Hen egg white lysozyme, lysostaphin, nisin, mutanolysin, and melittin were purchased from Sigma Aldrich. The following bacteria were used for antimicrobial activity assays and RNA isolation: Staphylococcus aureus ATCC 6538 (American Type Culture Collection, Manassas, VA, USA), Bacillus cereus ATCC 10876, Enterococcus faecalis ATCC 29212, Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC 27957, Streptococcus equi subsp. zooepidemicus ATCC 43079, Streptococcus iniae KCTC 3657 (Korean Collection for Type Cultures, Daejeon, Korea), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Salmonella typhimurium ATCC 14028. E. faecalis and streptococci were cultured in brain heart infusion broth (BD Bioscience) because of their slow growth in LB medium. All other bacteria were cultured in LB medium.
Pseudomonas aeruginosa
Manufactured by Korean Collection for Type Cultures
Pseudomonas aeruginosa is a type of bacteria that can be used as a reference strain for laboratory testing and research purposes. It is a Gram-negative, aerobic, rod-shaped bacterium.
Automatically generated - may contain errors
Lab products found in correlation
Escherichia coli, by Korean Collection for Type Cultures (2 mentions)
Staphylococcus aureus, by Korean Collection for Type Cultures (2 mentions)
Bacillus subtilis, by Korean Collection for Type Cultures (2 mentions)
Staphylococcus epidermidis, by Korean Collection for Type Cultures (2 mentions)
Mutanolysin, by Merck Group (1 mentions)
Lysostaphin, by Merck Group (1 mentions)
Nisin, by Merck Group (1 mentions)
Hen egg white lysozyme, by Merck Group (1 mentions)
Streptococcus iniae kctc 3657, by Korean Collection for Type Cultures (1 mentions)
Melittin, by Merck Group (1 mentions)
Bacillus cereus, by American Type Culture Collection (1 mentions)
Brain heart infusion broth, by BD (1 mentions)
Staphylococcus aureus atcc 6538, by American Type Culture Collection (1 mentions)
Enterococcus faecalis, by American Type Culture Collection (1 mentions)
Streptococcus agalactiae, by American Type Culture Collection (1 mentions)
Salmonella typhimurium, by Korean Collection for Type Cultures (1 mentions)
Acinetobacter baumannii, by Korean Collection for Type Cultures (1 mentions)
Brain heart infusion broth, by Thermo Fisher Scientific (1 mentions)
P aeruginosa, by Thermo Fisher Scientific (1 mentions)
Shigella sonnei, by Korean Collection for Type Cultures (1 mentions)
6 protocols using pseudomonas aeruginosa
1
Antimicrobial Assays and RNA Isolation
2
Antimicrobial Susceptibility Screening
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Four Gram-negative bacteria species (Escherichia coli KCTC 1682, Acinetobacter baumannii KCTC 2508, Pseudomonas aeruginosa KCTC 1637 and Salmonella typhimurium KCTC 1926) and three Gram-positive bacteria species (Staphylococcus aureus KCTC 1621, Bacillus subtilis KCTC 3028 and Staphylococcus epidermidis KCTC 1917) were procured from the Korean Collection for Type Cultures (KCTC). Multi-drug-resistant Gram-negative bacteria (Salmonella typhimurium CCARM 8003 and 8007, Escherichia coli CCARM 1229 and 1238, Pseudomonas aeruginosa CCARM 2002 and 2095 and Acinetobacter baumannii CCARM 12035 and 12036) were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM). To determine MICs, bacteria were cultured in Luria-Bertani (LB) medium for overnight at 37 °C. An aliquot of the culture was incubated in 1% peptone media at 37 °C until mid-log phase. 100 μl of serial 2-fold diluted peptides were treated in 96 well plates and added to 100 μl of 2 × 106 CFU/ml bacterial suspensions in 1% peptone media for 16 h at 37 °C. The lowest concentration of peptide that completely inhibited bacterial growth was determined to be the MIC.
3
Antimicrobial Efficacy of κ-CRG-R-PE Hydrogel
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The antimicrobial efficacy of κ-CRG-R-PE hydrogel was assessed against both gram-negative (Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacterial strains. Kanamycin served as the positive control in the experiments. The bacterial strains, cm aureus (KCTC 1621) and Pseudomonas aeruginosa (KCTC 1637), were procured from the Korean Collection for Type Cultures (KCTC).
4
Antibacterial Assay of Diverse Pathogens
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Antibacterial activity was measured by modifying the method of Brandt et al. (19 (link)). E. coli (KCTC 1682), Vibrio paraheamolyticus (KCTC 2729), Pseudomonas aeruginosa (KCTC 1750), Shigella sonnei (KCTC 2518), and Shigella flexneri (KCTC 22192) were purchased from the Korean Collection for Type Cultures (Daejeon, Korea). All samples (10 mg/mL) were dissolved in 50 mM potassium phosphate buffer (pH 7.0) and filtered through a sterilized 0.2 μm syringe filter (Millipore, Temecula, CA, USA). The bacterial cells were incubated in brain heart infusion broth (Oxoid Ltd., Basingstoke, England) for E. coli, P. aeruginosa, S. sonnei, and S. flexneri, and tryptic soy broth (17 g/L of pancreatic digest of casein, 3 g/L of papaic digest of soybean, 2.5 g/L of dextrose, 5 g/L of sodium chloride, and 2.5 g/L of dipotassium phosphate) with 3% NaCl for V. parahaemolyticus at 37°C for 16 h, diluted with 200 μL of a liquid medium inoculated at about 106 CFU/mL, and 100 μL of the samples were mixed with the bacteria in 96-well plates. Sterilized liquid medium (100 μL) was used as a control, and samples were cultured at 37°C for 24 h. Using a microplate reader, the absorbance values were compared at 600 nm during the incubation period, and data is expressed as growth inhibition rate (%).
5
Antioxidant and Antibacterial Assays
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All solvents used in this study were of high-performance liquid chromatography (HPLC) grade. Trifluoroacetic acid (extra-pure grade) was supplied by Alfa Aesar (Ward Hill, MA, USA). Quercetin-3,4′-O-diglucoside and quercetin-4′-O-monoglucoside were supplied by Polyphenols Laboratories AS (Sandnes, Norway). Gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-Tris (1-pyridyl)-5-triazine (TPTZ), ferric chloride, Folin Ciocalteu reagent and ampicillin used were purchased fromSigma–Aldrich (St. Louis, MO, USA), and 2,2-diphenyl-1-picrylhydrazyl from Wako Pure Chemicals (Osaka, Japan) were used for antioxidant assays. Stock solutions of quercetin (1 mg mL−1) and quercetin glucosides (4 mg mL−1) were prepared in methanol. All the solutions were stored at −20 °C and calibration standards were obtained by appropriate dilution of the stock solutions. The standard bacterial strains of the Gram negative bacteria E. coli (KCTC-1924), Pseudomonas aeruginosa (KCTC-2004), and of the Gram positive bacteria B. cereus (KCTC-1012) and Staphylococcus aureus (KCTC-1916) were obtained from the Korean Collection for Type Cultures (KCTC).
6
Antibacterial Activity Screening of Peptides
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Bacteria, including E. coli (KCTC 1682), Staphylococcus typhimurium (KCTC 1926), Pseudomonas aeruginosa (KCTC 1637), Bacillus subtilis (KCTC 3068), Staphylococcus epidermidis (KCTC 1917), and S. aureus (KCTC 1621) were procured from the Korean Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology (Deajeon, South Korea). MRSA strains (CCARM 3089, CCARM 3090, and CCARM 3095) were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM) at the Seoul Women's University (Seoul, South Korea). All bacteria were routinely grown at 37°C in Luria-Bertani broth. MICs were measured in 96-well microplates using the micro-broth dilution method. Bacteria were grown in Luria-Bertani broth at 37°C for 18 h and then diluted with 1% peptone to a final concentration of 2 × 10 6 colonyforming units per ml. Subsequently, 100 µl of a bacterial suspension and 100 µl of peptide solution were mixed together in a 96-well plate. The peptide solution was prepared by performing two-fold dilutions in 1% peptone, while the PGP-E solutions were prepared to final peptide concentrations of 0.06-128 µg ml -1 . The bacteria and peptide solution mixtures were incubated at 37°C for 18 h. The peptide MIC was defined as the lowest concentration of peptide that inhibited bacterial growth.
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