Mueller hinton mh

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Mueller-Hinton (MH) is a type of agar medium used in microbiology laboratories. Its core function is to support the growth of a wide range of bacteria, including both fastidious and non-fastidious organisms, for the purpose of antimicrobial susceptibility testing.

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Mueller-Hinton (18 citations) Mueller-Hinton Broth (MH; (4 citations) Mueller Hinton Agar (MH; (3 citations) Mueller-Hinton agar plates (MH, (2 citations) Mueller Hinton Broth (MH Broth; (2 citations)

9 protocols using mueller hinton mh

Antimicrobial Susceptibility Evaluation

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Antimicrobial susceptibility assays were performed according to a method described in our recent studies [9 (link),10 (link)]. Blank disks (6 mm, Oxoid, Basingstoke, UK) and Mueller–Hinton (MH) were purchased from Oxoid, Basingstoke, UK. An aliquot of 10 μL crude extract (500 μg/mL) was added onto each disk and the bacteriostatic effect on the corresponding strains evaluated by measuring the diameter of the inhibition zone after incubation at 37 °C for 12 h. A gentamicin disk (10 μg, Oxoid, Basingstoke, UK) was used as a positive control, while the methanol phase with water and chloroform phase with anhydrous ethanol were used as negative controls.
Broth dilution testing (microdilution) was carried out to determine MICs of the extracts according to the standard method issued by the Clinical and Laboratory Standards Institute, USA (CLSI, M100-S28, 2018). The standard solution of gentamicin (100 µg/mL) was purchased from the National Standard Material Information Center, Beijing, China [9 (link)].

Liu Y., Tang Y., Ren S, & Chen L. (2023). Antibacterial Components and Modes of the Methanol-Phase Extract from Commelina communis Linn. Plants, 12(4), 890.

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Antibacterial Efficacy of Herbal Extracts

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The antibacterial properties of heather herb and oak bark extracts were tested using the standard laboratory method to determine this susceptibility, that is, the agar disk diffusion test.
A standardized bacterial inoculum of each tested bacteria with 0.5 McFarland density was prepared and inoculated using a sterile cotton swab on Mueller-Hinton (MH) (Oxoid, UK) agar or Mueller-Hinton agar with 5% sheep blood (Oxoid) (MHBA). After inoculation, four sterile filter paper disks (5 mm in diameter) were placed on the agar surface and impregnated with 15 μL of heather herb or oak bark semi-solid extracts, which were prepared by evaporating the solvent and were quantitatively moved (dissolved with water up to 5 mL) to vials.
The MH and MHBA agar plates were incubated under suitable conditions in a thermostat (Memmert, Germany) for 18 h at 37°C. Twenty-four hours later, the antibacterial properties of the selected samples were analyzed by measuring the sterile area (diameter) around the disk.

Šukele R., Skadiņš I., Koka R, & Bandere D. (2022). Antibacterial effects of oak bark (Quercus robur) and heather herb (Calluna vulgaris L.) extracts against the causative bacteria of bovine mastitis. Veterinary World, 15(9), 2315-2322.

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Cross-immunity Assays for Bacteriocin Relatedness

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To determine the relatedness of the bacteriocins produced by the strains, cross-immunity assays were performed during which the producing strains were tested against each other by conducting spot bioassays using each producing strain as a producer and also as a target strain. More precisely, the two 96-well plates were stamped using a replica platter (Sigma-Aldrich, Germany) onto clean Tryptic Soy Agar (TSA) plates or Mueller Hinton (MH; Oxoid) plates; the plates were incubated aerobically overnight at 37°C, before UV irradiation for 45 min. These were then overlaid with the same media (0.75% w/v agar seeded with 0.25% of an overnight culture of the producing strains) and grown aerobically overnight. Cross-immunity was performed for each of the two 96-well plates separately. Strains with a distinct profile from the two 96-well plates were merged into one and cross-immunity was repeated as described-above leading to 80 strains with a distinct profile (Supplementary Table S1). The 80 strains were stored individually.

Angelopoulou A., Warda A.K., O’Connor P.M., Stockdale S.R., Shkoporov A.N., Field D., Draper L.A., Stanton C., Hill C, & Ross R.P. (2020). Diverse Bacteriocins Produced by Strains From the Human Milk Microbiota. Frontiers in Microbiology, 11, 788.

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Antibiofilm Activity of Essential Oils against Salmonella

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Salmonella enterica serovar Enteritidis ATCC 13076 (S. Enteritidis) strains were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). M63 and Mueller Hinton (MH) culture media were obtained from OXOID (Hampshire, UK). All reactions and antibiofilm activity assays were performed using Milli-Q water of 18.2 Ω resistivity, extracted from the Smart 2 Pure Kit (Thermo Fisher Scientific, Waltham, MA, USA). Prior to determining the antibiofilm activity of the EOs, the Salmonella strain was grown in M63 medium [54 (link)] at 37 °C.

Guillín Y., Cáceres M., Stashenko E.E., Hidalgo W, & Ortiz C. (2023). Untargeted Metabolomics for Unraveling the Metabolic Changes in Planktonic and Sessile Cells of Salmonella Enteritidis ATCC 13076 after Treatment with Lippia origanoides Essential Oil. Antibiotics, 12(5), 899.

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Microbial Susceptibility Testing Protocol

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Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined using a broth microdilution method (Kim et al., 2020 (link)). Each bacterial strain (1 ×10⁶ CFU/mL) was incubated with identified compounds at concentrations ranging from 0.01 to 2 mg/mL in a 96-well microplate for 24 h at 37°C. The concentration of the compound with no visible bacteria growth was regarded as the MIC. The wells with no visible bacterial growth were taken for colony counting and MBC was determined as the lowest concentration with no colony growth on a Mueller-Hinton (MH; Oxoid, Basingstoke, United Kingdom) agar plate.

Kim G., Xu Y., Zhang J., Sui Z, & Corke H. (2022). Antibacterial Activity and Multi-Targeting Mechanism of Dehydrocorydaline From Corydalis turtschaninovii Bess. Against Listeria monocytogenes. Frontiers in Microbiology, 12, 799094.

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Antibacterial Efficacy of ZnO against E. coli and S. aureus

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Two bacteria strains of differing Gram stain properties (Gram-negative Escherichia coli (E. coli) CCM 3954 and Gram-positive Staphylococcus aureus (S. aureus) CCM 3953) were used to assess the efficacy of antibacterial treatment using the different types of ZnO. Stock cultures were kept at −20 °C and a fresh culture was prepared the day before each new experiment. 1 mL of the stock culture was added to 1 Mueller Hinton (MH, Oxoid, Brno, Czech Republic) agar plate and incubated at 37 °C overnight. The following day, the growth on the agar surface was removed using sterile loop and added to 5 mL MH broth (Oxoid, Brno, Czech Republic) and diluted 1:1000 in MH broth to achieve a McFarland’s density of 0.5 (Den 1B Densitometer, BioSan, Riga, Latvia). This value is equivalent to approximately 1×10⁸ colony forming units per millilitre (cfu/mL).

Rutherford D., Jíra J., Kolářová K., Matolínová I., Mičová J., Remeš Z, & Rezek B. (2021). Growth Inhibition of Gram-Positive and Gram-Negative Bacteria by Zinc Oxide Hedgehog Particles. International Journal of Nanomedicine, 16, 3541-3554.

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Phenotypic Characterization of Klebsiella pneumoniae

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A total of ten K. pneumoniae clinical isolates were obtained from the strain collection at the Bacterial Molecular Genetics research group, Universidad El Bosque, Bogotá, Colombia. Control strains consisting of previously characterized K. pneumoniae isolates, the LM21 gfp strain [27] (link), and mutants lacking genes relevant to biofilm formation [28] (link) were also included. Unless otherwise indicated, all strains were grown at 37 °C in bacterial culture media Luria Bertani (LB; Oxoid) or Mueller-Hinton (MH; Oxoid), with agitation at 180 rpm, overnight for 16 hours. Media were supplemented with antibiotics (Sigma-Aldrich) when necessary: Meropenem (MER), amikacin (AMK), ciprofloxacin (CIP), trimethoprim-sulfamethoxazole (TS); the latter prepared at a 1:19 combination of two antibiotics that inhibit two steps in the synthesis of tetrahydrofolate. LB agar, supplemented with Congo red (Sigma-Aldrich), Coomassie blue (Sigma-Aldrich), and calcofluor (fluorescent brightener 28; Sigma-Aldrich), was used to assess colony morphology [29, (link)30] (link).

Posada L., Acosta I.F., Zárate L., Lorenzo P., Elrod M.G., & Zambrano M.M. (2020). Biofilm and persister cell fomation variability in clinical isolates of Klebsiella pneumoniae in Colombia. Universitas Scientiarum, 25(3), 545-571.

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Isolation of Plasmid-Containing Bacteria from Environmental Samples

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Plasmid-containing bacteria were isolated as follows: 5 g of BPS sample was resuspended in 50 ml of sterile physiological-saline solution and the suspension was shaken at 180 rpm for 1 h at room temperature. The bacteria were harvested from the supernatant and aliquots plated in different cycloheximide-containing media (200 μg/ml) as well as in antibiotic-containing Luria-Bertani (LB) medium (Sambrook et al., 1989 ) and incubated at 28 °C for 48 h. Eight different types of agar plates were used for colony isolation: LB, tryptone-yeast (TY; Beringer, 1974) , eosinmethylene-blue (EMB; Levine, 1918), MacConkey (Mck; Macconkey, 1905) , dextrose-agar (Dex; Sigma-Aldrich), Mueller-Hinton (MH; Oxoid Ltd, UK), glutamate-sucrose minimal medium (GS) (Del Papa et al., 1999) (link) and M9 minimal medium (M9) (Kempner and Miller, 1972) . When required, the following antibiotics were added to LB medium: 120 μg/ml neomycin (Nm), 400 μg/ml streptomycin (Str), 200 μg/ml ampicillin (Ap), 10 μg/ml tetracycline (Tc), 25 μg/ml kanamycin (Km), 50 μg/ml gentamicin (Gm), 20 μg/ml trimethoprim (Tp), 200 μg/ml erythromycin (Er), 20 μg/ml chloramphenicol (Cm), 5 μg/ml nalidixic acid (Nx), 50 μg/ml carbenicillin (Cb) or 100 μg/ml rifampicin (Rf).

Martini M.C., Albicoro F.J., Nour E., Schlüter A., van Elsas J.D., Springael D., Smalla K., Pistorio M., Lagares A, & Del Papa M.F. (2015). Characterization of a collection of plasmid-containing bacteria isolated from an on-farm biopurification system used for pesticide removal. Plasmid, 80.

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Detecting Efflux-Mediated Antibiotic Resistance

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The MIC values of meropenem and imipenem were detected with the two-fold agar dilution method (as recommended by the 2012 CLSI) in the presence and absence of efflux pump inhibitors to determine the contribution of efflux pumps to resistance. Mueller-Hinton (MH; Oxoid, Basingstoke, UK) agar containing carbonyl cyanide 3chlorophenylhydrazone (CCCP; Sigma-Aldrich, St. Louis, MO, USA) or phenyl-arginine-β-naphthylamide (PAβN; Sigma-Aldrich) was prepared. Seoul, Korea. To confirm the activity of Gram-negative bacteria on the RND pump, experiments were performed using MH agar supplemented with CCCP or PAβN. (Pagès et al., 2005) . To evaluate the effect of CCCP or PAβN on bacterial growth, all the bacteria were cultured in the MH agar containing the final concentration of CCCP (25 μg/mL) or PAβN (70 μg/mL). The criteria for a positive efflux phenotype were defined as an MIC value with CCCP or PAβN that decreased 4-fold or more compared to the MIC values without the efflux pump inhibitors (Shi et al., 2005) . Both the sensitive and resistant strains were used for the test, and A. baumannii ATCC17978 was selected as the quality control strain.

Ju Y., Kim Y., Chang C.L., Choi G., & Hyun K. (2021). Relationship between AdeABC Efflux Pump Genes and Carbapenem in Multidrug-resistant Acinetobacter baumannii. Journal of Experimental & Biomedical Sciences/Biomedical Science Letters, 27(2), 59-68.

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