The susceptibility of the retrieved isolates to different commercial antimicrobial agents (Oxoid), including ofloxacin (5 µg), amoxicillin (10 µg), cefotaxime (30 µg), tetracycline (30 µg), levofloxacin (5 µg), gentamicin (10 µg), norfloxacin (10 µg), tobramycin (10 µg), and colistin sulfate (25 µg) was evaluated using a disc diffusion method32 (link). The selected antimicrobial agents are representatives of the drugs used commonly in the aquaculture sector in Egypt and were selected according to the National Antimicrobial Resistance Monitoring System records. The test was performed using Muller Hinton agar plates (Oxoid, UK) and the plates were incubated at 37 °C for 24 h. The test was conducted according to the instructions of the Clinical Laboratory Standards Institute (CLSI) criteria33 .
Muller hinton agar plate
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, Italy
Muller-Hinton agar plates are a standardized microbiological growth medium used for antimicrobial susceptibility testing. They provide a consistent, reliable, and reproducible platform for evaluating the effectiveness of antimicrobial agents against bacterial isolates.
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Lab products found in correlation
Ampicillin, by Thermo Fisher Scientific (3 mentions)
Imipenem, by HiMedia (2 mentions)
Meropenem, by HiMedia (2 mentions)
Sensi disc, by BD (2 mentions)
Antimicrobial disks, by Thermo Fisher Scientific (1 mentions)
Bhi broth, by Bio-Rad (1 mentions)
Vitek 2 automated system, by bioMérieux (1 mentions)
Glycerol, by Thermo Fisher Scientific (1 mentions)
Mueller hinton broth (mhb), by Merck Group (1 mentions)
Imipenem, by Thermo Fisher Scientific (1 mentions)
Meropenem, by Thermo Fisher Scientific (1 mentions)
Amoxicillin, by Thermo Fisher Scientific (1 mentions)
Vancomycin, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Thermo Fisher Scientific (1 mentions)
Sulfamethoxazole, by Thermo Fisher Scientific (1 mentions)
Florfenicol, by Thermo Fisher Scientific (1 mentions)
Ciprofloxacin, by Thermo Fisher Scientific (1 mentions)
Erythromycin, by Thermo Fisher Scientific (1 mentions)
E test strip, by Liofilchem (1 mentions)
Gentamycin, by Bioanalyse (1 mentions)
20 protocols using muller hinton agar plate
1
Antimicrobial Susceptibility Profiling of Aquaculture Isolates
2
Bacterial Count in Sepsis Mice
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The bacterial count was determined as previously described [24] (link). Briefly, mice were euthanized 6, 12 or 24 h after sepsis induction. After harvesting the blood and peritoneal lavage using sterile PBS, aliquots of serial dilutions of these samples were plated on Muller-Hinton agar plates (OXOID, UK) and incubated at 37°C. CFUs were analyzed 18 h after the incubation.
3
Antimicrobial Susceptibility Testing of Enterococcus
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Enterococcus spp. isolates were tested for antimicrobial susceptibility against a panel of 12 antimicrobials by the disk diffusion method (Kirby Bauer Test) as described by the Clinical and Laboratory Standards Institute (CLSI 2018 , 2021). The following antimicrobials were tested: amikacin 30 mg, amoxicillin/clavulanic acid 30 (20 + 10) mg, ampicillin 10 μg, ceftriaxone 30 μg, chloramphenicol 30 μg, ciprofloxacin 5 μg, kanamycin 30 μg, sulphamethoxazole 25 μg, sulphamethoxazole/ trimethoprim 25 μg, tetracycline 30 μg, ticarcillin 75 μg, vancomycin 30 μg. This antimicrobial panel was selected to test the major groups of antimicrobials. Briefly, frozen isolates were thawed and cultured in BHI broth (Bio-Rad) at 35 to 37°C for 24 h. A portion of the culture broth was inoculated into 6 mL of 0.9% sterile physiological saline solution until a turbidity of 0.5 McFarland was reached (1.0 for vancomycin (Wongthong et al. 2015 (link))). Using a sterile swab, the solution was spread on Muller-Hinton agar plates (Oxoid). Antimicrobial disks (Oxoid) were placed on Muller-Hinton agar plates which were incubated at 37°C for 18 to 24 h. At the end of incubation, the diameters of the growth inhibitory zones were measured, and these were interpreted using specific CLSI tables whereby the bacterium is classified as susceptible, intermediately susceptible or resistant (CLSI 2021).
4
Antimicrobial Susceptibility of Salmonella
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The antimicrobial susceptibility test was performed according to the Clinical Laboratory Standards Institute (CLSI) guideline using the Kirby–Bauer disk diffusion method on Muller–Hinton agar plates (Oxoid, Basingstoke, England). Antimicrobials used in this study were ampicillin (10 μg), nalidixic acid (30 µg), sulfamethoxazole + trimethoprim (23.75 μg), chloramphenicol (30 μg), cephalothin (30 μg), amoxicillin + clavulanic acid (20/10 µg), streptomycin (10 μg), ciprofloxacin (5 μg), tetracycline (30 μg), gentamicin (10 μg), and amikacin (30 μg). Antimicrobial disks used in this study were all obtained from Sensi-Disc, Becton, Dickinson and Company. Escherichia coli ATCC 25922 strains were used as a control during the antimicrobial susceptibility test. Salmonella isolates were considered multidrug-resistant when they were resistant to two or more antimicrobials belonging to different classes. The interpretation of the susceptibility test result is based on the CLSI guideline [21 ].
5
Antibiotic Susceptibility of Gram-Negative Isolates
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All identified enterobacterales and non-fermenter isolates (Acinetobacter species and Pseudomonas spp) were assessed for antibiotic susceptibility by modified Kirby Bauer disc diffusion method on Muller–Hinton agar plates (Oxoid, UK) and the Vitek-2 automated system with the AST-gram-negative card (Biomérieux, Marcy-LÉtoile, France). The antibiotics used were ceftriaxone (30 μg), amikacin (30 μg), ciprofloxacin (5 μg), ampicillin (10 μg), cefoxitin (30 μg), amoxicillin-clavulanate (10 μg), gentamicin (10 μg), aztreonam (30 μg), tigecycline (15μg), cefepime (30 μg), piperacillin-tazobactam (10 μg), ceftazidime (30 μg), levofloxacin (5 μg), co-trimoxazole (30 μg), imipenem (10 μg) and meropenem (10 μg). The strains were characterized as susceptible, intermediate or resistant according to the Clinical and Laboratory Standard Institute (CLSI) guidelines.16
6
Antibiotic Susceptibility Testing Protocol
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Confirmed isolates tested using disk diffusion method on Muller-Hinton agar plates (Oxoid) for susceptibility to commonly used antibiotics (Oxoid, UK), amikacin (AK30, 30 mcg), amoxicillin/clavulanic acid (AMC30, 20/10 mcg), ampicillin (AMP 10, 10 mcg), cefotaxime (CTX30, 30 mcg), ceftriaxone (CR30, 30 mcg), cephalothin (KF30, 30 mcg), chloramphenicol (C30, 30 mcg), ciprofloxacin (CIP5, 5 mcg), gentamicin (GN10, 10 mcg), streptomycin (S10, 10 mcg), and tetracycline (TE30, 30 mcg). The results were interpreted according to CLSI [19 ].
7
Antibacterial Activity Screening of AgPtNPs
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A Kirby–Bauer disk diffusion susceptibility test was used to screen the antibacterial activity of AgPtNPs against different ATCC bacteria, and MDR and MS clinical isolates. Briefly, fresh colonies were used to prepare an inoculum at 0.5 McFarland turbidity. The bacterial suspension was homogeneously plated on Muller Hinton agar plates (Oxoid, Basingstoke, Hampshire, MA, USA). Different paper disks were placed on the agar plate, and loaded with 10 µL of AgPtNPs, AgNPs and PtNPs. A Vancomycin disk (Thermo Fisher Scientific, Waltham, MA, USA) was used for Gram positive bacteria and an ampicillin disk for Gram negative bacteria (Thermo Fisher Scientific, Waltham, MA, USA). Finally, plates were incubated at 37 °C for 24 h until the zone of inhibition was observed.
8
Anti-biofilm Properties of Essential Oils
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Four bacterial strains from American Type Culture Collection (ATCC, Rockville, MD, USA) and clinical isolates were used as controls to test the anti-biofilm properties of the EO and synergistic effect in combination with antibiotic drugs, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Staphylococcus epidermidis IG4, Staphylococcus aureus IG22. The last two strains were isolates from Department of Biomedical Science and Oncology—University of Bari. The isolates were identified by assimilation profiles using the biochemical tests performed with the commercial system API® (bioMérieux, Marcy l’Ete, Grenoble, France). Stocks were maintained at −80 °C in Triptic soy broth with 10–25% glycerol (Oxoid, Italy) solution. All strains were stored at −20 °C in glycerol stocks and were subcultured on Muller Hinton agar plates (Oxoid, Rodano, Italy) to ensure viability and purity before the beginning of study.
The bacterial species were cultured on Mueller Hinton agar (MHA, Oxoid), and each bacterial suspension was composed of 2–3 colonies of each strain taken from an MHA plate and dissolved in 2 mL of MHB (Mueller Hinton Broth, Sigma-Aldrich, St. Louis, MO, USA) [26 ].
The bacterial species were cultured on Mueller Hinton agar (MHA, Oxoid), and each bacterial suspension was composed of 2–3 colonies of each strain taken from an MHA plate and dissolved in 2 mL of MHB (Mueller Hinton Broth, Sigma-Aldrich, St. Louis, MO, USA) [26 ].
9
Antibacterial Potential of Bacterial Cell-Free Extracts
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Muller-Hinton agar plates (Oxoid) were prepared and seeded with log phase cells of P. mirabilis (105 CFU/mL). Then, 5 mm wells were created with a sterile crok pourer and loaded with 100 μL of CFE. Finally, the plates were incubated for 24 h at 37 °C and the inhibition zones were measured in millimeters. The antibacterial activity was done in triplicates to confirm the antibacterial activity [11 (link)].
10
Carbapenem Resistance Profiling
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The minimum inhibitory concentrations (MICs) of meropenem and imipenem were measured on Muller Hinton Agar plates (Oxoid, Basingstoke, UK) by the agar dilution method and interpreted according to CLSI breakpoint.15 meropenem and imipenem were purchased from Himedia, Mumbai (India). Antimicrobial profile against different anti-pseudomonas drugs was done for the carbapenem-resistant isolates using disc-diffusion method according to CLSI.15
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