Efficacy of NAP-Based Compounds
3
Enzymatic Assay for Screening 3-Nitro-2-Phenyl-2H-Chromene Analogues
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Adenosine 5′-triphosphate disodium salt hydrate (≥99%), chlorophenol red-β-d -galactopyranoside (CPRG), β-nicotinamide adenine dinucleotide phosphate hydrate (NADP+), magnesium chloride hexahydrate (≥99%), d -(+)-glucose (≥99%), dimethyl sulfoxide (ACS reagent (≥99.9%)), d -glucose-6-phosphate sodium salt, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES, ≥99.5%), imidazole (96.0%), Terrific broth modified, and triethanolamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from CellGro Technologies, LLC (Lincoln, NE, USA). Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (LmG6PDH) was purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA). NADPH tetrasodium salt (≥95%) was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). For the 3-nitro-2-phenyl-2H-chromene analogues, compounds 1 –7 were purchased from TimTec, LLC (Tampa, FL, USA), while compounds 8 –13 were purchased from ChemDiv, Inc. (San Diego, CA, USA). Ethylenediaminetetraacetic acid (EDTA) tetrasodium salt hydrate (98%), Luria-Bertani (LB) broth, lysozyme (type VI), protease inhibitor tablets (EDTA-free), glycerol, and all other chemicals were purchased from Fisher Scientific (Hampton, NH, USA).
4
Thiol Assay of Membrane Lipids
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Propyl Gallate (PG), L-α-phosphatidylcholine from egg yolk (egg PC), Reduced L-glutathione (GSH), Sodium Chloride (NaCl), Sodium Dodecyl Sulfate (SDS), Chloroform, Methanol, Ethylenediaminetetraacetic acid (EDTA), and Triton X-100 were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Measure-iT™ Thiol Assay Kit, a thiol-reactive fluorescence probe, was purchased from Molecular Probes (Eugene, OR, USA). 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (β-Py-C10-HPC) was purchased from Invitrogen by Thermo Fisher Scientific (Waltham, MA, USA). Zirconia-silica beads (0.1 mm diameter) were acquired from Biospec Products (Bartlesville, OK, USA). Luria-Bertani (LB) broth, Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), Phosphate-buffered Saline (PBS), and Tris-hydrochloride (1 M; Tris-HCl) were purchased from Fisher BioReagents (Pittsburgh, PA, USA). Ultrapure water was obtained using a Milli-Q filtration system (EDM Millipore; Billerica, MA, USA).
5
Induced Bacterial Protein Expression
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Protein expression was carried out in Luria–Bertani (LB) Broth (FisherBioReagents, Pittsburgh, PA). Briefly, starter cultures were grown for 16 h at 36 °C and inoculated at 1/100 into 1L of LB and grown at 37 °C. At an OD600 of 0.8 cultures were induced with 1 mM Isopropyl-β-d -thiogalactopyranoside (IPTG) (FisherBioReagents, Pittsburgh, PA). Cultures were grown for 4 h, pelleted at 4000× g and stored at −80 ºC until use.
6
Cultivation and Preservation of Bacterial Strains
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All strains used in this study (Table S7 ) were stored at −20 or −80 °C. Streptococcus, Enterococcus, Bacillus, and Staphylococcus strains were cultivated in BHI broth (Oxoid, Hampshire, UK). Escherichia coli for protein expression was grown in Luria-Bertani (LB) broth (Fisher Scientific, Waltham, MA, USA), supplemented with 50 µg/mL kanamycin (Sigma Aldrich, Saint Louis, MO, USA). Bacteria were incubated at 37 °C and shaken at 120 rpm, if necessary. Lactococcus lactis was cultivated in GM17 broth (Oxoid, Hampshire, UK) at 30 °C. The Streptococcus infantis phage 23TH and Streptococcus anginosus phage SA01 and their host strains Streptococcus infantis 23TH and Streptococcus anginosus SA01 were saliva isolates sourced from the APC Culture Collection (APC Microbiome Ireland, Cork, Ireland).
7
Fluorescent Nucleic Acid Staining Protocol
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SYBR Green I nucleic acid stain, 10,000× concentrate was purchased from Invitrogen, USA, and a working solution of 10× SYBR Green I was prepared in Milli-Q water. p-Phenylenediamine and chloroform were obtained from Acros Organic (Fair Lawn, NJ, USA) and a 10% (wt/vol) stock solution of p-phenylenediamine was prepared in Milli-Q water. Whatman® anodisc inorganic filter membrane (13 mm, 0.02 μm pore size) was obtained from GE Healthcare (Buckinghamshire, UK). Microscopic slide was obtained from VWR international (Radnor, PA, USA). Tryptic soy broth (TSB) and tryptic soy agar (TSA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Filtration system, cover glass and Luria Bertani (LB) broth were obtained from Fisher Scientific (Pittsburgh, PA, USA). Phosphate buffered saline (PBS) was purchased from Fisher Bioreagents (Fair Lawn, NJ, USA). Polycarbonate filter (20 um pore size, 47 mm diameter) was purchased from Maine Manufacturing (ME, USA). Milli-Q water was produced by QPAK® 2 purification system (EMD Millipore, Billerica, MA, USA).
8
Vibrio cholerae Growth Conditions
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All bacterial strains and plasmids used in this study are listed in Table S1 in the supplemental material. All V. cholerae strains were grown aerobically with aeration (225 rpm) at 37°C in Luria Bertani (LB) broth (Fisher Scientific, Waltham, MA) with a 1% NaCl concentration. For growth analysis on intestinal mucus, mucus sugars, and sialic acid derivatives as sole carbon sources, M9 minimal medium was supplemented with 0.02 mM MgSO4 and 0.1 mM CaCl2. M9 minimal medium supplemented with intestinal mucus (M9M) (30 µg/ml) or glucose (M9G) was used or N-acetylglucosamine, d -ribose, d -mannose, d -gluconate, or d -galactose (all 10 mM), or Neu5Ac (3 mM), 2-keto-3-deoxy-d -glycero-d -galacto-nononic acid (KDN) (4 mM), or N-glycolylneuraminic acid (Neu5Gc) (3 mM) (Sigma-Aldrich) was added to M9 to serve as the carbon source. Antibiotics were used at the following concentrations: streptomycin (Sm) at 200 µg/ml, ampicillin (Amp) at 100 µg/ml, chloramphenicol (Cm) at 25 µg/ml, and kanamycin at 50 µg/ml.
9
Characterization of Pseudomonas aeruginosa Strains
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PA strains PAO1 and its isogenic ΔfliD mutant were generous gifts from Professor Reuben Ramphal (University of Florida). CF clinical PA isolates CF1, CF2, CF26 and CF32 were collected while the Dr. Lau was on staff at the University of Cincinnati College of Medicine under the IRB # 04-7-16-2. All PA strains were cultured in plain Luria-Bertani (LB) broth (Fisher Scientific) with the exception of ΔfliD, which was grown in LB containing 75 μg/ml gentamicin (Life Technologies), at 37°C overnight. They were then stored at −80°C in 25% glycerol (Sigma-Aldrich). Before each experiment, bacteria were cultured from frozen stocks in 5 ml LB broth to stationary phase to OD at 600 nm ~ 3.0 by using a spectrophotometer, Genesys 10 UV (Thermo Scientific). Bacteria were then washed 3x with sterile PBS, diluted to appropriate concentrations for binding experiments.
Mucoidy phenotypes of CF clinical PA strains were determined by streaking the frozen stocks onto the Pseudomonas Isolation Agar plates. Motility of PA strains were determined by inoculating 1 μl of overnight bacterial culture (OD 600nm 3.0) onto the Brain Heart Infusion broth supplemented with 0.4% agar. Bacterial plates were incubated at 37°C with 5% CO2 for 48 - 72 hr for observation.
Mucoidy phenotypes of CF clinical PA strains were determined by streaking the frozen stocks onto the Pseudomonas Isolation Agar plates. Motility of PA strains were determined by inoculating 1 μl of overnight bacterial culture (OD 600nm 3.0) onto the Brain Heart Infusion broth supplemented with 0.4% agar. Bacterial plates were incubated at 37°C with 5% CO2 for 48 - 72 hr for observation.
10
E. coli Glycoconjugate Production Protocol
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For all experiments, E. coli CLM24 was cultured in Luria-Bertani (LB) broth (Fisher Scientific, UK) supplemented with appropriate antibiotics in the following concentrations: ampicillin 100 μg/mL, tetracycline 20 μg/mL, and spectinomycin 80 μg/mL. The addition of manganese chloride at the time of protein and PglB induction was at a final concentration of 4 mM, and made up as a 1 M stock fresh prior to each experiment. Cultures were incubated at 37°C shaking at 110 rpm for 16–20 hrs for large-scale preparation. For three-plasmid system glycoconjugate production, an overnight LB culture of E. coli CLM24 harbouring pGVXN114, pGVXN150: GtExoA, and pGab2 were subcultured in a 1 : 10 dilution of LB broth (Fisher Scientific) with antibiotics, and grown to mid log phase. pGVXN150: GtExoA was induced by the addition of 0.2% L-(+)-arabinose (Sigma-Aldrich, UK), and C. jejuni PglB was induced with 1 mM IPTG, followed by incubation for an initial 4 hours. Another addition of 0.4% L-(+)-arabinose was then added and cultures were incubated overnight.
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