Standard conditions or modifications from CLSI methods by 25% goat serum supplementation were performed to determine the MIC of tildipirosin, gamithromycin, oxytetracycline, and danofloxacin. Minimum inhibitory concentration tests were performed by the microdilution broth technique (Clinical and Laboratory Standards Institute 2009 ) using U-bottom 96-well microtiter plates. Serial two-fold dilutions of the antimicrobial agents were prepared starting from the stock solution. Broth dilutions were made using Mueller–Hinton broth (MHB) (Merck, Madrid, Spain) for CNS, S. aureus, and E. coli. To investigate Streptococcus spp., cation-adjusted Mueller–Hinton broth (Merck, Madrid, Spain) with 5% defibrinated horse blood (Thermo Fisher Scientific, Massachusetts, USA) was used. Concentrations of all antibiotics ranging from 0.03 to 128 mg/l were used. Inocula were prepared by diluting an overnight MHB culture in buffered saline solution to a density of 0.5 on the McFarland Turbidity Scale and finally diluting again 40-fold before testing. The U-bottomed microtiter plates were incubated at 37 °C and observed 24 h later. The MIC was defined as the lowest concentration of antibiotic at which bacterial growth was completely inhibited. The reference strains S. aureus (ATCC 29213) and E. coli (ATCC 25922) were used as controls.
Mueller hinton broth (mhb)
Manufactured by Merck Group
Sourced in Germany, United States, Spain, United Kingdom, Poland, Australia, Canada, Sao Tome and Principe, Portugal, Austria, France
Mueller-Hinton broth is a microbiology culture medium used for antimicrobial susceptibility testing. It is a standardized medium recommended by the Clinical and Laboratory Standards Institute (CLSI) for conducting antimicrobial disk diffusion and minimum inhibitory concentration (MIC) tests.
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Lab products found in correlation
Mueller hinton agar (mha), by Merck Group (76 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (19 mentions)
Tween 80, by Merck Group (12 mentions)
Sabouraud dextrose agar, by Merck Group (9 mentions)
Sabouraud dextrose broth (sdb), by Merck Group (9 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Ciprofloxacin, by Merck Group (9 mentions)
Sodium chloride (nacl), by Merck Group (8 mentions)
Phosphate buffered saline (pbs), by Merck Group (8 mentions)
Mannitol salt agar, by Merck Group (7 mentions)
Vancomycin, by Merck Group (7 mentions)
Methanol, by Merck Group (7 mentions)
Gallic acid, by Merck Group (7 mentions)
Resazurin, by Merck Group (7 mentions)
Gentamicin, by Merck Group (7 mentions)
Blood agar, by Merck Group (6 mentions)
Crystal violet, by Merck Group (6 mentions)
Quercetin, by Merck Group (6 mentions)
Ethanol, by Merck Group (6 mentions)
Acetic acid, by Merck Group (6 mentions)
383 protocols using mueller hinton broth (mhb)
1
Antimicrobial Susceptibility Testing Protocols
2
Gram-Positive S. aureus ATCC 29213 Cultivation
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The strain used in this study was Gram-positive S. aureus ATTC 29213. Bacteria were cultured in Mueller-Hinton broth (MHB; Sigma-Aldrich) and incubated at 37 °C for 24 h. The final density of the culture was 2–3 × 105 CFU/mL, which was confirmed by regular colony counts during the study. A fresh overnight culture was established for each experiment.
3
Antibiotic Susceptibility of Lactic Acid Bacteria
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A single colony of LAB was selected, inoculated into a 10‐ml tube containing Mueller–Hinton broth (Merck; CLSI, 2006 ), and incubated at 37°C. When turbidity of tube reached 0.5 McFarland, it was streak cultured on a plate containing 90% (w/v) Mueller–Hinton agar and 10% (w/v) MRS dehydrated broth (pH = 6.7). After implantation of the antibiotic disc on the plates, they were incubated at 37°C for 24–48 h.
Samples with a zone diameter of ≤15 mm are considered resistant. All antibiotic susceptibility tests were carried out in triplicate. Antibiotic susceptibility test was performed for amoxicillin (25 µg/disc), ampicillin (10 µg/disc), chloramphenicol (30 µg/disc), erythromycin (15 µg/disc), streptomycin (10 µg/disc), tetracycline (30 µg/disc), vancomycin (30 µg/disc), cefotaxime (30 µg/disc), kanamycin (30 µg/disc), meropenem (10 µg/disc), nalidixic acid (30 µg/disc), gentamycin (10 µg/disc), and ceftazidime (30 µg/disc) that all had been provided from Padtanteb Company. The results showed the percentage of sensitivity to the antibiotics.
Samples with a zone diameter of ≤15 mm are considered resistant. All antibiotic susceptibility tests were carried out in triplicate. Antibiotic susceptibility test was performed for amoxicillin (25 µg/disc), ampicillin (10 µg/disc), chloramphenicol (30 µg/disc), erythromycin (15 µg/disc), streptomycin (10 µg/disc), tetracycline (30 µg/disc), vancomycin (30 µg/disc), cefotaxime (30 µg/disc), kanamycin (30 µg/disc), meropenem (10 µg/disc), nalidixic acid (30 µg/disc), gentamycin (10 µg/disc), and ceftazidime (30 µg/disc) that all had been provided from Padtanteb Company. The results showed the percentage of sensitivity to the antibiotics.
4
Bioautography for Plant Extract Bioactivity
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The bioautography procedure as described by Begue and Kline [39 (link)] and refined for plant extracts by Masoko and Eloff [40 ] was used to identify bioactive chromatograms of plant extracts. The duplicate of TLC plates prepared were dried overnight under a stream of air to remove residual TLC solvents which may be harmful to bacteria. A 10 ml of overnight broth culture of test bacteria in Mueller Hinton broth (Merck) was centrifuged at 5300 x g for 20 min. The supernatant was discarded and the pellet was re-suspended in 2–4 ml of fresh broth and adjusted to make 0.5 McFarland standards which is equivalent to1.0 × 10−7 cfu/mL [41 ]. The dried chromatographic plates were sprayed with the test bacteria until they were completely wet in a Laminar flow cabinet (Labotec, SA). The plates were incubated overnight at 37 °C in a clean chamber at 100% relative humidity. After overnight incubation, plates were sprayed with a 2 mg/ml solution of INT (p-iodonitrotetrazolium violet, Sigma Chemicals). Plates were incubated and monitored for colour development at 2 h and further incubated overnight. Inhibition of growth of tested organisms was indicated by clear or yellow zones on chromatogram an indication of where reduction of INT to the coloured formazan did not take place.
5
Rapid ESBL-Production Detection Assay
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Micronaut-S beta-lactamase VII Plate (Merlin Diagnostika, Berlin, Germany) was used for the phenotypic detection of ESBL-production, including AmpC, metallo-beta-lactamase (MBL), and carbapenemase (KPC) according to the criteria described by 22 . A 50 μL aliquot of 0.5 McFarland-standardised microbial suspension of the isolate was initially vortexed in 10 mL of Mueller Hinton Broth (Merck, Darmstadt, Germany). Subsequently, 100 μL of this suspension was pipetted into each well of the plate. After that, the plate was incubated overnight at 37 °C. The reading was recorded using a Thermofisher Multiskan FC Spectrometer. The MIC analysis was automatically performed by the MCN6 Software (Sifin, Berlin, Germany).
6
Nanocarrier-Mediated Drug Delivery for MRSA Treatment
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Lecithin, monostearin, soybean oil, TMP/SMZ, phosphate-buffered saline (PBS), dialysis bag (10,000 nominal-molecular-weight cutoff (NMWCO)), acetonitrile (HPLC grade), di-potassium hydrogen phosphate, ethanol (EtOH), Hanks’ balanced salt solution (HBSS), blood agar, Mueller Hinton broth (MHB), hematoxylin and eosin (H&E), mannitol, Roswell Park Memorial Institute (RPMI)-1640 medium, penicillin and streptomycin (pen/strep) antibiotics, and fetal bovine serum (FBS) were purchased from Merck (Darmstadt, Germany). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). A 12-well Transwell® insert (0.4 μm pore size, 1.12 cm2 area) was purchased from Corning (Corning, NY, USA). MRSA ATCC 33591 was obtained from the culture collection of the Iranian Research Organization for Science and Technology (IROST), Tehran, Iran. Human embryonic kidney HEK 293 cells, human colon adenocarcinoma Caco-2 cells, and male Balb/c mice (6–8 weeks old) were purchased from the Pasteur Institute of Iran (Tehran, Iran).
7
Formulation and Evaluation of Antimicrobial Topical Gel
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Citral, Tween® 80, Mueller Hinton agar, Mueller Hinton Broth, and nutrient dextrose agar were supplied from Merck Chemicals Co. (Germany). Poloxamer® 407 and Miglyol® 812 were obtained from Sigma-Aldrich Company (USA) and Sasol Company (Germany), respectively. Glyceryl palmitostearate (Precirol® ATO-5) was kindly donated by Gattefossé Company (France).
8
Analytical-Grade Compounds for Research
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All the compounds used in this work were of analytical grade, with a purity higher than 95%. The standard substances for GC identification and determination, the chemicals for the antioxidant capacity assays, and the reagents for soybean lipoxygenase inhibition were purchased from Sigma–Aldrich, Spain. The following culture media for bacteria and yeasts were provided by VWR Chemicals, Spain: Mueller Hinton Agar (MHA), Mueller Hinton Broth (MHB), Roswell Park Memorial Institute (RPMI-1640), Sabouraud Dextrose Agar (SDA), tryptic soy broth (TSB) and yeast peptone dextrose (YPD).
Solvents of analytic grade and buffers were purchased from Merck (Madrid, Spain). Type I (18 MΩ cm) deionized water (MilliQ-Reference, Millipore, Madrid, Spain) was used throughout in this work.
Solvents of analytic grade and buffers were purchased from Merck (Madrid, Spain). Type I (18 MΩ cm) deionized water (MilliQ-Reference, Millipore, Madrid, Spain) was used throughout in this work.
9
Antimicrobial Peptide Synthesis and Evaluation
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L. monocytogenes ATCCbaa751 and E. coli ATCC25922, were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). NovaPEG Rink Amida resin, Fmoc-protected amino acids, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro-borate (TBTU), Piperidine, N,N-Diisopropylethylamine (DIEA), Ninhydrin, Dimethylformamide (DMF), Trifluoroacetic acid (TFA), 1,2-Ethanedithiol (EDT), Triisopropylsilane (TIS) and Mueller Hinton Broth (MHB) were purchased in Merck (Darmstadt, Germany). Cholesterol, Epikuron 200® and dioleoyl-phosphatidyl-ethanolamine (DOPE) were obtained from Avanti Polar Lipids (Alabaster, Alabama, USA). Eudragit® E-100 was purchased in Evonik (Darmstadt, Germany). Ampicillin (Fersinsa Gb) was supplied by Tecnoquímicas S.A. (Cali, Colombia) and Gentamicin solution (GENFAR®, Cali, Colombia) was purchased from a local pharmacy. The datasets used and materials using during this study are available from the corresponding author on reasonable request.
10
Bactericidal Effect of Enzymes on Pathogens
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The bactericidal effect of enzymes on planktonic cells of each strain from each of S. aureus and P. aeruginosa species was independently evaluated as described previously [10 (link)]. The experiment was done using the minimum effective concentration of each enzyme and lower concentrations (Trypsin: ≤ 1 μg/ml, DNase I: ≤ 150 U/ml, β-glucosidase: ≤ 8 U/ml).
Briefly, 50 μl of Mueller Hinton broth (Merck, Germany) was added to each microtiter plate well (Tissue culture plate 96 wells, SPL, Korea). The enzyme was loaded to each well. Finally, 50 μl of bacterial suspension with a final inoculum of 106 CFU/ml was added to each well. The microtiter plate was then incubated for 20 h at 37° C. Plates were inspected based on bacterial growth. The lowest enzyme concentration that visibly inhibited microbial growth was defined as the minimum inhibitory concentration (MIC) [36 ]. The minimum bactericidal concentration (MBC) was determined by pipetting 10 μl of each well with a clear suspension onto a TSA. After incubation at 37° C for 24 h, the plates were inspected for the presence of colonies. Inoculated MHB without enzyme and MHB plus enzyme with no bacteria were considered as control groups.
Briefly, 50 μl of Mueller Hinton broth (Merck, Germany) was added to each microtiter plate well (Tissue culture plate 96 wells, SPL, Korea). The enzyme was loaded to each well. Finally, 50 μl of bacterial suspension with a final inoculum of 106 CFU/ml was added to each well. The microtiter plate was then incubated for 20 h at 37° C. Plates were inspected based on bacterial growth. The lowest enzyme concentration that visibly inhibited microbial growth was defined as the minimum inhibitory concentration (MIC) [36 ]. The minimum bactericidal concentration (MBC) was determined by pipetting 10 μl of each well with a clear suspension onto a TSA. After incubation at 37° C for 24 h, the plates were inspected for the presence of colonies. Inoculated MHB without enzyme and MHB plus enzyme with no bacteria were considered as control groups.
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