Before conducting the challenge test, OFSP puree was assessed microbiologically for E. coli and S. aureus and then microwaved and assessed again. The initial enumeration of E. coli and S. aureus in the puree was carried out as described in previous studies [22 ]. Puree sample (25 g) was homogenized with 0.85 % NaCl and serial dilutions were prepared up to 10−6. A volume of 0.1 mL from each dilution was spread in triplicate onto Brilliance E. coli/Coliform agar (Oxoid, Hampshire, England) and incubated at 37°C for 24 hours for enumeration of E. coli. Similarly, 0.1 mL of each dilution was spread in triplicate onto Baird parker agar (Oxoid, Hampshire, England) and incubated at 37°C for 48 hours for the enumeration of S. aureus. Enumeration was done for plates with 30-300 colonies. All microbial counts were expressed as mean base-10 logarithms of colony forming units per gram (log cfu/g). Data points were expressed as means from the triplicate analysis. The results indicated high levels of E. coli and S. aureus in puree before treatment with preservatives. E. coli and S. aureus were not detected after microwaving OFSP puree as shown in Table 1 . This formed the basis for the level of inoculation of E. coli and S. aureus into the puree.
Brilliance e coli coliform agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Brilliance E. coli/coliform agar is a selective and differential culture medium used for the detection and enumeration of Escherichia coli (E. coli) and total coliforms in water and food samples. The agar contains chromogenic and fluorogenic substrates that allow for the visual identification of target microorganisms based on colony color and fluorescence under UV light.
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Lab products found in correlation
Baird parker agar, by Thermo Fisher Scientific (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
Buffered peptone water (bpw), by Thermo Fisher Scientific (1 mentions)
Violet red bile glucose agar, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
2100n turbidity meter, by HACH (1 mentions)
Pannoramic midi 2 scanner, by 3DHISTECH (1 mentions)
Caseviewer software 2, by 3DHISTECH (1 mentions)
Dbs100, by American Type Culture Collection (1 mentions)
7 protocols using brilliance e coli coliform agar
1
Microbiological Assessment of OFSP Puree
2
Microbial Enumeration in Food Samples
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The standard pour plate technique was employed to estimate TVC (total viable count), EBC (Enterobacteriaceae count), and ECC (E. coli count). A 25 g food sample was placed into 225 mL sterilised BPW (buffered peptone water) (Oxoid, Hampshire, UK). The setup was homogenised in a stomacher for 3 min. Serial dilutions up to 10−8 were prepared and plated in triplicates in nutrient agar (Oxoid, Hampshire, UK). For TVC and ECC, nutrient agar (Oxoid, Hampshire, UK) and Brilliance E. coli/coliform agar (Oxoid, Hampshire, UK) were used and plates were incubated at 37 °C for 48 h. For EBC, violet-red bile glucose agar (Oxoid, Hampshire, UK) was used and plates were incubated at 37 °C for 24 h.
3
Coliform and E. coli Detection in Samples
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The presence of coliforms and E. coli was examined by plating 0.1 mL of each sample on Brilliance E. coli/coliform agar (Oxoid, Hampshire, England) according to the method by Sylvia et al. [29 (link)]. The plates were incubated at 37°C for 24 hours. Dark blue colonies were enumerated as E. coli while pink colonies were recorded as total coliforms.
4
Isolation and Quantification of E. coli from Shellfish Waters
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The bacterial strain of Escherichia coli, which was used in this work as a water quality indicator, was isolated from the shellfish washing waters by membrane filtration. From the collected samples 200 μL were passed through a 0.45 μm pore-sized filter (cellulose acetate/nitrate membranes by Sigma-Aldrich), using a vacuum pump VP series (KNF Lab). These membranes were aseptically placed up on plates with Brilliance E. coli/Coliform Agar (Oxoid) selective media, thus ensuring that no air bubbles were trapped. The plates were incubated at 37 °C for 20 -24 hours and E. coli colonies with purple-blue colour were picked for further use. Specifically, the isolated E. coli were spiked into the sterile industrial washing water to achieve the desired initial bacterial loading for each experimental run. The standard E. coli ATCC 23716 (American Type Culture Collection, Rockville, MD, USA) strain was also used. The freeze-dried cultures were rehydrated and reactivated according to the manufacturer's instructions. The concentration of bacterial cells in the shellfish processing water ranged from 10 4 -10 6 CFU mL -1 , as estimated by measuring its optical density at 600 nm on a Cary100 UV-Vis double-beam (Varian, Inc.) spectrophotometer.
5
Detecting and Quantifying E. coli in Water
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The detection and quantification of E. coli in the processing water was performed using the serial dilution pour plate agar technique. Serial dilutions of the reaction solution were performed in sterile 0.8% (w/v%) NaCl (Fisher Scientific, UK) aqueous solution and 200 μL of each dilution (including neat sample) were pipetted and spread onto Brilliance E. coli/Coliform Agar (Oxoid) plates, a selective culture medium. The plates were incubated at 37 •C for 20-24 h before viable counts were determined. E. coli colonies appeared with purple colour, while coliforms colonies had a pinkish colour. For the undiluted samples, 1 mL of sample was spread over five 90 mm Petri dishes (i.e. 200 μL of sample per Petri dish).
This was done to reduce the detection limit to 1 CFU mL -1 for the undiluted samples (Paleologou et al., 2007; Rincón and Pulgarin, 2004) .
The turbidity was measured on a HACH 2100N turbidity meter, while conductivity and pH were measured by a portable conductivity and pH meter (± 0.1 pH accuracy), respectively, by Hanna Instruments.
This was done to reduce the detection limit to 1 CFU mL -1 for the undiluted samples (Paleologou et al., 2007; Rincón and Pulgarin, 2004) .
The turbidity was measured on a HACH 2100N turbidity meter, while conductivity and pH were measured by a portable conductivity and pH meter (± 0.1 pH accuracy), respectively, by Hanna Instruments.
6
Murine Model of Citrobacter rodentium Infection
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The C. rodentium strain DBS100 (ATCC 51459; American Type Culture Collection) was used as described69 (link) with some amendments. Briefly, C. rodentium cultures were grown in Luria-Bertani (LB) medium to an OD600 of 1–1.5. Mice were orally inoculated with LB broth or 2 × 109 CFU C. rodentium, and their weight was monitored daily. Fecal samples were collected on indicated days post-infection, serially diluted in PBS and plated on Brilliance™ E. coli/coliform Agar (Thermo Fisher). C. rodentium colonies, from at least three dilutions per sample, were enumerated and normalized to the weight of feces.
For histopathology, 0.5 cm of the distal colon was immediately placed in 10% formalin and processed by the DTU Histology Core for hematoxylin and eosin (H&E) staining. Images were acquired on a Pannoramic MIDI II scanner (3DHISTECH) and visualized with CaseViewer software 2.4 (3DHISTECH). The severity of the infection was assessed in a blinded manner.
For histopathology, 0.5 cm of the distal colon was immediately placed in 10% formalin and processed by the DTU Histology Core for hematoxylin and eosin (H&E) staining. Images were acquired on a Pannoramic MIDI II scanner (3DHISTECH) and visualized with CaseViewer software 2.4 (3DHISTECH). The severity of the infection was assessed in a blinded manner.
7
In Vivo C. rodentium Infection
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Starter cultures of C. rodentium strain DBS100 (ATCC 51459; American Type Culture Collection) were grown overnight at 37°C in Luria-Bertani (LB) medium. The cultures were then used at 5% vol/vol to inoculate a sterile LB medium. The cultures were grown at 37°C to an OD600 of 0.8–1, and the CFU count was determined from the OD600 measurement using the following formula: CFU/ml = (5 × 108) × (OD) – (3 × 107). Subsequently, the bacteria were collected by centrifugation at 4,000 g for 10 min. The bacterial pellet was then resuspended in LB medium to give at 2 × 109 CFU/100 μl. To infect adult mice, the mice were orally gavaged with either 100 μl of C. rodentium or LB control. The mice were weighed before oral gavage and once daily until the termination of the experiment. At day 12 after infection, all mice were euthanized and dissected to collect organs and fecal samples.
The collected fecal samples were weighed and dissolved in PBS and then serially diluted. The serial dilutions were plated on Brilliance E. coli/coliform Agar (CM0956; Thermo Fisher Scientific) and incubated overnight at 37°C. C. rodentium colonies were identified as being pink colonies and enumerated, while E. coli colonies were identified as purple colonies. CFU/g stool was then calculated as previously described (Bouladoux et al., 2017 (link)).
The collected fecal samples were weighed and dissolved in PBS and then serially diluted. The serial dilutions were plated on Brilliance E. coli/coliform Agar (CM0956; Thermo Fisher Scientific) and incubated overnight at 37°C. C. rodentium colonies were identified as being pink colonies and enumerated, while E. coli colonies were identified as purple colonies. CFU/g stool was then calculated as previously described (Bouladoux et al., 2017 (link)).
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