Listeria was isolated according to ISO 11290 method with partial modifications. Isolation was divided into two step enrichments. For fecal samples, about 2–5 g of feces were collected into 10 mL Half Fraser broth; for environmental swabs, 1 mL of samples were collected into 9 mL Half Fraser broth; for food samples, 25 g of sample was added to 225 mL Half Fraser broth, and incubated at 30°C for 24 h. Then 1 mL of primary enrichment cultures were added to 5 mL Fraser broth and incubated at 37°C for 48 h. Two loopful of secondary enrichment broths were steaked on the Chromogenic Listeria Agar (Oxoid, Basingstoke, UK), incubated at 37°C for 48 h. Five presumptive positive colonies were selected for each samples, and each colony was identified by PCR for Listeria genus-specific gene prs and species-specific gene lmo0733. Primers were shown in Table S1.
Chromogenic listeria agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Chromogenic Listeria Agar is a selective and differential culture medium used for the isolation and identification of Listeria species, particularly Listeria monocytogenes, from food, environmental, and clinical samples.
Automatically generated - may contain errors
Lab products found in correlation
Api listeria test, by bioMérieux (2 mentions)
Half fraser broth, by Thermo Fisher Scientific (1 mentions)
Chromocult coliform agar, by Merck Group (1 mentions)
Palcam agar, by Thermo Fisher Scientific (1 mentions)
Fraser broth, by Thermo Fisher Scientific (1 mentions)
Fraser supplement, by Thermo Fisher Scientific (1 mentions)
Listeria selective supplement, by Thermo Fisher Scientific (1 mentions)
Api listeria system, by bioMérieux (1 mentions)
Listeria differential supplement, by Thermo Fisher Scientific (1 mentions)
5 protocols using chromogenic listeria agar
1
Isolation and Identification of Listeria
2
Isolation and Identification of Listeria
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Listeria strains were isolated according to the ISO 11290 method with modifications. The intestinal feces were introduced into 10 mL Half-Fraser broth and incubated at 30 °C for 24 h. Subsequently, 0.5 mL of the primary enrichment cultures were transferred to 4.5 mL Fraser broth and incubated at 37 °C for 48 h. A loopful of secondary enrichment was streaked onto Chromogenic Listeria Agar (Oxoid, Basingstoke, UK) and incubated at 37 °C for 24–48 h. After incubation, colonies suspected of being Listeria spp. based on color and morphology were selected for identification. Bacterial colonies were identified using 16S rDNA amplification and sequencing and the API Listeria test (bioMérieux, Marcyl’Etoile, France). All confirmed Listeria isolates were stored in BHIB containing 15% glycerol at −80 °C.
3
Isolation and Identification of Listeria
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Listeria strains were isolated according to the ISO 11290 method with modifications. The intestinal feces were introduced into 10 mL Half-Fraser broth (Oxoid Ltd., Basingstoke, UK) and incubated at 30 °C for 24 h. Subsequently, 0.5 mL of the primary enrichment cultures were transferred to 4.5 mL Fraser broth and incubated at 37 °C for 48 h. A loopful of secondary enrichment was streaked onto Chromogenic Listeria Agar (Oxoid Ltd., Basingstoke, UK) and incubated at 37 °C for 24–48 h. After incubation, the colonies suspected of being Listeria spp. based on color and morphology were selected and identified by API Listeria test (bioMérieux, Marcyl’Etoile, France) [25 (link)].
4
Antimicrobial Activity of Biopolymers
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The bacteria strains used in the antimicrobial experiment were obtained from the Spanish Type Culture Collection (CECT) of Valencia University: Listeria innocua (CECT 910) and Escherichia coli (CECT 45).
An adaptation of the broth dilution method was used to study the antimicrobial activity of the biopolymers. Falcons were prepared with 10 mL of Muller–Hinton broth. Then, 1.5 × 1.5 cm pieces of each biopolymer were cut and introduced into the prepared medium. Finally, the preparations were inoculated with Listeria innocua and Escherichia coli (~103 CFU·mL−1).
Counts of the microorganisms were carried out at 0, 24, 48, and 72 h on a plate of Chromogenic Listeria Agar (Oxoid CM1084) for L. innocua and Chromocult® Coliform Agar (Merck, Darmstadt, Germany) for E. coli. The plates were incubated at 37 °C with readings at 24–48 h. The results were expressed as log CFU·ml−1.
An adaptation of the broth dilution method was used to study the antimicrobial activity of the biopolymers. Falcons were prepared with 10 mL of Muller–Hinton broth. Then, 1.5 × 1.5 cm pieces of each biopolymer were cut and introduced into the prepared medium. Finally, the preparations were inoculated with Listeria innocua and Escherichia coli (~103 CFU·mL−1).
Counts of the microorganisms were carried out at 0, 24, 48, and 72 h on a plate of Chromogenic Listeria Agar (Oxoid CM1084) for L. innocua and Chromocult® Coliform Agar (Merck, Darmstadt, Germany) for E. coli. The plates were incubated at 37 °C with readings at 24–48 h. The results were expressed as log CFU·ml−1.
5
Listeria monocytogenes Detection in Cheese
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In accordance with ISO 11290-1:2004, detection of L. monocytogenes was carried out by suspending 10 g of cheese in 90 mL of Fraser Broth (CM0895, Oxoid Ltd.) with Half Fraser Supplement (SR0166, Oxoid Ltd.) and incubating for 24 h at 30°C. Subsequently, 0.1 mL of the first enrichment broth was suspended in 10 mL of Fraser Broth (CM0895, Oxoid Ltd.) with Fraser Supplement (SR0156, Oxoid Ltd.) and then incubated for 24 h at 37°C. One loopfull of the enrichment broth was then streaked onto PALCAM Agar (CM0877, Oxoid Ltd.) added with PALCAM Selective Supplement (SR0150, Oxoid Ltd.) and onto Chromogenic Listeria Agar (CM1084, Oxoid Ltd.) supplemented with Listeria Selective Supplement (SR0226, Oxoid Ltd.) and Listeria Differential Supplement (SR0244, Oxoid Ltd.). Both plates were incubated for 48 h at 37°C, then Listeria monocytogenes presumptive colonies were biochemically identified by using the API Listeria System according to manufacturer's instructions (bioMérieux).
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