A single bacterial colony of each pure culture was transferred into 250 mL Erlenmeyer flasks containing 100 mL Luria-Bertani broth (LB) (Sigma-Aldrich, USA) with a sterile loop and incubated for 6 days at 28 ± 1 °C in a shaking incubator at 150 rpm [36 ,37 ,38 (link)]. Then, sub-cultured bacteria suspensions were transferred into 50 mL sterile Falcon tubes. The supernatant and pellets were carefully separated from the suspension by centrifugation twice at 20,000 rpm for 15 min at 4 °C. The final supernatant fractions were clarified by passing through a 0.22 μm millipore filter [39 (link),40 (link)]. The presence of bacterial cells in the filtrated suspensions was checked by streaking a drop of each suspension on the NBTA medium. The remaining bacterial cell pellets were purified by washing with sterile distilled water twice. The resulting bacterial pellets were re-suspended in 5 mL of sterile distilled water. The total number of cells was calculated by using a spectrophotometer (OD600, 600 nm) and adjusted to a final concentration of 4 × 107 cells/mL by diluting with sterile distilled water [37 ]. The cell-free supernatants and cell suspensions were stored for one week at 14 °C prior to their use in the experiment.
Luria bertani broth lb
Manufactured by Merck Group
Sourced in United States, Germany, Italy, United Kingdom
Luria-Bertani broth (LB) is a widely used nutrient-rich medium for the growth and cultivation of a variety of bacteria, including Escherichia coli. It provides the essential nutrients and growth factors required for the optimal growth and maintenance of bacterial cultures.
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Lab products found in correlation
Chloramphenicol, by Merck Group (3 mentions)
Dmem hg, by Thermo Fisher Scientific (2 mentions)
Tetracycline, by Merck Group (2 mentions)
Trypticase soy agar tsa, by Merck Group (2 mentions)
Elastin congo red ecr, by Merck Group (1 mentions)
Escherichia coli, by Merck Group (1 mentions)
Ureaplasma urealyticum, by Hardy Diagnostics (1 mentions)
Streptococcus agalactiae, by American Type Culture Collection (1 mentions)
Escherichia coli e coli, by American Type Culture Collection (1 mentions)
Kanamycin, by Thermo Fisher Scientific (1 mentions)
E coli bl21 de3, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Promega (1 mentions)
Escherichia coli dh5α, by Thermo Fisher Scientific (1 mentions)
S aureus atcc 43300, by American Type Culture Collection (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Staphylococcus aureus atcc 29213, by American Type Culture Collection (1 mentions)
Pseudomonas aeruginosa atcc 27853, by American Type Culture Collection (1 mentions)
Rhamnose, by Merck Group (1 mentions)
Hepes buffer solution, by Thermo Fisher Scientific (1 mentions)
0.22 μm pore size filter, by Merck Group (1 mentions)
50 protocols using luria bertani broth lb
1
Bacterial Culture Preparation and Purification
2
Evaluating Pomegranate Elastase Inhibition
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The estimation of the effects of P. granatum extracts on the elastolytic activity of P. aeruginosa PAO1 was established based on protocols published by Vasavi et al. (24 (link)). Elastin Congo red (ECR; Sigma, St. Louis, USA) assay was used to measure elastase activity. PAO1 was grown in Luria-Bertani broth (LB; Sigma Aldrich, USA) at 37°C and the different concentrations of methanolic extracts (0.5; 1; 2.5; 5 and10 mg/ml). PAO1 culture was centrifuged and 100 μl supernatant was added to 900 μl of Elastin Congo Red buffer (100 mM Tris, 1 mM CaCl2, pH 7.5) containing 20 mg of elastin Congo red and incubated with shaking at 37°C for 3 h. After centrifugation for 15 min at 13,000 rpm, 200 μl of the supernatant were recovered in 96-well flat-bottom ELISA plates. The optical density was measured at 495nm using a specific ELISA reader (France).
3
Cultivation and Characterization of Clinically Relevant Bacteria
4
Biofilm Experiments with E. coli Strains
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The Escherichia coli strains BW25113 and BW25113-∆wrbA [5 (link)] were used as model systems for the bacterial biofilms. The strains were stored at −80 °C in suspensions containing 20% glycerol and 2% peptone and were routinely grown in Luria-Bertani broth (LB, Sigma-Aldrich, Saint Louis, MO, USA; Merck KGaA, Darmstadt, Germany) at 30 °C for 16 h.
5
E. coli Biofilm Model Systems
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The bacterial biofilm model systems used in the study were Escherichia coli strains BW25113 and BW25113-∆wrbA [25 (link)]. The strains were stored in suspensions containing 20% glycerol and 2% peptone at a temperature of −80 °C. For regular cultivation, the strains were grown in Luria-Bertani broth (LB, Sigma-Aldrich/Merck KGaA, Darmstadt, Germany) at a temperature of 30 °C for a duration of 16 h.
6
Recombinant Protein Expression in E. coli
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Luria-Bertani broth (LB) was obtained from Sigma-Aldrich. Kanamycin was obtained from Gibco. IPTG (isopropyl β-D-1-thiogalactopyranoside) was obtained from Promega. Glucose was obtained from Vetec. Escherichia coli DH5α (Invitrogen Carlsbad, CA, USA) was used as the host for the cloning procedures. E. coli BL21 (DE3) (Invitrogen, Carlsbad, CA, USA) was used for expressing L-asparaginase.
7
Bacterial Biofilm Cultivation Protocol
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The well characterized Escherichia coli K-12 wild-type strain (ATCC 25404) was used as a model system for the bacterial biofilms. The strain was stored at –80°C in suspensions containing 20% glycerol and 2% peptone, and was routinely grown in Luria-Bertani broth (LB, Sigma-Aldrich) at 30°C for 16 h.
8
Cellular Morphology of P. mirabilis
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Cellular morphology of P. mirabilis strains from clinical and environmental origins was observed by TEM and the frequency of their appendages was determined in the total observed cells. The strains were grown in Luria-Bertani broth (LB, Sigma-Aldrich, Munich, Germany) at 37 °C in logarithmic phase. The cells were harvested by centrifugation at 327 g (Eppendorf, Hamburg, Germany) for 10 min, resuspended in 10 μmol l−1 Tris–HCl buffer at pH 7.4 and negatively stained with 2 % aqueous uranyl acetate. The specimens were examined on a transmission electron microscope model FEI CM10 (FEI, Eindhoven, Holland) at an accelerating voltage of 80 kV.
9
Biofilm Formation of Candida and E. coli
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The microbial strains Candida albicans SC5314 (ATCC MYA-2876) and Escherichia coli K-12 wild-type strain (ATCC 25404) were selected as model systems for fungal and bacterial biofilms respectively. C. albicans and E. coli strains were stored at − 80 °C in suspensions containing 50% glycerol and 2% peptone, and were routinely grown in amino acid-free yeast nitrogen base (YNB, Sigma-Aldrich) supplemented with 0.5% glucose (YNBG, Conda) and Luria-Bertani broth (LB, Sigma-Aldrich), respectively, for 16 h at 30 °C.
10
E. coli B ATCC 11303 Growth in LB Broth
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Escherichia coli B ATCC 11303 was cultured at 37 °C for 18 h in Luria-Bertani broth (LB; Sigma-Aldrich, St-Louis, Missouri, MO, USA) in an orbital shaker at a speed of 225 rpm.
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