In this work, we used Bacillus cereus ATCC14579, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, and Staphylococcus aureus ATCC25923 (ATCC, USA) as gut pathogenic representatives for screening of antibacterial activity of the extracts using disc-diffusion and broth microdilution assays. For disc diffusion assay, a single colony of each bacteria was selected, sub-cultured into 5 mL NB (HiMedia, India), and incubated for 24 h at 37 °C. Each culture was then added into NSS until the turbidity reach McFarland’s standard No. 0.5. The inoculated NSS (approximately 1.5 × 108 cells/mL) was swabbed on freshly prepared NA. Sterile 6 mm diameter filter paper discs (Whatman, UK) were impregnated with 2 and 5 mg extracts before placing on the bacterial swabbed NA. After the incubation at 37 °C for 24 h, the antibacterial activity was evaluated by measuring the IZD. We used ampicillin (10 μg/disc), ceftriaxone (30 μg/disc), chloramphenicol (30 μg/disc), and rifampicin (5 μg/disc) (Oxoid, UK) as positive controls for inhibition of Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, respectively.
Staphylococcus aureus
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Staphylococcus aureus is a species of Gram-positive bacteria commonly found in the human respiratory system and on the skin. It is a common laboratory organism used for various microbiological applications.
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Lab products found in correlation
Escherichia coli, by Thermo Fisher Scientific (7 mentions)
Pseudomonas aeruginosa, by Thermo Fisher Scientific (4 mentions)
Candida albicans, by Thermo Fisher Scientific (3 mentions)
Enterococcus faecalis, by Thermo Fisher Scientific (2 mentions)
Nutrient broth, by Thermo Fisher Scientific (2 mentions)
Streptococcus pyogenes, by Thermo Fisher Scientific (2 mentions)
Rifampicin, by Thermo Fisher Scientific (1 mentions)
Ceftriaxone, by Thermo Fisher Scientific (1 mentions)
Bacillus cereus, by American Type Culture Collection (1 mentions)
Escherichia coli atcc 25922, by American Type Culture Collection (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol, by Thermo Fisher Scientific (1 mentions)
Pseudomonas aeruginosa, by American Type Culture Collection (1 mentions)
Staphylococcus aureus, by American Type Culture Collection (1 mentions)
Thioglycollate medium, by Thermo Fisher Scientific (1 mentions)
Salmonella typhimurium, by American Type Culture Collection (1 mentions)
Streptococcus mutans, by American Type Culture Collection (1 mentions)
Salmonella enteritidis, by American Type Culture Collection (1 mentions)
Helicobacter pylori, by American Type Culture Collection (1 mentions)
Culti loops, by Thermo Fisher Scientific (1 mentions)
16 protocols using staphylococcus aureus
1
Antibacterial Activity Screening of Extracts
2
Standardized Microbial Strain Preparation
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Strains used in this study were obtained from the Infectious and Tropical Diseases Hospital “Victor Babes,” Bucharest, and they were as follows: Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli (with antigens grown in nutrient broth, Oxoid) and, respectively, from Romvac Company's microbiology lab collection: Streptococcus mutans (ATCC 55670), Salmonella typhimurium (RO 05 TL3/2014), Salmonella enteritidis (TL 248), and Helicobacter pylori (ATCC 49503). Helicobacter pylori was cultured in BHI medium (Bacto) and Candida albicans in Sabouraud liquid medium (HiMedia). Clostridium difficile was grown in thioglycollate medium (Oxoid). Cultures were incubated at 37°C, washed twice with sterile PBS at pH 7.2, and inactivated with 0.5% formaldehyde for 18 hours, after which the suspensions were adjusted to 0.05 at OD600, corresponding to a cell density of approximately 1 × 105 CFU/mL.
3
Standardized Microbial Strain Evaluation
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In this study, four bacterial and one yeast strain were tested. The following American Type Culture Collection (ATCC) in the form of Culti-Loops standard strains were purchased from Oxoid (Basingstoke, UK): Candida albicans ATCC 10231, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. Microorganism cultures were maintained in Mueller-Hinton broth (MHB) (Oxoid, Basingstoke, UK) at 4 °C until use. MHB equilibrated with Tris-buffered saline (Sigma-Aldrich, Prague, Czech Republic) was used as the culture medium (for E. faecalis, the MHB was enriched with 1% of glucose). For inoculum standardization, the turbidity of the microorganism suspension was adjusted to a 0.5 McFarland standard (1.5 × 108 CFU/mL) using a Densi-La-Meter II (Lachema, Brno, Czech Republic) spectrophotometric device.
4
Cultivation of Indicator Microorganisms
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LAB isolates were grown on de Man, Rogosa & Sharpe (MRS) agar (Laboratories Conda S.A., Madrid, Spain) at 37°C. The indicator strains were obtained from the American Type Culture Collection (ATCC) and cultivated as follows: Staphylococcus aureus (ATCC-6538) on brain heart infusion (BHI) agar (Oxoid, Basingstoke, UK); Escherichia coli (ATCC- 8739), Bacillus subtilis (ATCC-6633) and Pseudomonas aeruginosa (ATCC-9027) on Luria Bertani (LB) agar (Oxoid, Basingstoke, UK); Candida albicans (ATCC 10231) on yeast mold (YM) agar (Difco, Franklin Lakes, NJ, USA).
5
Antimicrobial Susceptibility Testing of Bacterial and Fungal Strains
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In this study, 20 bacterial strains and one yeast were tested. The following American Type Culture Collection (ATCC) standard strains were purchased from Oxoid (United Kingdom) for analysis: Candida albicans ATCC 10231, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus (ATCC 25923, ATCC 29213, ATCC 33591, ATCC 33592, ATCC 43300, ATCC BAA 976), and S. epidermidis ATCC 12228. Ten clinical isolates of antibiotic-sensitive as well as antibiotic-resistant S. aureus strains (SA1, SA2, SA3, SA4, SA5, SA6, SA7, SA8, SA9, SA10) were provided by University Hospital in Motol (Prague, Czech Republic). Microorganism cultures were stored in MHB at 4 °C until use. Prior to antimicrobial tests, microorganisms were re-cultured at 37 °C for 24 h (48 h for C. albicans).
6
Isolation and Characterization of Wound Microbiota
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Samples of the wound‐populating microbiota were collected from transport media during isolation of cellular constituents.5 All collected media was filtered through 0.22 μm filters, and filters were intensively scraped in PBS. To obtain pooled soluble antigens, the resultant bulk microbiota samples or Staphylococcus aureus (Invitrogen, Carlsbad, CA) were heat inactivated at 95°C for 1 h. Concentration of the resultant antigenic mixture was assessed calorimetrically using 660 reagent (Thermo/Fisher, Waltham, MA).
7
Phagocytosis Assay for E. coli and S. aureus
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SL2 cells were soaked with 1.25 μg of dsRNAs in 96 well plate for 3 days prior to phagocytosis assays. Fluorescein-labeled E. coli (K-12 strain), and Staphylococcus aureus (Wood strain, without protein A) (Invitrogen), were washed 3 times with PBS by centrifugation and sonicated for 3 times at 50 kHz for 20 s. Twenty micrograms of bacterial particles were added to each well at 4°C, the plates were centrifuged 300xg for 3 min at 4°C, and incubated in a 27°C water bath for 20 or 40 min (for E. coli and S. aureus, respectively). Cells and bacteria were removed from plates at 4°C by pipetting 300 μL of a 0.1% Trypan Blue solution in PBS pH 5.4 to quench the fluorescein from extracellular bacteria. The mount of bacteria inside live cells was quantified by flow cytometry (Ramet et al., 2002 (link)).
8
Immortalized Meningioma Cell Cultures
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Human WHO grade I meningioma derived, hTERT immortalized (Ben-Men-I) cells
[15 (link)] were cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate (Sigma-Aldrich). Primary porcine meningothelial cells (PMECs) were cultured as described previously by us
[16 (link)]. Monocyte-like U-937 cells (Sigma-Aldrich, 85011440) were cultured in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate (Sigma-Aldrich). SH-SY5Y cells were cultured in DMEM supplemented with 15% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate. Ben-Men-I cells were treated with 0, 50, or 100 ng/mL of IL-6 (Sigma-Aldrich, SRP3096) or IL-8 (Sigma-Aldrich, I1645). For uptake studies, cells were incubated for 4 or 14 h with Alexa Fluor 488-labeled, heat- or chemically-killed Staphylococcus aureus (Invitrogen, S-23371) or apoptotic cells.
[15 (link)] were cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate (Sigma-Aldrich). Primary porcine meningothelial cells (PMECs) were cultured as described previously by us
[16 (link)]. Monocyte-like U-937 cells (Sigma-Aldrich, 85011440) were cultured in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate (Sigma-Aldrich). SH-SY5Y cells were cultured in DMEM supplemented with 15% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate. Ben-Men-I cells were treated with 0, 50, or 100 ng/mL of IL-6 (Sigma-Aldrich, SRP3096) or IL-8 (Sigma-Aldrich, I1645). For uptake studies, cells were incubated for 4 or 14 h with Alexa Fluor 488-labeled, heat- or chemically-killed Staphylococcus aureus (Invitrogen, S-23371) or apoptotic cells.
9
Neutrophil Activation and Phagocytosis
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To address neutrophils activation and phagocytic capacities we used pHrodo-labeled BioParticles and coated with Staphylococcus aureus (S. aureus) or Zymosan antigens (Invitrogen). The staining protocol is detailed in the supplementary methods section.
10
Antimicrobial Activity Screening of Compounds
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The test compounds were investigated for their antimicrobial potential using American Type Culture Collection strains (ThermoScientific, Waltham, MA, USA): Streptococcus pyogenes ATCC 19615, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC27853, Candida albicans ATCC 10231 and Candida parapsilosis ATCC 22019.
The antimicrobial activity of these samples was initially evaluated using the Kirby Bauer disk diffusion method, and when antimicrobial activity was observed, the broth dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungal concentration (MFC).
All tests were conducted in accordance with the guidelines of the Clinical Laboratory and Standard Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST), with minor changes based on the results of our previous investigations [102 (link),103 (link),104 (link),105 ].
The antimicrobial activity of these samples was initially evaluated using the Kirby Bauer disk diffusion method, and when antimicrobial activity was observed, the broth dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungal concentration (MFC).
All tests were conducted in accordance with the guidelines of the Clinical Laboratory and Standard Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST), with minor changes based on the results of our previous investigations [102 (link),103 (link),104 (link),105 ].
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