Mueller hinton agar plate

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Mueller–Hinton agar plates are a type of laboratory culture medium used for the growth and isolation of bacteria. The plates provide a standardized and nutritious environment for the cultivation of various microorganisms, allowing for the assessment of their susceptibility to antimicrobial agents.

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Mueller Hinton (11 citations) Mueller-Hinton (MH) agar (3 citations)

30 protocols using mueller hinton agar plate

Antimicrobial Susceptibility Testing of Bacterial Isolates

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Antimicrobial susceptibility testing was done by disk diffusion method as recommended by the Clinical Laboratory Standards Institute (CLSI) guidelines (Wayne, 2012 ). A bacterial suspension of 0.50 McFarland standard turbidity was prepared from pure culture. An inoculum was then plated on Mueller-Hinton Agar plates (HiMedia, Mumbai, India) and the following antibiotic disks were set: Tetracycline (2 μg), ciprofloxacin (5 μg), gentamicin (10 μg), or trimethoprim/sulphamethoxazole (1.25/23.75 μg) (Oxoid, Hampshire, UK). The plates were incubated aerobically at 37°C for 18–24h. The diameters of the respective zone of inhibitions were measured and interpreted following CLSI 2012 guidelines (Wayne, 2012 ). Disk approximation method based on CLSI guidelines was used to confirm ESBL production and for selected isolates ESBL production was further identified using VITEK® 2 system (BioMérieux, Marcy l'Etoile, France) and in addition the MIC for cefepime, carbapenems and colistin were determined in all selected isolates.
Using STATA version 11 the two-sample test of proportion was done to compare the rates of resistance between ESBL isolates from fish and those from environment. A p-value < 0.05 was used to indicate a significant difference at 95% confidence interval.

Moremi N., Manda E.V., Falgenhauer L., Ghosh H., Imirzalioglu C., Matee M., Chakraborty T, & Mshana S.E. (2016). Predominance of CTX-M-15 among ESBL Producers from Environment and Fish Gut from the Shores of Lake Victoria in Mwanza, Tanzania. Frontiers in Microbiology, 7, 1862.

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Antibiotic Susceptibility Profiling of LAB

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The antibiotic susceptibility of the LAB strains was determined using a MIC
Test Strip (Liofilchem^® MTS^TM, Roseto degli
Abruzzi, Italy). Overnight cultures of LAB were adjusted to 0.5 McFarland
standard and diluted to 5×10⁵ CFU/mL. Subsequently, 0.1 mL
of the LAB suspension was plated onto Mueller–Hinton agar plates
(HiMedia). The MIC Test Strips containing ampicillin, chloramphenicol,
erythromycin, gentamicin, and tetracycline (Liofilchem^®MTS^TM) were positioned at the center of the plate and
incubated for 24 h at 37°C. These antibiotics were chosen based on
their inclusion in the European Food Safety Authority (EFSA) list. The MIC
of each antibiotic was determined by evaluating the ellipsoid zones of
inhibition of bacterial growth and determining the point of intersection
between these zones and the concentration mark on the test strip.
Susceptibility or resistance was assessed in accordance with the microbial
cutoff values recommended by EFSA
(2012).

Foongsawat N., Sunthornthummas S., Nantavisai K., Surachat K., Rangsiruji A., Sarawaneeyaruk S., Insian K., Sukontasing S., Suwannasai N, & Pringsulaka O. (2023). Isolation, Characterization, and Comparative Genomics of the Novel Potential Probiotics from Canine Feces. Food Science of Animal Resources, 43(4), 685-702.

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Phenotypic Profiling of S. aureus Isolates

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S. aureus strains were isolated from chromogenic agar medium (ChromAgar; BioMérieux, Marcy-L'Etoile, France). Gram staining was performed; the presence of catalase, coagulase and DNase was confirmed, as described elsewhere [14] . Antimicrobial susceptibility testing was performed for erythromycin, gentamicin, amikacin, tetracycline, chloramphenicol, cotrimoxazole and ciprofloxacin with the disk diffusion method according to recommendations of the Clinical Laboratory Standard Institute [15] on Mueller-Hinton agar plates (Himedia, Mumbai, India) at 37°C. Methicillin resistance was tested using a cefoxitin disk on Mueller-Hinton agar [16] (link).

Bouchiat C., El-Zeenni N., Chakrakodi B., Nagaraj S., Arakere G, & Etienne J. (2015). Epidemiology of Staphylococcus aureus in Bangalore, India: emergence of the ST217 clone and high rate of resistance to erythromycin and ciprofloxacin in the community. New Microbes and New Infections, 7, 15-20.

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Antibiotic Susceptibility of Colistin-Resistant Isolates

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Colistin-resistant isolates were subjected to antibiotic susceptibility testing by Kirby-Bauer disc diffusion method on Mueller Hinton Agar plates (HiMedia, India) against the ampicillin, ampicillin/clavulanic acid, amikacin, azithromycin, cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, ertapenem, imipenem, tazobactam/piperacillin, tetracycline, and trimethoprim.
Minimum inhibitory concentrations (MICs) were determined by E-test (trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid, and imipenem) and broth microdilution (chloramphenicol, ciprofloxacin, colistin, gentamicin, kanamycin, polymyxin B, rifampicin, nalidixic acid, tigecycline, and tetracycline). E. coli ATCC 25922 (antibiotic-susceptible), K. pneumoniae ATCC 700603 (ESBL-producing), and E. coli NCTC 13846 (colistin-resistant) isolates were used as quality control strains. Results were interpreted as per CLSI (2018) guidelines and for tigecycline, EUCAST (2018) guidelines were followed. The susceptibility profile of K. pneumoniae isolates was determined using the WHONET (v20.8.21) database software.

Azam M., Gaind R., Yadav G., Sharma A., Upmanyu K., Jain M, & Singh R. (2021). Colistin Resistance Among Multiple Sequence Types of Klebsiella pneumoniae Is Associated With Diverse Resistance Mechanisms: A Report From India. Frontiers in Microbiology, 12, 609840.

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Antimicrobial Susceptibility Testing of MRSA and MSSA

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MRSA and methicillin sensitive and S. aureus (MSSA) isolates were subjected to disc diffusion antimicrobial sensitivity test on Mueller-Hinton agar plates (HiMedia, Mumbai, India) as per the previous protocol described by Bauer et al. [14 ]. The six different antimicrobial discs (HiMedia, Mumbai, India) used in this study were ampicillin, amoxicillin-sulbactam, ceftrioxone, enrofloxacin, methicillin, and pencillin.

Hamid S., Bhat M.A., Mir I.A., Taku A., Badroo G.A., Nazki S, & Malik A. (2017). Phenotypic and genotypic characterization of methicillin-resistant Staphylococcus aureus from bovine mastitis. Veterinary World, 10(3), 363-367.

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Antibiotic Susceptibility of Bacterial Isolates

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As the isolates have the potentiality to be used in the field for bioremediation, therefore it is important to check their susceptibility to have the way of remedy for any accidental human infection. In this study, we were used commonly available antibiotics viz., Levofloxacin (5 μg/disc), Doxycycline (30 μg/disc), Neomycin (30 μg/disc), Carbenicillin (100 μg/disc), Ceftazidime (10 μg/disc), Azithromycin (15 μg/disc), Vancomycin (30 μg/disc), Ampicillin (10 μg/disc), Tetracycline (10 μg/disc), Penicillin-G (10 μg/disc), Cefixime (5 μg/disc), Ciprofloxacin (10 μg/disc) to check the resistance or susceptibility of the bacterial isolates. Blank paper disc (6 mm) was used as a negative control. Zone of inhibition were measured on Mueller Hinton agar plates (HIMEDIA, India) and noted after 36 h of incubation at 37 °C.

Hossain M.F., Akter M.A., Sohan M.S., Sultana D.N., Reza M.A, & Hoque K.M. (2021). Bioremediation potential of hydrocarbon degrading bacteria: isolation, characterization, and assessment. Saudi Journal of Biological Sciences, 29(1), 211-216.

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Antibiotic Susceptibility Profiling of Bacterial Isolates

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Antibiotic susceptibility of each isolate was determined by the disk diffusion method [19 (link)]. The bacterial cultures maintained in nutrient agar slant at 4°C were subcultured in nutrient broth to obtain the working cultures approximately containing 1 × 10⁶ CFU/mL. Mueller Hinton agar plates (Hi-Media, Mumbai, India) were swabbed with each bacterial strain and the antibiotic disks were placed on the plates. Disks of cephalexin (30 μg/disc) (CP), ciproflax (5 μg/disc) (CFx), endrofloxaxin (10 μg/disc) (Ex), and cefixime (5 μg/disc) (CFIx) were used. Plates were incubated overnight at 37°C. Clear, distinct zone of inhibition was visualized surrounding the disks. The antimicrobial activity was determined by measuring the zone of inhibition expressed in mm. The sensitivity and resistance of each isolate were determined by the criteria of the National Committee for Clinical Laboratory Standards (1997).

Sreerag R.S., Jayaprakas C.A., Ragesh L, & Kumar S.N. (2014). Endosymbiotic Bacteria Associated with the Mealy Bug, Rhizoecus amorphophalli (Hemiptera: Pseudococcidae). International Scholarly Research Notices, 2014, 268491.

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Antibiotic MIC Determination in P. aeruginosa

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The minimum inhibitory concentration (MIC) values of several antibiotics from among those most prevalently used in CF patients were determined by E-TEST (Biomérieux, Marcy-l'Etoile, France). The antimicrobial concentration ranges of 0.016 to 256 µg/mL were tested for gentamicin, tobramycin, azithromycin, levofloxacin, ciprofloxacin, and colistin. Antimicrobial-agent-coated test strips were placed on 1% Mueller-Hinton agar plates (Himedia Laboratories) supplemented with 50 µM DIM (DMSO as solvent) or an equivalent volume of DMSO and uniformly spread with a lawn of P. aeruginosa pre-grown for 24 h in the presence of 50 µM DIM. The MIC value of every antibiotic was assessed according to the manufacturer’s instructions after incubation for 24 h at 37 °C.

Golberg K., Markus V., Kagan B.E., Barzanizan S., Yaniv K., Teralı K., Kramarsky-Winter E., Marks R.S, & Kushmaro A. (2022). Anti-Virulence Activity of 3,3′-Diindolylmethane (DIM): A Bioactive Cruciferous Phytochemical with Accelerated Wound Healing Benefits. Pharmaceutics, 14(5), 967.

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Screening of Imipenem-resistant Isolates for MBL

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Imipenem-resistant isolates were screened for producing MBL. The double disk method was used to detect this enzyme. Colonies from overnight cultures on blood agar plates were suspended in Mueller-Hinton broth and the turbidity standardized to equal that of a bacterial concentration of 1:100 suspensions of the 0.5 McFarland standards. Then the suspension was streaked onto Mueller-Hinton agar plates (Hi Media, Mumbai, India). A disc of Imipenem alone (10 μg) and Imipenem (10 μg) in combination with EDTA (750 μg/disc) was placed at the distance of 20 mm (centre to centre). After overnight incubation at 35 °C, a ≥ 7 mm increase in the inhibition zone of diameter around Imipenem-EDTA discs, as compared to imipenem discs alone, interpreted as indicative of MBL production [18 ].

Solomon F.B., Wadilo F., Tufa E.G, & Mitiku M. (2017). Extended spectrum and metalo beta-lactamase producing airborne Pseudomonas aeruginosa and Acinetobacter baumanii in restricted settings of a referral hospital: a neglected condition. Antimicrobial Resistance and Infection Control, 6, 106.

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Antibiotic Resistance Profiling of Clinical Isolates

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Bacterial strains were isolated from the various sample specimens at the clinical microbiology laboratory of the hospital. All isolates were subjected to antibiotic susceptibility testing by Kirby-Bauer disc diffusion method on Mueller Hinton Agar plates (HiMedia, India) against the ceftazidime, cefotaxime, cefepime, amikacin, gentamicin, ciprofloxacin, levofloxcine, piperacillin-tazobactam, ampicillin-sulbactam, imipenem and meropenem as well as nitrofurantoin for urine samples. Minimum inhibitory concentrations (MICs) were determined by E-test (meropenem and imipenem) and broth microdilution (colistin). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. We interpreted these in accordance with the guideline document M100-S30 established by Clinical and Laboratory Standards Institute (CLSI-2017) [10 ]. Initial screening for detection of carbapenemases was done by the modified carbapenem inactivation method (mCIM) [10 ].

Sanikhani R., Akbari M., Hosseinzadeh M., Siavash M., Badmasti F, & Solgi H. (2024). Outbreak of colistin and carbapenem-resistant Klebsiella pneumoniae ST16 co-producing NDM-1 and OXA-48 isolates in an Iranian hospital. BMC Microbiology, 24, 59.

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