Jasmonic acid, yeast extract, ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-triazine), 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), α-amylase, pancreatin, pepsin, lipase, α-glucosidase, bile extract, lipoxygenase, linoleic acid, angiotensin converting enzyme, o-phthalaldehyde, p-nitrophenyl acetate, dimethyl sulfoxide, 3,5-dinitrosalicylic acid, p-nitrophenol, protocatechuic acid, syringic acid, vanillic acid, sinapic acid, salicylic acid, caffeic acid, and hydroxybenzoic acid, cyclohexamide, resazurin were purchased from Sigma-Aldrich (Poznan, Poland). The COX Activity Assay kit was purchased from Cayman Chemical company (Ann Arbor, MI, USA). Penicillin and streptomycin were purchased from Life Technologies, Warsaw, Poland. Mueller–Hinton broth, and Mueller–Hinton agar were also obtained (Biomaxima, Lublin, Poland). All other chemicals were of analytical grade.
Mueller hinton agar (mha)
Manufactured by Biomaxima
Sourced in Poland
Mueller–Hinton agar is a solid growth medium used in microbiology for the cultivation and antimicrobial susceptibility testing of bacteria. It is a commonly used agar for the standard disk diffusion antimicrobial susceptibility test.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Merck Group (3 mentions)
Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Sabouraud dextrose agar, by Biomaxima (2 mentions)
Baird parker agar, by Biomaxima (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Protocatechuic acid, by Merck Group (1 mentions)
Vanillic acid, by Merck Group (1 mentions)
Sinapic acid, by Merck Group (1 mentions)
Syringic acid, by Merck Group (1 mentions)
Hydroxybenzoic acid, by Merck Group (1 mentions)
Bile extract, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Salicylic acid, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
α amylase, by Merck Group (1 mentions)
O phthalaldehyde, by Merck Group (1 mentions)
19 protocols using mueller hinton agar (mha)
1
Comprehensive Analysis of Bioactive Compounds
2
Minimum Bactericidal and Fungicidal Concentrations Determination
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MBCs or MFCs were determined using the broth dilution from the MIC tests in subcultures in Mueller–Hinton Agar (BioMaxima, Lublin, Poland) medium for bacteria or Mueller–Hinton Agar medium supplemented with 2% glucose (MHA +2% glucose) plate for fungi. After incubation, 5 μL of the broth media cultured with bacteria or fungi were removed from each well of the plates used in the MIC experiments. Next, they were culturd onto the surface of the MHA medium (BioMaxima, Lublin, Poland) plate for bacteria or MHA +2% glucose plate for yeasts. The inoculated plates were incubated in the conditions mentioned above. After incubation, a visual reading of MBC or MFC values was made. The MBC or MFC was observed as the lowest concentration of the tested compounds with the absence of any visible bacterial (MBC) or fungal (MFC) growth on the MHA (for bacteria) or MHA +2% glucose (for fungi) plates. Three replicates for each strain and compound were performed.
All the experiments were repeated three times and representative data were presented. In this study, the MFC/MIC ratios were also calculated in order to determine the bactericidal and fungicidal (MBC/MIC and MFC/MIC ≤ 4) or bacteriostatic and fungistatic (MBC/MIC and MFC/MIC > 4) effect of the examined Schiff bases [54 (link),55 (link)].
All the experiments were repeated three times and representative data were presented. In this study, the MFC/MIC ratios were also calculated in order to determine the bactericidal and fungicidal (MBC/MIC and MFC/MIC ≤ 4) or bacteriostatic and fungistatic (MBC/MIC and MFC/MIC > 4) effect of the examined Schiff bases [54 (link),55 (link)].
3
Antimicrobial Activity of Essential Oils
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The experiments were performed using Mueller–Hinton agar (Biomaxima, Lublin, Poland) plates (90 mm diameter, 14.2 mm height, Noex, Komorniki, Poland). The agar layer was 5 mm thick. Standard paper discs (diameter of 6 mm, 0.5 mm of thickness) were placed in a 48-well plate (Thermo Fisher Scientific, Waltham, MA, USA), soaked with 0.2 mL of each EOs or saline (as control of bacterial growth) (NaCl, Stanlab, Lublin, Poland), wrapped with tape and kept refrigerated for 30 min. The abovementioned suspensions of all strains (two references and fourteen clinical) (0.5 McFarland) were cultured onto the plates. To evaluate the antimicrobial activity of liquid fractions of all EOs, the paper discs were placed onto the agar, while to assess the activity of vapour fractions onto the plate lids. The plates were sealed with parafilm and incubated for 24 h at 37 °C. Microbial growth inhibition zones were measured (in mm) afterwards using a ruler. Zones of partial growth inhibition were evaluated (in mm) if no total inhibition was observed. In the case of unequal zones, a shorter diameter was included. Each experimental setting was performed in triplicate, and the mean diameter was calculated.
4
Antimicrobial Activity of Silver Nanoparticles
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The test of the antimicrobial activity of silver nanoparticles in alginate films was carried out using a total of 79 bacterial strains, including 74 isolates collected from animals and 5 type strains (Table 1 ). Bacterial isolates were transferred to sterile saline solution to prepare 0.5 MacFarland suspensions, which were then streaked onto Mueller–Hinton agar (Biomaxima, Lublin, Poland). Prior to the experiment, the foils were sterilized under UV light for 20–30 min. Then, 10 × 10 mm squares were cut with a surface sterilized scissors and applied onto the surface of bacterial cultures. The cultures were incubated at 37 ± 1 °C for 24 h. Afterwards the results were read by measuring the diameters of bacterial growth inhibition zones around the foil fragments. Two diameters were read and the final result was expressed as a mean of the two reads (mm). All experiments were conducted in three replicates.
5
Antibiotic Susceptibility Profiling of S. aureus
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Susceptibility to antibiotics was tested on Mueller Hinton agar (Biomaxima) by using the Kirby-Bauer disc diffusion method, and commercially prepared discs of 30 μg vancomycin, 30 μg teicoplanin, and 5 μg methicillin (Liofilchem, Italy), according to CLSI guidelines [35 ]. Plates were incubated at 35 °C for 24 h. S. aureus strains identified as methicillin resistant by disc diffusion assay were then tested by oxacillin agar screening. Specifically, a colony suspension was spotted on MH agar supplemented with 4% NaCl and oxacillin at 6 μg/ml (Sigma), according to CLSI recommendations. After incubation for 24 h, any growth was interpreted as a positive result for oxacillin resistance [36 ]. MIC values for vancomycin and teicoplanin (Sigma) were tested by broth microdilution method, according to CLSI guidelines [37 ]. The final concentrations of vancomycin and teicoplanin ranged from 0.125 to 256 μg/ml.
6
Microbial Strain Preparation for Assays
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The Acinetobacter baumannii ATCC BAA-1605, Enterococcus faecium ATCC 700221, Escherichia coli ML-35 ATCC 43827, Klebsiella aerogenes ATCC 13048, Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 33591, Candida albicans ATCC 10231, Candida glabrata ATCC 15126, strains were acquired from the American Type Culture Collection (ATCC). All the strains were stored at −80 °C in Roti-Store cryo vials and before the tests were transferred into fresh Mueller–Hinton broth (MHB, Biocorp, Warsaw, Poland) for bacteria or RPMI-1640 (Sigma-Aldrich, Steinheim, Germany) for fungi and incubated for 24 h at 37 °C. Then, the cultures were seeded on the Mueller–Hinton agar (BioMaxima, Lublin, Poland) or Sabouraud dextrose agar (SDA, BioMaxima, Lublin, Poland) plates, respectively, and incubated as just mentioned. These agar cultures were used for further microbiological assays. Cell densities for all assays were adjusted spectrophotometrically (Multiskan GO Microplate Spectrophotometer, Thermo Fisher Scientific, Vantaa, Finland) at 600 and 530 nm for bacteria and fungi, respectively.
7
Caffeine and Antibiotic Interaction Assay
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Caffeine (1,3,7-trimethylxanthine) and antibiotics novobiocin sodium salt, cefepime hydrochloride, gentamycin sulfate salt hydrate, azithromycin dihydrate, and ticarcillin disodium salt, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Refer to Figure 5 for the chemical structures of the compounds. For UV-Vis spectroscopy and isothermal titration calorimetry (ITC) measurements, 100 mM sodium phosphate buffer (pH 6.8) was used. The buffer was filtered through a 0.2 μm pore Millex Millipore filter and degassed before experiments. Caffeine stock solutions were prepared in the buffer or deionized water at concentrations of approximately 10−1 M, and stored at 4 °C in darkness. Antibiotic stock solutions were prepared by dissolving their weight amounts in the buffer or deionized water immediately before the experiments.
Solid media for bacterial cultivation and testing included tryptone soya agar (TSA, Oxoid Ltd., Basingstone, UK) and Mueller-Hinton agar (BioMaxima, Lublin, Poland). Cation-adjusted Mueller-Hinton broth (CA-MHB) for antimicrobial susceptibility testing using the broth microdilution method was purchased from Beckton Dickinson (BD Difco™ BBL™; Franklin Lakes, NJ, USA).
Solid media for bacterial cultivation and testing included tryptone soya agar (TSA, Oxoid Ltd., Basingstone, UK) and Mueller-Hinton agar (BioMaxima, Lublin, Poland). Cation-adjusted Mueller-Hinton broth (CA-MHB) for antimicrobial susceptibility testing using the broth microdilution method was purchased from Beckton Dickinson (BD Difco™ BBL™; Franklin Lakes, NJ, USA).
8
Preparation of Bacterial and Fungal Cultures
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All strains were stored at −80°C in ROTI Store cryo vials. Before the tests the strains were transferred to fresh Mueller-Hinton broth (Biocorp) for bacteria or RPMI-1640 (Sigma-Aldrich) for fungi and incubated for 24 hours at 37°C while being shaken at 120 rpm. The cultures were then seeded on Mueller-Hinton agar (BioMaxima) or Sabouraud dextrose agar (BioMaxima) plates and incubated as described. These agar cultures were used for further microbiological assays. Cell densities for all assays were adjusted spectrophotometrically (Multiskan GO Microplate Spectrophotometer, Thermo Fisher Scientific) at 600 and 530 nm for bacteria and fungi, respectively.
9
Antimicrobial Effectiveness of Bacterial Cellulose
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The 0.5 MF suspensions of the microorganisms were prepared and cultured onto Mueller–Hinton agar (Biomaxima, Lublin, Poland) plates using sterile cotton swabs. The soaked BC discs were placed onto the inoculated plates. The plates were incubated for 24 h at 37 °C and zones of growth inhibitions were measured in mm. Each extract was examined once for each strain.
10
Gentamicin Susceptibility Testing of Staphylococci
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The 24 h cultures of staphylococci strains on Columbia agar plates (Becton, Dickinson and Company, Heidelberg, Germany) were used for the method purpose. Firstly, the bacterial suspension at a density of 0.5 McFarland (densitometer Densitomat II, BioMerieux, Warsaw, Poland) was prepared in sterile 0.9% NaCl (Chempur, Piekary Slaskie, Poland). Next, using a sterile swab stick, the inoculum was seeded on the on the Mueller–Hinton agar (Biomaxima, Lublin, Poland) by streaking the swab three times over the agar surface. A disc impregnated with 10 µg gentamicin (Becton, Dickinson and Company, Sparks, MD, USA) was placed on the inoculated agar plate and incubated for 18 h at 35 °C. After the time period, the inhibition zones were measured and interpreted with the European Committee on Antimicrobial Susceptibility Testing breaking points table [78 ].
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