A total of 302 samples including frozen chicken meat (n = 130) and fresh chicken meat (n = 172) were purchased from 20 supermarkets and wet markets every 3 months from April 2014 to April 2015. Each sample was homogenized, and the number of Campylobacter in 10 g sample homogenate was enumerated using a three-tube MPN combining with PCR method [21 (link)]. In brief, a ten-fold serial dilution series of each homogenates were prepared. Then 1 ml of each original homogenate or the diluted homogenate was transferred into each of the three tubes containing 9 ml of Bolton Enrichment Broth (OXOID, Basingstoke, England) and incubated at 42 °C for 48 h under microaerophilic condition. After incubation, total bacterial DNA was extracted and PCR amplification of 16s rDNA was performed to detect Campylobacter positive tubes. For statistical analysis, the differences in frequencies were analyzed by Chi square test.
Bolton enrichment broth
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Bolton Enrichment Broth is a culture medium designed for the enrichment and isolation of Campylobacter species. It provides the necessary nutrients and growth factors to support the cultivation of these fastidious microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Karmali agar, by Thermo Fisher Scientific (1 mentions)
Ccda selective supplement, by Thermo Fisher Scientific (1 mentions)
Bolton broth, by Thermo Fisher Scientific (1 mentions)
Campylobacter blood free agar, by Thermo Fisher Scientific (1 mentions)
Campygen kit, by Thermo Fisher Scientific (1 mentions)
Laked horse blood, by Thermo Fisher Scientific (1 mentions)
Gaspak ez campy container system, by BD (1 mentions)
4 protocols using bolton enrichment broth
1
Campylobacter Enumeration in Chicken Meat
2
Prevalence of Campylobacter in Poultry, Cattle, and Humans
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The study was conducted from March 2017 to September 2019. A total of 286 samples were collected from various sources; chickens (n = 195), cattle (n = 47) and human (n = 44) in Zagazig City, Sharkia Governorate, Egypt. The samples collected from broiler chickens included cloacal swabs (n = 75), breast meat (n = 40), caecal parts (n = 40) and liver (n = 40) were collected from retail outlets, while raw milk samples were collected from retail shops. Moreover, human stool samples were taken from gastroenteritis patients from clinical laboratories. Samples were collected in a sterile Bolton enrichment broth (Oxoid, UK) and transported in an ice box within 3 h to the laboratory for bacteriological analysis. The animal study was endorsed by the committee of Animal Welfare and Research Ethics, Faculty of Veterinary Medicine, Zagazig University. Regarding the human samples, it was approved by the research ethical committee of Faculty of Medicine, Zagazig University and the work was conducted in compliance with the Ethics of the World Medical Association (Declaration of Helsinki) for studies involving humans. Written informed permissions were taken from patients taking part in the research study after a complete explanation of the aim of the study.
3
Isolation and Identification of Campylobacter
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In the laboratory, caecal samples were streaked directly onto two selective solid media: Karmali agar (Oxoid, UK) and Campylobacter blood-free agar (Oxoid) with CCDA selective supplement (Oxoid). The swabs from carcasses were placed in 5 ml of Bolton enrichment broth (Oxoid, UK) supplemented with 5% lysed horse blood and modified Bolton broth supplement. The cultures from both types of samples were incubated at 41.5 o C for 48 h under microaerobic conditions using the CampyGen kit (Oxoid). Campylobacter bacteria were isolated and identified according to the ISO 10272-1:2006 standard. Briefly, after the enrichment step, the cultures from swab samples were plated onto Karmali agar (Oxoid) and Campylobacter blood-free agar (Oxoid) with CCDA selective supplement (Oxoid) and incubated at 41.5 o C for 48 h under microaerobic conditions. The plates with caecal and carcass bacterial cultures were then examined for morphologically typical Campylobacter colonies (grayish, often with a metallic sheen, flat, and moist with a tendency to spread) and from each sample, one presumptive Campylobacter isolate was confirmed by PCR assay as previously described (Wieczorek et al. 2013) . Furthermore, the isolated strains were identified as C. jejuni or C. coli by PCR (Wieczorek and Osek 2005) .
4
Isolation of Campylobacter from Water
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The presence of Campylobacter in the water samples was investigated following the method described by Walters et al. (2007) . Briefly, 200 ml of the water sample was filtered through a 0.45 µm Millipore membrane and the filter was placed in 30 ml of Bolton Enrichment Broth (Oxoid) containing selective supplement (Oxoid SR183E) and 5% (v/v) laked horse blood (Oxoid). The broth cultures were incubated under microaerophilic conditions (5-15% oxygen) using a GasPak EZ Campy container system (BD Diagnostic Systems, New Jersey), at The enriched cultures were also grown on Blood-free Campylobacter selective agar (Oxoid) followed by incubation at 42 o C under microaerophilic conditions as described above for 48 hours. Presumptive Campylobacter colonies were identified based on colony morphology, i.e. grey and moist colonies.
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