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.
Sabouraud dextrose agar
Manufactured by Biomaxima
Sourced in Poland
Sabouraud dextrose agar is a culture medium used for the isolation and cultivation of fungi. It provides nutrients and growth factors necessary for the optimal growth of fungal organisms.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Merck Group (2 mentions)
Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Mueller hinton agar (mha), by Biomaxima (2 mentions)
Mueller hinton broth (mhb), by Biocorp (1 mentions)
D glucose, by Merck Group (1 mentions)
Rpmi medium, by Merck Group (1 mentions)
Chloramphenicol, by Biomaxima (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Lb broth, by Biomaxima (1 mentions)
4 protocols using sabouraud dextrose agar
1
Microbial Strain Preparation for Assays
2
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.
3
Microdilution Fungal Susceptibility Assay
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The microdilution method described in the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), version 10.0-valid from 4 February 2020, was used to determine the minimum inhibitory concentrations (MICs) of the tested agents against fungal strains suspended at populations of 2 × 105 CFU (colony-forming units)/mL. The final concentrations of the tested compounds ranged from 0.1 to 51.2 µg/mL. MICs were determined visually and confirmed by spectrophotometer in an RPMI medium (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with MOPS (Sigma-Aldrich, Saint Louis, MO, USA) and D-(+)-glucose (Sigma-Aldrich, Saint Louis, MO, USA) at the lowest concentration of tested agents that showed no visible microbial growth after 24 h. In turn, the minimum fungicidal concentration (MFC) was determined by plating each sample (10 µL) on Sabouraud Dextrose agar with the chloramphenicol (Biomaxima, Lublincity, Poland).
4
Microbiological Identification of Mastitic Milk
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Samples of mastitic milk were collected from farms from July 2021 to March 2022. The collected samples were submitted to standard microbiological procedures for initial multiplication and preliminary species identification: strains were isolated and then cultured on solid and liquid media. Specialized microbiological media (Biomaxima, Poland) were used to culture the strains: nutrient broth for general microbial culture, Mannitol Salt Lab-Agar, Edwards Lab-Agar with the addition of bovine blood, Chromogenic Uri-Color Lab-Agar, MacConkey Lab-Agar, Salmonella-Shigella Lab-Agar, Chromogenic Candida Lab Agar, Rose Bengal Lab-Agar, TBX Lab-Agar, Chromogenic Coliform Lab-Agar, LB Broth, Rogosa LS Lab-Agar, Reinforced Clostridial Medium, Legionella CYE Lab-Agar Base, Listeria acc., Oxford Lab-Agar Base, Enterococcus Confirmatory Lab-Agar, Eijkman Lactose Medium, Prototheca Isolation Medium according to Pore (1973),31 (link) and Sabouraud dextrose agar (all from Argenta, Poland). Subsequently, the selected colony samples were subjected to identification on a MALDI-TOF MS (Bruker, Poznań, Poland).
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