The minimum inhibitory concentrations (MICs) were determined according to the clinical laboratory Standards Institute (CLSI) guideline (2015) using micro titer plate method. In this method, colonies of lactobacilli from TSA were suspended in Muller Hinton broth (Merck, Germany) and the turbidity of suspension was adjusted to 0.5 McFarland and subsequently diluted in Muller Hinton broth (1:100) to reach a final concentration of 1 × 106 CFU/ml. Dilutions of cephalothin (Sigma-Aldrich), cefazolin, amikacin, ceftriaxone and ceftazidime (Exir, Broujerd, Iran) were made in distilled water. The antibiotics were prepared at different concentrations ranged from 0.125 to 512 μg/ml. Each well was filled with 100 μl of each dilution of the antibiotic and 100 μl of bacterial suspension. Each plate included positive controls (bacteria without an antimicrobial), negative controls (medium only). Micro titer plates were incubated at 37°C for 24 h. MIC was determined as the minimum antibiotic concentration that inhibited the visible growth (19 ). All tests were carried out in duplicates.
Muller hinton broth
Manufactured by Merck Group
Sourced in Germany, United States, Italy
Muller-Hinton broth is a microbiological culture medium used for the growth and isolation of various bacteria. It provides the necessary nutrients and conditions for the cultivation of a wide range of microorganisms. The broth is commonly used in antimicrobial susceptibility testing to determine the sensitivity of bacteria to different antibiotics.
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Lab products found in correlation
Muller hinton agar, by Merck Group (5 mentions)
Ciprofloxacin, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
96 well round bottom plate, by Corning (3 mentions)
Human serum, by BioIVT (3 mentions)
Glycine, by Merck Group (2 mentions)
Chitosan, by Merck Group (2 mentions)
Glacial acetic acid, by Merck Group (2 mentions)
Gentamicin, by Merck Group (2 mentions)
Nutrient broth, by Merck Group (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Human serum albumin (hsa), by Merck Group (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Fungizone amphotericin, by Thermo Fisher Scientific (1 mentions)
Antimycotic, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Glutaraldehyde ga, by Merck Group (1 mentions)
Low molecular weight marker protein, by GE Healthcare (1 mentions)
39 protocols using muller hinton broth
1
Antibiotic Susceptibility of Lactobacilli
2
Colocasia esculenta Tuber Powder-based Biomaterials
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Colocasia esculenta tuber (CET) powder was obtained from PT. Sentra Biogen Bandung, Indonesia. Poly(ethylene oxide) (PEO, average Mw = 600 kDa), chitosan (CS, ≥ 75% deacetylation degree), bovine serum albumin (BSA), and trypan blue solution were purchased from Sigma Aldrich (Saint Louis, MO, USA). Glacial acetic acid, ethanol, glycine, glutaraldehyde (GA, 25% solution in H2O), Muller Hinton Agar, and Muller Hinton Broth were purchased from Merck (Darmstadt, Germany). Low-molecular-weight marker protein was purchased from GE Healthcare (Little Chalfont, UK). Minimum Essential Medium (MEM), penicillin-streptomycin, fetal bovine serum (FBS), trypsin-EDTA, fungizone amphotericin, gentamicin, phosphate buffered saline (PBS), antibiotic, and antimycotic were purchased from Gibco (Grand Island, USA). Human skin fibroblast cell line (BJ cell, ATCC CRL-2522), Gram-positive Staphylococcus aureus (S. aureus, ATCC 6538), and Gram-negative Escherichia coli (E. coli, ATCC 8939) were purchased from American Type Culture Collection (Rockville, MD, USA). PrestoBlue reagent was purchased from Invitrogen (Carlsbad, CA).
3
Antimicrobial Activity of Ag-NPs, Oil, and Extract
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The minimum inhibitory concentration (MIC) values of Ag-NPs, oil, and extract were determined by broth microdilution assay. The Ag-NPs were serially diluted two-fold with deionized water in concentrations ranging from 5.00 to 7.812 μg/mL. The oil and extract were serially diluted two-fold with 10% dimethyl sulfoxide (DMSO) (Merck, Germany) containing 1.00 - 1.56 mg/mL of oil. After shaking, 100 mL of diluted Ag-NPs, oil, and extract was added to each well of 96-well microtiter plates. Cation-adjusted Muller-Hinton broth (Merck, Germany) was used as the broth medium. Microbial suspensions were adjusted to 0.5 MacFarland and diluted to 1 × 106 CFU/mL, then 100 mL of the suspension was added to each well and incubated at 35 ± 2°C for 24 hours. MIC values were determined as the lowest concentration of compound that inhibited bacteria after 24 hours (17 (link), 18 ).
4
Antimicrobial Efficacy of S. macrantha EOs
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Antibacterial activity of S. macrantha EOs at different growth stages was evaluated using the broth micro-dilution method based on the estimation of the minimum inhibitory concentration (MIC) [31 ]. The stock solutions of the EOs were prepared in dimethylsulphoxide (DMSO) (Sigma Aldrich, Darmstadt, Germany) and two-fold serial dilutions were inserted in 96-well microtitre plates. The bacterial suspensions in Muller Hinton broth (Merck, Darmstadt, Germany) were standardized to 106 CFU/µL and then added to microtiter plates with EO concentrations from 2.5 to 160 µg/L. Gentamicin (Sigma Aldrich, Darmstadt, Germany) and DMSO were used as positive and negative controls, respectively. The microtiter plates were incubated at 37 ± 2 °C for 72 h. The first plate that contained no visibility grown bacteria was considered as the MIC.
5
Biofilm Eradication Concentration Evaluation
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The minimum biofilm eradication concentration (MBEC) values of levofloxacin, amikacin, meropenem, tigecycline and cefepime in A. baumannii isolates were measured using the broth microdilution method.19 (link) First, the isolates were cultivated in the sterile 96-well polystyrene microtiter plates for an overnight at 37°C to allow for the biofilm formation. The biofilms were then exposed to the concentrations of 2–4,096 µg/mL of levofloxacin, 4–8,192 µg/mL of amikacin, 2–8,192 µg/mL of meropenem, 0.5–2048 µg/mL of tigecycline and 16–16,384 µg/mL of cefepime for an overnight at 37°C.Then, the wells were washed with sterile PBS three times, and incubated with Muller Hinton Broth (Merck, Darmstadt, Germany) for an overnight at 37°C. The MBEC was proposed as any viable cell was not recovered from the biofilm material or, ie, OD of 570nm (OD570) was <0.1. All tests were repeated in triplicate.
6
Antibacterial Efficacy of CO-SLN and CO
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The minimum inhibitory concentration (MIC) of CO-SLN and CO were investigated using micro broth dilution assay in a 96- well plate. Based on the preliminary study results, the CO-SLN (80 − 50 µg/ml) and CO (170 − 140 µg/ml) were serially diluted in sterile Muller-Hinton broth (Merck, Germany) in each well. The final concentration of E. coli was adjusted to 106 CFU/mL for each well and incubated at 37 ° C for 20 h. The MIC values were determined as the lowest concentration of drugs that inhibited bacterial growth. To determine the Minimum Bactericide Concentration (MBC), 100 µL content of no growth wells were cultured in Muller-Hinton agar (Merck, Germany) and were incubated at 37 °C for 24 h. The MBCs were reported as the lowest concentration that resulted in killing 99.9% of bacterial cells.
7
Determining Minimum Inhibitory Concentration
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Minimum inhibitory concentration (MIC) is the lowest concentration of an antibiotic able to inhibit visible microorganism growth after 24 hours of incubation. We used E-test method to determine MIC. For this purpose, all A. baumannii samples were cultured on Blood Agar medium and incubated for 24 hours at 37˚C. Then two or three colonies of the cultured samples were transferred to Mueller–Hinton Broth (Merck) and placed in the incubator to get 1.5 × 108 colony-forming units (CFU)/mL. In the next step, each sample was cultured on three Muller–Hinton Broth plates. Two E-test strips containing different antibiotics were placed on each plate. After 24 hours of incubation, MIC number of each antibiotic was read on the strip.
8
Cell Viability and Cytotoxicity Assay Protocol
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RPMI-1640 medium was purchased from Biosera, England, fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin – streptomycin, trypsin, trypsin – EDTA were purchased from PAA Laboratories, Austria. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT), Bovine serum albumin (BSA), trypan blue dye, ammonium sulfate, Dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich, USA. β- Mercaptoethanol, sodium dodecyl sulfate, tris (hydroxymethyl) aminomethane, glycine, hydrochloric acid, sodium carbonate, Skim milk powder, Muller Hinton broth, peptone, agar, agarose, and acrylamide were purchased from Merck, Germany.
9
Biofilm Eradication Concentrations of Antibiotics
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The minimum biofilm eradication concentration (MBEC) values of levofloxacin, amikacin, meropenem, tigecycline, colistin and cefepime in A. baumannii isolates were measured using the broth microdilution method. First, the isolates were cultivated in the sterile 96-well polystyrene microtiter plates for an overnight at 37°C to allow for the biofilm formation. The biofilms were then exposed to the concentrations of 2 to 4096 µg/mL of levofloxacin, 4 to 8192 µg/mL of amikacin, 2 to 4096 µg/mL of meropenem, 0.5 to 2048 µg/mL of tigecycline and 16 to 16,384 µg/mL of cefepime for an overnight at 37°C. Then, the wells were washed with sterile PBS three times, and incubated with Muller Hinton Broth (Merck, Germany) for an overnight at 37°C. The MBEC was proposed as any viable cell was not recovered from the biofilm material or i.e. OD570 was less than 0.1. All tests were repeated in triplicate.21 (link)
10
Determination of Antibiotic MICs
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All tests were carried out in duplicates. Minimum inhibitory concentrations (MICs) were carried out according to the clinical laboratory standards institute (CLSI) guidelines by using microtiter plate method. To this reason, colonies from TSA were suspended in Muller Hinton broth (Merck, Germany) and adjusted to 0.5 McFarland and 1:100 diluted in Muller Hinton broth to reach to a final concentration of 1×10⁶ CFU/mL.
Dilutions of ciprofloxacin and imipenem (Exir, Iran) were made in distilled water and phosphate buffer 0.01 M, respectively. The antibiotics were prepared at different concentrations ranged from 0.25 to 512 µg/mL. Each well filled with 100 µL of different dilution of the antibiotic and 100 µL of bacterial suspension. Microtiter plates were incubated at 37 ° C for 24 hours. MIC was determined as the minimum antibiotic concentration that inhibited the visible growth (12 ).
Dilutions of ciprofloxacin and imipenem (Exir, Iran) were made in distilled water and phosphate buffer 0.01 M, respectively. The antibiotics were prepared at different concentrations ranged from 0.25 to 512 µg/mL. Each well filled with 100 µL of different dilution of the antibiotic and 100 µL of bacterial suspension. Microtiter plates were incubated at 37 ° C for 24 hours. MIC was determined as the minimum antibiotic concentration that inhibited the visible growth (12 ).
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