Mh agar

Manufactured by Thermo Fisher Scientific

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MH agar is a microbiological culture medium used for the growth and isolation of a wide range of bacteria. It is a non-selective and nutritious agar formulation that supports the growth of many different types of bacteria.

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31 protocols using mh agar

Conjugation and Transformation of mcr and blaVIM-1

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The ability of the plasmids harboring the mcr genes and the bla_VIM-1 gene to conjugate was tested through conjugation experiments. The conjugation was performed in Mueller Hilton (MH) broth (OXOID, Hampshire, UK) using E. coli A15^rAzi as the recipient.
Transconjugants for ENCL_3849 were selected on MH agar (OXOID, Hampshire, UK) plates supplemented with sodium azide (150 mg/L) (Sigma-Aldrich, St. Louis, MO, USA) and ampicillin (1000 mg/L) (Sigma-Aldrich, St. Louis, MO, USA). For ENCB_IB2020, transconjugants were selected on MH agar plates supplemented with sodium azide (150 mg/L), meropenem (2 mg/L) (Sigma-Aldrich, St. Louis, MO, USA) and colistin (2 mg/L) (Sigma-Aldrich, St. Louis, MO, USA). The presence of bla_VIM-1, and mcr-like genes in the transconjugants was confirmed through PCR. MICs for transconjugants were performed using the broth-microdilution method. Isolates that failed to transfer the mcr genes of interest through conjugation were subjected to transformation; plasmids were extracted using Qiagen Maxi kit (Qiagen, Hilden, Germany) and the competent E. coli DH5α cells were used as the recipient. Transformants were selected on MH agar (OXOID, Hampshire, UK) with 2 mg/L colistin. Transformants were confirmed to be MCR producers through PCR.

Marchetti V.M., Bitar I., Sarti M., Fogato E., Scaltriti E., Bracchi C., Hrabak J., Pongolini S, & Migliavacca R. (2021). Genomic Characterization of VIM and MCR Co-Producers: The First Two Clinical Cases, in Italy. Diagnostics, 11(1), 79.

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Culturing Campylobacter jejuni and Escherichia coli

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The C. jejuni NCTC11168 was kindly provided by Chinese Center for Disease Control. C. jejuni strains were routinely cultured on Mueller-Hinton (MH) agar (Oxoid, Basingstoke, UK) or blood agar plates at 42°C under microaerophilic conditions. E. coli DH5α was grown aerobically in Luria-Bertani medium at 37°C. The Ham’s F-12 nutrient powder mixture (SH30010, Thermo Fisher, Waltham, MA, USA) and Ham’s F-12m nutrient powder mixture (RR13033.01, Thermo Fisher, Waltham, MA, USA) lacking leucine were used for leucine biosynthesis test.

Hao H., Li F., Han J., Foley S.L., Dai M., Wang X., Wang Y., Huang L., Sun Y., Liu Z, & Yuan Z. (2017). Cj1199 Affect the Development of Erythromycin Resistance in Campylobacter jejuni through Regulation of Leucine Biosynthesis. Frontiers in Microbiology, 8, 16.

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Antimicrobial Efficacy of Ciprofloxacin Brands

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The antimicrobial efficacy of five brands of ciprofloxacin sold in the Bishoftu markets was evaluated on SE, STM and other non-serotyped Salmonella isolates using the agar-disc diffusion method following the Performance Standards for Antimicrobial Susceptibility Testing guidelines recommended by CLSI (2020).23 MH agar (Oxoid, UK) was used for sensitivity analysis as it shows good batch-to-batch uniformity.26 This medium is also effective to grow only the target bacterial species for study by inhibiting the growth of other alternative bacterial species.26 The disc diffusion method is based on the determination of the zone of inhibition (ZI) proportional to the bacterial susceptibility to the antimicrobial present in the disc. The diameter of this ZI around the antimicrobial disc depends on the concentration of antibiotics in the disc and its diffusibility.27 The ZI diameter breakpoints around the discs were measured to the nearest whole millimeter using a digital caliper, and the isolates were classified as susceptible (≥31), intermediate (21–30), and resistant (≤20) according to the interpretative standards where the breakpoints for ciprofloxacin are based on a dosage regimen of 400 mg IV or 500mg orally administered every 12 h.23

Geleta K, & Tufa T.B. (2023). In vitro Efficacy Evaluation of Leading Brands of Ciprofloxacin Tablets Found in Bishoftu City Against Salmonella Isolates, Central Ethiopia. Infection and Drug Resistance, 16, 1433-1440.

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Antimicrobial Activity Determination

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The MICs of the synthesized compound were determined by agar dilution method according to the Clinical Laboratory and Standards Institute Guidelines (CLSI, 2015) [55 (link),64 (link)]. Briefly, the tested strains were incubated overnight in tryptic soy broth (TSB) (Oxoid, United Kingdom) and then diluted in Muller–Hinton (MH) broth (Oxoid, United Kingdom) to turbidity approximating to the equivalent of 0.5 McFarland standard [65 (link)]. The suspensions were further diluted with sterile saline (1:10) and standardized inoculums (approximately 10⁴ CFU per spot) were spotted on the surfaces of MH agar (Oxoid, United Kingdom) plates containing different concentrations of tested compounds and a control plate. The MICs were the lowest concentrations that inhibit growth on the plates after incubation at 37 °C for 20 h.

Almalki A.J., Ibrahim T.S., Taher E.S., Mohamed M.F., Youns M., Hegazy W.A, & Al-Mahmoudy A.M. (2022). Synthesis, Antimicrobial, Anti-Virulence and Anticancer Evaluation of New 5(4H)-Oxazolone-Based Sulfonamides. Molecules, 27(3), 671.

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Antimicrobial Susceptibility Testing of Salmonella Isolates

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Fourteen Salmonella isolates were collected by the National Antimicrobial Resistance Monitoring System (NARMS) in year 2004–2005. All isolates were streaked onto Mueller-Hinton (MH) agar (Oxoid, Cambridge, UK). A single colony was selected and subsequently inoculated in MH broth (Oxoid, Cambridge, UK), and incubated for 16–18 h at 37°C with shaking (250 rpm). All isolates were subjected to susceptibility testing via the Sensititre^TM semi-automated antimicrobial susceptibility system (TREK Diagnostic Systems, Inc.) using a custom-made panel including amikacin, gentamicin, kanamycin, streptomycin, ampicillin, amoxicillin-clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, sulfamethoxazole/sulfisoxazole, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin, nalidixic acid, and tetracycline [21 ]. The isolates were subjected to preliminary biochemical screening to distinguish the different serogroups using serogroup-specific antisera (Difco Laboratories, Detroit, MI) and serotyping was used to identify serovars at the National Veterinary Services Laboratories, APHIS, USDA (Ames, IA).

Gupta S.K., Sharma P., McMillan E.A., Jackson C.R., Hiott L.M., Woodley T., Humayoun S.B., Barrett J.B., Frye J.G, & McClelland M. (2019). Genomic comparison of diverse Salmonella serovars isolated from swine. PLoS ONE, 14(11), e0224518.

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Preparation of Muller-Hinton Agar Plates

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12-well polystyrene plates (NUNC, Roskilde, Denmark) were cast with Muller-Hinton (MH) agar (Oxoid). Briefly, 7.6 g MH agar powder was dissolved in 200 mL water, autoclaved, and poured into each well of the 12-well plates and allowed to solidify.

Hakonen B., Lönnberg L.K., Larkö E, & Blom K. (2014). A Novel Qualitative and Quantitative Biofilm Assay Based on 3D Soft Tissue. International Journal of Biomaterials, 2014, 768136.

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Antimicrobial Resistance Mechanisms Study

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The instruments that were used for the experiments included a VITEK-2 automated microbial analyzer and Sakuma MIT-P bacterial multipoint inoculator (Sakuma Co., Ltd., Matsudo, Japan). A quantitative polymerase chain reaction (qPCR) kit was purchased from Takara Biotechnology Co., Ltd., Dalian, China. Reference standards of amikacin, netilmicin and imipenem were purchased from The European Pharmacopoeia (EP; Strasbourg, France). Luria-Bertani and Mueller-Hinton (MH) broths and MH agar were purchased from Oxoid Ltd. (Nepean, ON, Canada). AdeB and 16S rRNA primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). RNA extraction kits were purchased from Thermo Fischer Scientific, Inc. (Waltham, MA, USA). Quantitative and reverse transcription PCR kits were purchased from GeneCopoeia, Inc. (Rockville, MD, USA), diethyl pyrophosphate from Tiangen Biotech Co., Ltd. (Beijing, China), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and acetone from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Zhu W., Wang H, & Zhang J.P. (2017). A comparison of adeB gene expression levels under conditions of induced resistance by different drugs in vitro in Acinetobacter baumannii. Experimental and Therapeutic Medicine, 13(5), 2177-2182.

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MRSA and VRSA Detection via Disk Diffusion

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The antimicrobial susceptibility of the isolated S. aureus toward cefoxitin and vancomycin was performed by the Kirby-Bauer disk diffusion method to detect MRSA and VRSA isolates respectively. From the previously isolated S. aureus, a loopful from each BHI culture slants was inoculated into Mueller-Hinton broth (MH broth, Oxoid) followed by incubation at 37 °C for 24 h. The concentrations of these suspensions were adjusted to be equal to the 0.5 McFarland standards by adding a sterile saline. Test and standard tubes were compared against a white background with a contrasting black line and complete adjustment of the suspension concentrations was done by spectrophotometer to reach to an optical density of 0.10 at 625 nm (1 × 10⁸ CFU/ml). A swab spreading of each 0.5 McFarland S. aureus-MH broth concentration was done onto the surface of the Mueller–Hinton agar medium (MH agar, Oxoid) supplemented with 5% defibrinated sheep blood, and then cefoxitin (30 µg, Oxoid) and vancomycin (30 μg, Oxoid) sensitivity disks were impregnated onto the surfaces of the MH agar. Plates were then incubated at 37 °C for 24 h, the zones of inhibition were measured, and the susceptibilities to both antibiotics were determined according to interpretive criteria provided by the National Committee for Clinical Laboratory Standards [25] .

, & Omara S.T. (2017). MIC and MBC of Honey and Gold Nanoparticles against methicillin-resistant (MRSA) and vancomycin-resistant (VRSA) coagulase-positive S. aureus isolated from contagious bovine clinical mastitis. Journal of Genetic Engineering & Biotechnology, 15(1), 219-230.

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Conjugative Transfer of fosA3 Gene

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The conjugation experiments were carried out to determine the mobility of fosA₃ gene from azide-sensitive isolates as donors to azide-resistant E. coli J53 as the recipient. Overnight cultures of 0.05 ml of the donor and 0.45 mL of the recipient strains were added to 3 mL of fresh Mueller Hinton (MH) broth (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom), then incubated and gently stirred at 37°C for 12 hours. 0.1 mL of the mixtures were plated on MH agar (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom) containing glucose-6-phosphate (25 mg/L), sodium azide (100 mg/L) and fosfomycin (Zambon Group, Milan, Italy) (32 mg/L) and incubated for 48 hours for selection. The conjugative transfer frequency was calculated as the ratio of the number of conjugants to the number of donors.

Li Y., Zheng B., Li Y., Zhu S., Xue F, & Liu J. (2015). Antimicrobial Susceptibility and Molecular Mechanisms of Fosfomycin Resistance in Clinical Escherichia coli Isolates in Mainland China. PLoS ONE, 10(8), e0135269.

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Evaluation of Anti-Quorum Sensing Compounds Against P. aeruginosa

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The strains of P. aeruginosa used in this study were clinical isolates collected from Mansoura University Hospital and obtained from different sources: burn, wound, urine, and pus. All the clinical isolates were collected after approval from the administrative authorities (Research Ethics Committee) in the Faculty of Pharmacy, Mansoura University, Egypt, on 13/10/2014, with the code number 214-70. The isolates were stored at −70°C till use. Farnesol (Sigma number 277541) and tyrosol (Sigma number 79058) were stored at −20°C before preparation of 500 mM stock solution in DMSO or water for Farnesol and tyrosol, respectively. Farnesol stock solution was prepared immediately prior to each experiment and it was added to the final concentration just at the time of inoculation, where control cultures received an equivalent amount of DMSO [24 (link)]. Muller-Hinton (MH) broth, MH agar, and antibiotic sensitivity disks were obtained from Oxoid.

Hassan Abdel-Rhman S., Mostafa El-Mahdy A, & El-Mowafy M. (2015). Effect of Tyrosol and Farnesol on Virulence and Antibiotic Resistance of Clinical Isolates of Pseudomonas aeruginosa. BioMed Research International, 2015, 456463.

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