Mueller hinton agar

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

Mueller-Hinton (MH) agar is a microbiological growth medium used for the cultivation and antimicrobial susceptibility testing of certain bacteria. It provides the necessary nutrients and growth conditions for the proliferation of specific microbial species.

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38 protocols using mueller hinton agar

Antifungal and Antibacterial Susceptibility Testing

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Fluconazole, voriconazole, chloramphenicol, Mueller-Hinton (MH) agar and Sabouraud Dextrose Agar (SDA) were obtained from Thermo Fisher (Oxoid Limited, Hampshire, UK). All the organic solvents were purchased from BDH laboratory supplies (Merck Ltd, Lutterworth, UK).

Harley B.K., Neglo D., Tawiah P., Pipim M.A., Mireku-Gyimah N.A., Tettey C.O., Amengor C.D., Fleischer T.C, & Waikhom S.D. (2021). Bioactive triterpenoids from Solanum torvum fruits with antifungal, resistance modulatory and anti-biofilm formation activities against fluconazole-resistant candida albicans strains. PLoS ONE, 16(12), e0260956.

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Construction and Characterization of OXA-48 Producing E. coli

Cited 2 times

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Mueller-Hinton (MH) agar and broth were purchased from Thermo Fisher Scientific (East Grinstead, United Kingdom). Luria-Bertani (LB) broth, LB agar, ampicillin, amoxicillin, cefepime, CAZ, imipenem, meropenem, piperacillin, and tazobactam were obtained from Sigma-Aldrich (St. Louis, MO). Nitrocefin was purchased from Merck (Darmstadt, Germany). All strains used and constructed within this study are listed in Table 1. The characteristics of the clinical E. coli strain 50579417 harboring the OXA-48 plasmid (p50579417_3_OXA-48) have been described previously (61 (link), 62 (link)). The plasmid p50579417_3_OXA-48 was conjugated into rifampin-resistant E. coli TOP10 and subsequently isolated using a plasmid mini-purification kit (Qiagen, Germany). E. coli MG1655 (DA4201) was electroporated with p50579417_3_OXA-48 as published previously (63 (link)). Transformants positive for bla_OXA-48 were checked by PCR using REDTaq ready mix (Sigma-Aldrich, St. Louis, MO) and preOXA-48 primers (61 (link)).

Fröhlich C., Sørum V., Thomassen A.M., Johnsen P.J., Leiros H.K, & Samuelsen Ø. (2019). OXA-48-Mediated Ceftazidime-Avibactam Resistance Is Associated with Evolutionary Trade-Offs. mSphere, 4(2), e00024-19.

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Comparative Antifungal Susceptibility Assay

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Fluconazole, Mueller-Hinton (MH) agar, voriconazole, Sabouraud Dextrose Agar (SDA), and chloramphenicol were bought from Thermo Fisher (Oxoid Limited, Hampshire, UK). Nystatin and caspofungin were bought from Sigma Aldrich (St. Louis, MO, United States).

Harley B.K., Neglo D., Aggrey M.O., Quagraine A.M., Orman E., Jato J., Mireku-Gyimah N.A., Amengor C.D, & Fleischer T.C. (2022). Antifungal Activities of Phytochemically Characterized Hydroethanolic Extracts of Sclerocarya birrea Leaves and Stem Bark against Fluconazole-Resistant Candida albicans Strains. BioMed Research International, 2022, 4261741.

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Bacterial Antibiotic Susceptibility Testing

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Mueller-Hinton (MH) agar and broth were purchased from Thermo Fisher Scientific (East Grinstead, UK). Luria-Bertani (LB) broth, LB agar, yeast extract, agar, terrific broth, ampicillin, amoxicillin, cefepime, ceftazidime, chloramphenicol, imipenem, meropenem, piperacillin, 2,3,5 tri-phenyl tetrazolium, sodium chloride, and maltose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tryptone was obtained from Oxoid (Hampshire, UK). All strains used and constructed within this study are listed in Table S1.

Fröhlich C., Gama J.A., Harms K., Hirvonen V.H., Lund B.A., van der Kamp M.W., Johnsen P.J., Samuelsen Ø, & Leiros H.K. (2021). Cryptic β-Lactamase Evolution Is Driven by Low β-Lactam Concentrations. mSphere, 6(2), e00108-21.

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Antifungal Drug Susceptibility Assay

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Sabouraud Dextrose Agar (SDA), Mueller-Hinton (MH) agar, RPMI 1640 media, XTT assay reagent, voriconazole and chloramphenicol were obtained from Thermo Fisher (Oxoid Limited, Hampshire, UK). Other drugs used in the experiment include fluconazole (Pfizer Inc., New York, NY, United States), nystatin and caspofungin (Sigma Chemical Co., St. Louis, MO, United States).

Harley B.K., Quagraine A.M., Neglo D., Aggrey M.O., Orman E., Mireku-Gyimah N.A., Amengor C.D., Jato J., Saaka Y, & Fleischer T.C. (2022). Metabolite profiling, antifungal, biofilm formation prevention and disruption of mature biofilm activities of Erythrina senegalensis stem bark extract against Candida albicans and Candida glabrata. PLOS ONE, 17(11), e0278096.

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Antibiotic Resistance Profile Determination

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Mueller Hinton (MH) agar and broth were purchased from Thermo Fisher Scientific (East Grinstead, UK). Luria-Bertani (LB) broth, LB agar, yeast extract, agar, terrific broth, ampicillin, amoxicillin, cefepime, ceftazidime, chloramphenicol, imipenem, meropenem, piperacillin, 2,3,5 tri-phenyl tetrazolium, sodium chloride and maltose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tryptone was obtained from Oxoid (Hampshire, UK) All strains used and constructed within this study are listed in Table S1.

Fröhlich C., Gama J.A., Harms K., Hirvonen V.H.A., Lund B.A., Kamp M.W.v.d., Johnsen P.J., Samuelsen Ø., & Leiros H.S. (2020). Cryptic β-lactamase evolution is driven by low β-lactam concentrations.

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Campylobacter jejuni Strain 81-176 Cultivation

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Campylobacter jejuni strain 81–176 was cultured as previously described^{39 (link)}. Briefly, a loop of frozen C. jejuni glycerol stock was streaked onto Columbia Agar with 5% Sheep Blood or Mueller–Hinton (MH) agar (Oxoid, Basingstoke, Hampshire, UK) and incubated for 18 h at 41 °C under microaerophilic conditions of 10% CO₂, 5% O₂, and 85% N₂. Subsequently, several colonies of C. jejuni were inoculated into 100 mL fresh Mueller–Hinton broth and incubated at 41 °C under microaerophilic conditions to reach mid-log phase (determined by growth curve analysis). A mid-log culture of C. jejuni was centrifuged and washed with Dulbecco phosphate-buffered saline (DPBS), and diluted in DPBS to an OD 600 nm of 0.01 which corresponds to approximately 2.0 × 10⁷ CFU/ml. The suspended bacteria were lysed as previously described^{13 (link)}. Briefly, the bacteria were heat-killed at 65 °C for 30 min and then sonicated on ice (six 15-s pulses interspersed with 30-s pauses). The protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Rochester, USA). One milliliter of lysate (2.0 × 10⁷ CFU/ml) contained approximately 8.6 µg protein. The lysate was stored at −80 °C until use.

Taha-Abdelaziz K., Yitbarek A., Alkie T.N., Hodgins D.C., Read L.R., Weese J.S, & Sharif S. (2018). PLGA-encapsulated CpG ODN and Campylobacter jejuni lysate modulate cecal microbiota composition in broiler chickens experimentally challenged with C. jejuni. Scientific Reports, 8, 12076.

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Culturing and Transforming Campylobacter jejuni

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C. jejuni strains were routinely cultured on Mueller Hinton (MH) agar (Oxoid) supplemented with 5% defibrinated horse blood (Thermo Scientific) and 5 μg/ml trimethoprim (Tp). Defined mutants and complemented strains were selected on 10 μg/ml chloramphenicol (Cm) or 50 μg/ml kanamycin (Km), as appropriate. C. jejuni cultures were grown in standard microaerophilic conditions (5% CO₂, 5% H₂, 85% N₂, 5% O₂) at 42 °C, unless otherwise indicated. Electrocompetent Escherichia coli and C. jejuni used in cloning were prepared and transformed as previously described [14] (link). Bacterial strains and plasmids used in this study are detailed in Table 1.

Esson D., Gupta S., Bailey D., Wigley P., Wedley A., Mather A.E., Méric G., Mastroeni P., Sheppard S.K., Thomson N.R., Parkhill J., Maskell D.J., Christie G, & Grant A.J. (2017). Identification and initial characterisation of a protein involved in Campylobacter jejuni cell shape. Microbial Pathogenesis, 104, 202-211.

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Antimicrobial Susceptibility Profiling Protocol

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Susceptibility against antimicrobial drugs was determined by disk diffusion protocol using Mueller-Hinton (MH) agar (Oxoid, UK). The inhibitory zones around these antimicrobial discs were measured using a millimeter (mm) unit utilizing a metric ruler, and the results were read [43 , 44 ]. Ten antibiotic disks (Merseyside, U.K.) used included amoxicillin 20 µg, clavulanic acid 10 µg (AUG 30C), trimethoprim (TM, 15 µg), ampicillin10 µg\ sulbactam 10 µg (SAM, 20 C), tetracycline (T, 30 µg), erythromycin (E, 10 µg), cefixime (CFM 5 µg), doxycycline (DXT, 30 µg), imipenem (IPM, 10 µg), chloramphenicol (C 30 µg), and streptomycin (S 25 µg). Multidrug resistance (MDR) was detected according to the work of Magiorakos et al. [45 (link)]. The isolates resistant against three or more separate antimicrobial classes are considered as MDR. The multiple antibiotics resistance (MAR) index was calculated by dividing (a): the number of antimicrobial drugs resistant of isolate by (b): the total number of antimicrobial drugs, where the same isolate which exposed the results more than 0.2 was considered high risk [46 (link)].

, & Ayyal Al‐Gburi N.M. (2020). Isolation and Molecular Identification and Antimicrobial Susceptibility of Providencia spp. from Raw Cow's Milk in Baghdad, Iraq. Veterinary Medicine International, 2020, 8874747.

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Characterization of Campylobacter jejuni Strains

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C. jejuni 81-176 is a well characterized sequenced strain used widely in infection studies, C. jejuni NCTC11168 is the type strain of C. jejuni. Strain C19 was isolated from a chicken and was acquired from the laboratory of Professor Seamus Fanning. NCTC11168 cadF- and flpA- were acquired from Professor Steffen Backert and Dr Nick Dorrell respectively. 81-176 flgI-, 81-176 flgE- and 81-176 fliD- as well as the pRY107 plasmid were acquired from Professor Patricia Guerry. All C. jejuni strains were cultured on Mueller Hinton (MH) agar (Oxoid) at 37°C under microaerophilic conditions created using Campygen gas packs (Oxoid). C. jejuni stock cultures were maintained using MH broth (Oxoid) supplemented with 20% glycerol and stored at −80°C. To revive stocks of C. jejuni strains, bacteria were streaked with a single use sterile inoculation loop (Sarstedt) on MH agar and incubated under microaerophilic conditions for 48 h at 37°C. For liquid cultures, C. jejuni strains were equalized to specific optical densities in MH broth and incubated under microaerophilic conditions at 37°C, shaking at 200 rpm. NCTC11168 cadF-, NCTC11168 flpA- or strains transformed with plasmid pRY107 were cultured with the addition of 50 μg/ml kanamycin. 81-176 flgI-, 81-176 flgE- and 81-176 fliD- were cultured with the addition of 10 μg/ml chloramphenicol.

Scanlan E., Ardill L., Whelan M.V., Shortt C., Nally J.E., Bourke B, & Ó Cróinín T. (2017). Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni. Molecular microbiology, 104(1), 92-104.

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