Antibacterial and antifungal tests were performed by agar well diffusion method as described by Tagg and McGiven [12 ] and broth microdilution assay using sterile Mueller–Hinton media (Bio-Rad, France) for bacterial strains and potato dextrose agar (Bio-Rad,France) for antifungal tests. Fifteen milliliters of the molten agar (45 °C) were poured into sterile petri dishes (Ø 90 mm). Working cell suspensions were prepared and 100 μl were evenly spread onto the surface of the agar plates of Mueller-Hinton agar (Oxoid Ltd., UK) for bacteria, or potatoes dextrose agar medium (Oxoid Ltd., UK) for fungi. Once the plates had been aseptically dried, 06 mm wells were punched into the agar with a sterile Pasteur pipette. The ClEO were dissolved in dimethylsulfoxide (DMSO)/water (1/1) and sterile water to a final concentration of 50 mg/ml. Thus, 50 μl were placed into the wells and the plates were incubated at 37 °C for 24 h for bacterial strains and 72 h for fungi at 28 °C. Gentamicin (10 μg/wells), Amphotericin B (PubChem CID: 5,280,965) at 20 μg/wells and DMSO served as positive and negative control. Antimicrobial activity was evaluated by measuring the diameter of circular inhibition zones around the well. Tests were performed in triplicate.
Mueller hinton media
Manufactured by Bio-Rad
Sourced in France
Mueller–Hinton media is a type of agar commonly used in microbiology laboratories for antimicrobial susceptibility testing. It provides a standardized growth medium for the cultivation of various bacterial species.
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6 protocols using mueller hinton media
1
Antimicrobial Screening of Cl EO
2
Antimicrobial Efficacy of AgNPs
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The antibacterial activity of AgNPs was investigated against clinical bacteria strains including Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Micrococcus luteus. The reference strains of the Candida species (Candida albicans, Candida tropicalis, and Candida glabrata) and the dermatophytes species (Trichophyton rubrum, Microsporum canis, and Trichophyton interdigitale) were obtained by clinical isolation and identification using conventional methods at the dermatophytes laboratory services, La Rabta Hospital, Tunis, Tunisia. For the antibacterial and antifungal assays, Mueller–Hinton media (BioRad, France) and the Sabouraud chloramphenicol agar were used, respectively.
3
Antimicrobial Activity of Silver Nanoparticles
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The antimicrobial activity of silver nanoparticles was assessed against clinical pathogens at a Tunisian public hospital, including Gram-negative bacterial species (Pseudomonas aeruginosa, Salmonella typhi, Escherchia coli) and Gram-positive bacteria (Staphylococcus aureus). Antifungal activity was determined using two fungal species: Candida albicans and Candida tropicalis. Mueller–Hinton media (BioRad, Mitry-Mory, France) and potato dextrose agar were used for the antibacterial and antifungal assays, respectively.
4
Antibacterial Assay of LmPS Extract
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Antibacterial tests were performed as described by Ben Hsouna et al. [5 (link)] and the broth microdilution test by the sterile Mueller–Hinton media (Bio-Rad, Marnes-la-Coquette, France). One hundred μL was evenly spread on the surface of MH agar plates (Oxoid Ltd., Basingstoke, UK). Wells were dug into the agar using a sterile Pasteur pipette. The final concentration was 50 mg/mL of LmPS dissolved in distilled water. Then, 50 μL of extract was placed into the wells and the plates were incubated for 24 h at 37 °C. Streptomycin (20 μg/well) and distilled water were used as positive and negative controls, respectively. The antimicrobial effect was checked measuring the diameter of the circular inhibition zones of the wells. The tests were carried out in triplicate.
5
Antimicrobial Activity of Z. jujuba Extracts
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The various methanol extracts from Z. jujuba were dissolved in sterile water. Before use and for the detection of the antimicrobial activity, each extract was diluted to 50 μg/mL and sterilized by filtration through a 0.2 μm pore size filter.
Antibacterial tests were performed by agar well diffusion methods as described by Dharajiy et al. (2016 ). Broth microdilution assay using sterile Mueller‐Hinton media (BioRad, France) for bacterial strains and yeast malt extract agar YMA (Bio‐Rad, France) for antifungal tests were used. A fresh cell suspension (0.1 mL) adjusted to 107 CFU/mL for bacteria and 105 cell/mL for fungus were inoculated onto the surface of agar plates. Afterward, wells 6 mm diameter, were punched in the inoculated agar medium and 30 μL of the extract was added to each well. Negative controls consisted of using 30 μL water. The plate was allowed to stand for 40 min at 4°C to permit the extract diffusion followed by incubation at 37°C for 24 h for bacteria. The antibacterial activities were evaluated by measuring the zones of inhibition (clear zone around the well) against the test microorganisms. All tests were repeated three times (Essghaier et al., 2014 (link)).
Antibacterial tests were performed by agar well diffusion methods as described by Dharajiy et al. (2016 ). Broth microdilution assay using sterile Mueller‐Hinton media (BioRad, France) for bacterial strains and yeast malt extract agar YMA (Bio‐Rad, France) for antifungal tests were used. A fresh cell suspension (0.1 mL) adjusted to 107 CFU/mL for bacteria and 105 cell/mL for fungus were inoculated onto the surface of agar plates. Afterward, wells 6 mm diameter, were punched in the inoculated agar medium and 30 μL of the extract was added to each well. Negative controls consisted of using 30 μL water. The plate was allowed to stand for 40 min at 4°C to permit the extract diffusion followed by incubation at 37°C for 24 h for bacteria. The antibacterial activities were evaluated by measuring the zones of inhibition (clear zone around the well) against the test microorganisms. All tests were repeated three times (Essghaier et al., 2014 (link)).
6
Antimicrobial Evaluation of Dissolved Oils
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Antibacterial and antifungal assays were performed by the agar well diffusion method [64 (link)] and broth microdilution method using sterile Mueller–Hinton media (Bio-Rad, France) for bacterial strains and potato dextrose agar (Bio-Rad, France) for antifungal tests. A fresh cell suspension (0.1 mL) adjusted to 107 CFU/mL for bacteria and 106 spores/mL for fungus was inoculated onto the surface of agar plates. Thereafter, wells with 6 mm in diameter were punched in the inoculated agar medium with sterile Pasteur pipette, and 50 μL of the dissolved oil was added to each well. Negative controls consisted of 50 μL DMSO, used to dissolve the oil (1 V/2 V). The plate was allowed to stand for 2 h to permit the diffusion of the oil followed by incubation at 37°C for 24 h for bacterial strains and 72 h for fungi at 28°C. The antimicrobial activity was evaluated by measuring the inhibition zones (clear areas around the wells) against the test microorganisms. All tests were carried out in triplicate.
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