The DDST was performed to determine the synergy between a disk of amoxicillin-clavulanic acid (AMC) (20/10 µg) and 30 µg disks of each ceftazidime and cefotaxime, aztreonam, and ceftriaxone placed at a distance of 20 mm (center to center) from the AMC disk [30 ]. The test inoculum (0.5 McFarland turbidity) was spread onto MHA (HiMedia) using a sterile cotton swab. A disk of AMC (20 µg amoxicillin + 10 µg clavulanic acid) was placed on the surface of MHA (HiMedia); then 30 μg disks of each cefotaxime, ceftazidime, aztreonam, and ceftriaxone were placed in such a way that each disk was at a distance ranging between 16 and 20 mm from the AMC disk (center to center). The plate was incubated at 37°C overnight. Any distortion or increase in the zone toward the disk of AMC was considered as positive for the ESBL production by Clavulanic acid (provided by the AMC disk) and the subsequent action of the extended-spectrum cephalosporin or aztreonam [31 (link)]. If no distortion occurs, then the result is negative for ESBL by the AMC.
Mueller hinton agar (mha)
Manufactured by HiMedia
Sourced in India, United States, Brazil, Kenya, Germany, France, United Kingdom
Mueller-Hinton agar is a microbiological growth medium used for the cultivation and antimicrobial susceptibility testing of a wide range of bacteria. It provides the necessary nutrients for the growth of various bacterial species.
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Lab products found in correlation
Mueller hinton broth (mhb), by HiMedia (26 mentions)
Ciprofloxacin, by HiMedia (18 mentions)
Ceftazidime, by HiMedia (15 mentions)
Nutrient broth, by HiMedia (13 mentions)
Meropenem, by HiMedia (13 mentions)
Ampicillin, by HiMedia (12 mentions)
Gentamicin, by HiMedia (12 mentions)
Amikacin, by HiMedia (11 mentions)
Cotrimoxazole, by HiMedia (11 mentions)
Imipenem, by HiMedia (11 mentions)
Nutrient agar, by HiMedia (10 mentions)
Ceftriaxone, by HiMedia (10 mentions)
Piperacillin tazobactam, by HiMedia (10 mentions)
Potato dextrose agar (pda), by HiMedia (10 mentions)
Cefotaxime, by HiMedia (9 mentions)
Sabouraud dextrose agar, by HiMedia (8 mentions)
Levofloxacin, by HiMedia (8 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Cefoxitin, by HiMedia (7 mentions)
Cefixime, by HiMedia (7 mentions)
297 protocols using mueller hinton agar (mha)
1
Detection of ESBL Production via DDST
2
Antibacterial Activity of RME and RME-AgNPs
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The antibacterial activity of RME and RME-AgNPs was studied by Kirby-Bauer Disk Diffusion Susceptibility Test17 (link) against Gram-negative bacteria Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 27853), Serratia marcescens (ATCC 14756), and Klebsiella pneumoniae (ATCC 13883) and Gram-positive bacteria Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6633). The bacterial strains were grown on a Mueller–Hinton agar (MHA; HiMedia, India) medium at 37 °C for 18 h. MHA plates were prepared by inoculating bacterial strains with a final inoculum size of ∼106 CFU mL−1. Sterile blank antimicrobial susceptibility disks were placed on agar plates, loaded with 50 μL of sterile suspension solution with a final concentration of 500 μg mL−1 and incubated at 37 °C for 24 h. The activity was determined by measuring the diameter of the inhibition zone in millimetres (mm). Antibiotic ampicillin (10 mg mL−1) was used as a positive control, while sterile deionized water was used as a negative control. This assay was performed in replica for each bacterial strain, and the data are expressed as mean ± standard deviation (SD).
3
Determining MRSA Antibiotic Resistance via E-test
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All the confirmed methicillin-resistant staphylococci via the PCR genotyping method were subjected to an E-test to determine the minimum inhibition concentration (MIC) required to inhibit/kill the bacteria [81 ]. To perform an E-test, a bacterial cell suspension was made in normal saline solution (0.85%) and the turbidity was set equivalent to a 0.5 McFarland [83 ]. A sterile cotton swab was dipped into the broth culture tube and rotated several times to get adequate amount of culture; it was then uniformly applied on the surface of the MHA (Hi-media, Maharashtra, India) plate. The antibiotic cefoxitin (0.016–256 mcg/mL) and oxacillin (0.016–256 mcg/mL) (Hi-media, Maharashtra, India) strips were placed on the MHA agar plate, using a sterile forceps, by gently pressing the antibiotic strips to ensure their complete contact with the surface of the agar plate. The inoculation was performed within 10–15 min of the inoculum being prepared in normal saline. The plates were then incubated at 37 ℃ for 16–20 h, and then examined for the MIC value from the scale in terms of µg/mL where the ellipse edge intersects the strip. For S. aureus, oxacillin ≥ 4 and cefoxitin ≥ 8 were considered as resistant, and for CoNS, oxacillin ≥ 0.5 was considered as resistant [88 ]. For quality control, S. aureus ATCC 29213 was used.
4
Kirby-Bauer Disk Diffusion Antimicrobial Susceptibility Testing
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Disk diffusion method by Kirby-Bauer technique as recommended by the Clinical and Laboratory Standards (CLSI) guidelines of 2020 [15 ]. One to two colonies of freshly growth of test bacteria were suspended into sterile 0.85 % normal saline and turbidity were adjusted equivalently to 0.5McFarland Standard solution. Then, using sterile bacteriological cotton swab, the entire surfaces of Mueller Hinton agar (MHA; HiMedia, India) plates were swabbed to obtain evenly lawns. Antibiotic disks of amoxicillin/clavulanic acid 30 μg (AMC; HiMedia, India) were seeded on each plate of MHA within 15 min. MHA plates were incubated at 37 °C in ambient air for 18 h. After incubation, zones of inhibitions around AMC disks were measured in millimetre using ruler and were interpreted to susceptible, intermediate and resistance as recommended by the CLSI, 2020.
5
ESBL Detection via Combination Disk Test
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ESBLs producing isolates were detected using the combination double disk test (CDDT) as a standard disk diffusion assay on Mueller Hinton agar (Himedia, India) (11 (link)). ESBLs presence was assayed using the following antibiotic disks: ceftazidime (CAZ) (30 μg), ceftazidime (30 μg) plus clavulanic acid (CA) (10 μg), cefotaxime (CTX) (30 μg), cefotaxime (30 μg) plus clavulanic acid (10 μg), and cefpodoxime (30 μg), cefpodoxime (30 μg) plus clavulanic acid (10μ g) (MAST Chemical Co, England). The disk with CA and without CA was placed on the inoculated surface of the Mueller–Hinton agar (Himedia, India) plate by the standard disk diffusion method. The plates were then incubated overnight at 37°C in ambient air. An increase of ≥ 5 mm in zone diameter of CAZ, CPD and/or CTX tested in combination with CA (CAZ-CA, CPD-CA and/or CTX-CA) versus CAZ, CPD and/or CTX alone was considered positive for ESBLs. E. coli ATCC 25922 and K. pneumoniae 700603 were used as control strains for detection of ESBLs producing isolates.
6
Bioactive Compound Analysis Protocol
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Analytical grade chemicals of 2, 2‐diphenyl‐1‐picrylhydrazyl (DPPH), Folin–Ciocalteu’s reagent, gallic acid, quercetin, β‐carotene, bovine serum albumin (BSA), sodium carbonate (Na2CO3), aluminium chloride (AlCl3), sodium nitrite (NaNO2), catalase and sodium hydroxide (NaOH) were purchased from Sigma‐Aldrich (Kobian Kenya Ltd.). Mueller‐Hinton Agar (MHA) was purchased from Himedia Laboratories Pvt. Ltd. (F&S Scientific, Nairobi, Kenya). Chemicals of analytical grade were used in all analyses.
7
Antimicrobial Activity Assessment
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Gentamicin and Ciprofloxacin (Microxpress, a division of Tulip Diagnostics (P), Ltd.) antibiotic discs were used as standard drugs for antimicrobial activity. Mueller Hinton Agar (MHA) (HiMedia Laboratories Pvt. Ltd., Mumbai), Nutrient Broth (HiMedia Laboratories Pvt. Ltd., Mumbai), DPPH (HiMedia Laboratories Pvt. Ltd., Mumbai), Barium Chloride (Thermo Fisher Scientific, India Pvt. Ltd., Mumbai), and Dimethyl Sulfoxide (Thermo Fisher Scientific, India Pvt. Ltd., Mumbai) were also used.
8
Isolation and Identification of Multidrug-Resistant Bacteria from Hospital Wastewater
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We wanted to investigate resistance, particularly among Gram-negative bacteria from hospital wastewater sources; we selected four Gram-negative isolates and one Gram-positive isolate. The team allowed the wastewater bacterial population to grow in four selective media to isolate five distinct bacterial species. Escherichia coli and Klebsiella spp. isolates were identified using MacConkey Agar media, while Staphylococcus aureus, Vibrio cholerae, and Pseudomonas aeruginosa isolates were selectively grown in Mannitol Salt Agar, TCBS Agar, and Cetrimide Agar media, respectively. These selective media possess exceedingly stringent specifications to screen exclusively for specific strains. Each one of the isolates was sub-cultured three times to eliminate any false-positive identification and to obtain a pure culture of the bacteria of interest. These media along with Mueller Hinton Broth (MHB) and Mueller Hinton Agar (MHA) media were purchased from HiMedia Laboratories Pvt Ltd., Maharashtra, India. Some 500 mL of pure culture broth was added to an equal amount of 50% glycerol, and stored at −80 °C for longer shelf life.
9
Antioxidant and Antimicrobial Assays
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2,2-Diphenyl-1-picryl-hydrazyl (DPPH) and ascorbic acid were from Wako Pure Chemical Industries, Ltd., Osaka, Japan, and Qualigens Fine Chemicals Pvt., Ltd., Mumbai, India. The chemicals used in the analysis were of analytical reagent grade, and all the glassware used were from Borosil Glassworks Ltd., Mumbai, India. Cyclophosphamide was obtained as a gift from Manipal College of Medical Sciences, Phulbari, Pokhara, Nepal. All bacterial media such as Mueller Hinton Agar (MHA) and nutrient broth were from HiMedia, Mumbai, India.
10
ESBL Detection in CTX-resistant E. coli
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The samples categorized as CTX-resistant E. coli were cultured in individual Petri dishes containing nutrient agar (Himedia). Three growth colonies were selected from each sample (three isolates per sample) and individually diluted in 0.85% NaCl to achieve a turbidity equivalent to 0.5 McFarland standard. A sterile cotton swab was immersed in the solution and then spread on Mueller-Hinton agar (MHA; Himedia). On an MHA Petri dish, an amoxicillin/clavulanate (20/10 μg/disk, respectively) disk (Oxoid, UK) and a CTX (30 μg/disk) disk (Oxoid) were placed at the center with a distance of 2.5 cm between them. Incubation was conducted at 37°C for 16–24 h, and the increase in the inhibition zone between both disks was considered an indicator of ESBL production [20 (link)].
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