Upon arrival at the laboratory, the faecal samples were either processed the same day or stored at 4 °C for up to 2 days. Samples were cultured on Brilliance E. coli/coliform Selective Agar (Oxoid) with and without 1 mg/L cefotaxime (BECSA+ and BECSA−) (Sigma) and incubated overnight at 37 °C to determine the presence of ESBL-E/K. In addition, a cotton swab with faecal material was incubated overnight at 37 °C in 2 mL of Luria Bertani broth (MP Biomedicals) supplemented with 1 mg/L cefotaxime. The following day, 10 µL of the enrichment broth was streaked on BECSA+ and incubated overnight at 37 °C. If plates showed suspected growth of ESBL-E/K (after direct plating and/or enrichment), three colonies per sample (blue, pink and/or purple) were selected for further testing (see Meijs et al. for a more detailed description) [14 (link)]. Pink coloured colonies were further analysed to determine bacterial species using Matrix Assisted Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) (Bruker). Presumptive positive ESBL-E/K isolates were characterized by multilocus sequence typing (MLST) [15 (link), 16 (link)], and ESBL-genes were typed using polymerase chain reaction (PCR) and sequencing; see Additional file 1 : Table S1.
Brilliance e coli coliform selective agar
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Brilliance E. coli/Coliform selective agar is a microbiological culture medium used for the detection and enumeration of Escherichia coli (E. coli) and other coliform bacteria. It contains selective agents that inhibit the growth of non-target microorganisms, allowing for the identification of target colonies based on their color.
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Lab products found in correlation
Plate count agar, by Thermo Fisher Scientific (4 mentions)
Dbs100, by American Type Culture Collection (3 mentions)
Monoclonal anti il 22 clone 8e11, by Genentech (2 mentions)
Il 22 fc pro312045, by Genentech (2 mentions)
Luria bertani broth, by MP Biomedicals (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Mannitol salt agar, by Thermo Fisher Scientific (1 mentions)
Brain heart infusion agar, by bioMérieux (1 mentions)
Cefotaxime, by Merck Group (1 mentions)
Filtering, by Merck Group (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Microflex lt, by Bruker (1 mentions)
Fty720, by Cayman Chemical (1 mentions)
12 protocols using brilliance e coli coliform selective agar
1
Detecting ESBL-producing Enterobacteriaceae from Fecal Samples
2
Enumeration of Coliforms and E. coli in Water
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Enumeration of total coliforms and E. coli in water samples were carried out using commercially available chromogenic medium, Brilliance™ E. coli/coliform selective agar (Oxoid, CM1046). The agar was prepared according to manufacturer instruction and the molten medium was kept at +50 °C in a water bath. After that, 1 ml of the water sample was pipetted into an empty sterile Petri dish after being thoroughly mixed and covered with approximately 15–20 ml of the molten medium. The plates were gently swirled thoroughly to mix the water sample and the medium and incubated for 24 h at 37 °C. After 24 h incubation, purple and pink colonies were counted as E. coli (Fig. 2 ) and expressed as colony forming units (CFU)/1 ml of water.![]()
Purple and pink colonies on Brilliance E. coli
3
Microbial Quality Assessment of Samples
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Total plate counts and total coliform counts were performed for all samples using the pour plate method. Serial dilutions of ten-fold units of each sample were plated on Plate Count Agar (Oxoid) and incubated for 48 h at 30 °C. Following incubation, plates with colonies ranging from 30 to 300 were counted and expressed in CFU/cm2. The total coliform count was performed in the same way using Brilliance E. coli/ Coliform selective agar (Oxoid). The results of both counts were interpreted using guidelines set by the Ghana Standards Authority and the European Commission Regulation on the microbial criteria for food stuffs [36 , 37 ]. Limits to total coliform counts were not specified in these documents hence limits set for Enterobacteriaceae counts in both documents were adopted for total coliform counts.
4
Bacterial Antimicrobial Efficacy of Treated Masks
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An overnight bacteria suspension (109 CFU/mL) was diluted to 104 CFU/mL in sterile water. To each mask type (treated and not treated)—3 spots, with diameters of 0.5 mm by 2 µL of the suspension 104 CFU/mL, were applied. The bacterial concentration of each spot at Time 0 (T0) was evaluated, seeding serial dilution aliquots of the starting suspension in order to estimate the number of CFUs per microliter of the applied suspension. Coated masks were exposed (described in Section 2.4 ) for 1, 5, and 15 min. At the end of the exposure, each piece of tissue was removed with sterile tweezers and placed on a selective growth medium to test microorganism growth [59 ], using Brilliance™ E. coli/coliform selective agar (Oxoid, Detroit, MI, USA) for Escherichia coli and Mannitol Salt Agar (Oxoid, USA) for Staphylococcus aureus. The plates were incubated at 37 ± 1 °C for 18–24 h. The number of bacterial concentrations, at 1, 5, and 15 min, were determined, counting bacterial colonies present in the agar plate, and the number of CFU was reported as CFU/mL. The percentage of surviving bacteria was calculated with respect the T0 microbial load.
5
Quantifying Culturable Bacteria in Water and Sediment
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Total number of culturable heterotrophic bacteria was determined by pour-plate method on Brain Heart Infusion agar (bioMérieux). Series of logarithmic dilutions (10−1 to 10−4) of water and sediment samples were prepared in sterile saline, added to the medium and the plates incubated at 25 °C for 72 h. Coliform bacteria were counted on Brilliance E. coli/Coliform Selective Agar (Oxoid). Serial dilutions (up to 1:1000) were inoculated on the surface of the medium and the plates were incubated at 37 °C for 24 h.
6
Microbial Quality Evaluation of Samples
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Total plate counts and total coliform counts were performed for all samples using the pour plate method.
Serial dilutions of ten-fold units of each sample were plated on Plate Count Agar (Oxoid) and incubated for 48 hours at 30°C. Following incubation, plates with colonies ranging from 30-300 were counted and expressed in CFU/cm 2 . The total coliform count was performed in the same way using Brilliance E. coli/ Coliform selective agar (Oxoid). The results of both counts were interpreted using guidelines set by the Ghana Standards Authority and the European Commission Regulation on the microbial criteria for food stuffs (35, 36) . Limits to total coliform counts were not speci ed in these documents hence limits set for Enterobacteriaceae counts in both documents were adopted for total coliform counts.
Serial dilutions of ten-fold units of each sample were plated on Plate Count Agar (Oxoid) and incubated for 48 hours at 30°C. Following incubation, plates with colonies ranging from 30-300 were counted and expressed in CFU/cm 2 . The total coliform count was performed in the same way using Brilliance E. coli/ Coliform selective agar (Oxoid). The results of both counts were interpreted using guidelines set by the Ghana Standards Authority and the European Commission Regulation on the microbial criteria for food stuffs (35, 36) . Limits to total coliform counts were not speci ed in these documents hence limits set for Enterobacteriaceae counts in both documents were adopted for total coliform counts.
7
Microbial Enumeration of Food Samples
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Total plate counts and total coliform counts were performed for all samples using the pour plate method.
Serial dilutions of ten-fold units of each sample were plated on Plate Count Agar (Oxoid) and incubated for 48 hours at 30°C. Following incubation, plates with colonies ranging from 30-300 were counted and expressed in CFU/cm 2 . The total coliform count was performed in the same way using Brilliance E. coli/ Coliform selective agar (Oxoid). The results of both counts were interpreted using guidelines set by the Ghana Standards Authority and the European Commission Regulation on the microbial criteria for food stuffs (36, 37) . Limits to total coliform counts were not speci ed in these documents hence limits set for Enterobacteriaceae counts in both documents were adopted for total coliform counts.
Serial dilutions of ten-fold units of each sample were plated on Plate Count Agar (Oxoid) and incubated for 48 hours at 30°C. Following incubation, plates with colonies ranging from 30-300 were counted and expressed in CFU/cm 2 . The total coliform count was performed in the same way using Brilliance E. coli/ Coliform selective agar (Oxoid). The results of both counts were interpreted using guidelines set by the Ghana Standards Authority and the European Commission Regulation on the microbial criteria for food stuffs (36, 37) . Limits to total coliform counts were not speci ed in these documents hence limits set for Enterobacteriaceae counts in both documents were adopted for total coliform counts.
8
Quantifying CTX-resistant E. coli in Water
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The water samples were diluted by serial ten-fold dilution method to obtain countable plates or the samples were concentrated using filtering (0.22 µm; Sigma-Aldrich, US) before plating. For enumeration of E. coli and other coliform bacteria, chromogenic medium Brilliance™ E. coli/coliform Selective Agar (Oxoid, UK) with and without cefotaxime (CTX, 2 mg/L; Sigma-Aldrich, US) was used and plates were incubated at 37 °C overnight. All samples were tested in triplicates. The proportion of CTX-resistant E. coli and other coliform bacteria out of the total counts of E. coli/coliforms were calculated and Fisher's exact test (with p-value < 0.05 indicating significant difference) was used to evaluate influence of wastewater treatment process (Municipal-WWTP-In vs Municipal-WWTP-Out) on CTXresistant bacteria selection.
Up to 40 colonies of presumptive CTX-resistant E. coli were collected from the different plates of each water sample trying to encompass as much diversity as possible. The selected colonies were subcultured on MacConkey agar (Oxoid, UK) and stored at -80 °C. Species identification was verified using MALDI-TOF MS (Microflex LT, Bruker Daltonics, Germany) and only E. coli isolates were subjected to further typing.
Up to 40 colonies of presumptive CTX-resistant E. coli were collected from the different plates of each water sample trying to encompass as much diversity as possible. The selected colonies were subcultured on MacConkey agar (Oxoid, UK) and stored at -80 °C. Species identification was verified using MALDI-TOF MS (Microflex LT, Bruker Daltonics, Germany) and only E. coli isolates were subjected to further typing.
9
Microbial Enumeration in Food Samples
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Total plate counts and total coliform counts were performed for all samples using the pour plate method. Serial dilutions of ten-fold units of each sample were plated on Plate Count Agar (Oxoid) and incubated for 48 hours at 30°C. Following incubation, plates with colonies ranging from 30-300 were counted and expressed in CFU/cm 2 . The total coliform count was performed in the same way using Brilliance E. coli/ Coliform selective agar (Oxoid). The results of both counts were interpreted using guidelines set by the Ghana Standards Authority and the European Commission Regulation on the microbial criteria for food stuffs (13, 14) . Limits to total coliform counts were not speci ed in these documents hence limits set for Enterobacteriaceae counts in both documents were adopted for total coliform counts.
10
C. rodentium Infection and IL-22 Modulation
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For C. rodentium infection a single colony of strain DBS100 (ATCC 51459; American Type Culture Collection) was transferred to Luria–Bertani (LB) broth and grown to log phase followed by centrifugation and resuspension in PBS. Mice were orally gavaged with 200μl of PBS containing 2x109C. rodentium. To determine bacterial load, intestinal tissue pieces or faecal pellets were weighed and homogenized in sterile PBS and serial dilutions were plated onto Brilliance E. coli/coliform Selective Agar (Fisher Scientific) or LB agar plates (liver and spleen) for measurement of colony-forming units (CFU). For neutralization of IL-22, mice were injected intraperitoneally (i.p.) three times per week with 150μg/mouse per dose monoclonal anti-IL-22 (clone 8E11, Genentech) or mouse IgG1 isotype control (BioXCell/2BScientific). Where indicated, mice were injected i.p. three times per week with 125μg/ml per dose IL-22–Fc (PRO312045, Genentech) or mouse IgG2a isotype control (BioXCell/2BScientific).
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