This study provides a rapid and robust protocol for the isolation of Campylobacter from food product. It overcomes several drawbacks of existing methods, and offers advantages for the subsequent detection, and characterization of isolates. Rinse aids in the field are often 10, 000 times larger than the samples used in food safety detection assays, such as PCR.
There's a need to reduce the error of sub-sampling in order to ensure the results are representative of the population of interest. This protocol has been used to isolate over 50 strains of a Campylobacter, which have subsequently been utilized in genomic studies. Our data and analysis contributed to understanding the molecule mechanisms of Campylobacter host and production.
And such in size, where facilitated the development of novel therapeutic strategies for managing Campylobacterlosis. This protocol offers the opportunity to reduce sub sampling by factor of 13, and also reduces the time required to isolate Campylobacter from raw meat by 24 hours, or within an 8 hour workday. Future studies can apply this methodology for the isolation of Campylobacter, not only from food products, but from environmental samples as well.
This can help eliminate false negative results, and provide more accurate quantification of the number of contaminated samples. To begin, cut 450 grams of chicken pieces from the package, and place them in a stomach or bag. Add 200 milliliters of 0.1%buffered peptone water to the stomach bag containing the chicken.
Manually massage or palpate the sample from the outside of the stomach or bag for 2 minutes. Using a motorized pipette controller, collect all of the chicken rinse from the filtered side of the bag, and dispense it into sterile centrifuge bottles. Then centrifuge the chicken rinse at 10, 000 G for 10 minutes at room temperature.
Using a motorized pipette controller with a 25 milliliter disposable serological plastic pipette. Collect the supernatant carefully, and discard it. For enrichment of Campylobacter, add 25 milliliters of late horse blood to the sterilized 500 milliliters of Bolton broth.
Reconstitute 1 vial of antibiotic supplement in 5 milliliters of 50%ethanol, and add the reconstituted antibiotic supplement to the Bolton broth. Then re-suspend the pellet in 50 milliliters of Bolton broth containing lake horse blood and antibiotics. Place the samples inside a sealed container that maintains a gas mixture of 85%nitrogen, 10%carbon dioxide, and 5%oxygen.
Incubate the samples at 42 degrees Celsius for 24 hours. To begin, place Brucella agar plates with lids opened in a bio safety cabinet for drying. Place a cellulose acetate filter at the center of a Brucella agar plate.
Pipette 4 drops per filter, and 20 microliters per drop of Campylobacter enriched sample onto the filter. After incubating the drops at room temperature for 15 minutes, carefully remove the filters, then incubate the plates at 42 degrees Celsius for approximately 24 hours. After incubation, pick characteristic Campylobacter colonies with specific traits.
Street colonies onto Brucella agar plates for purification. For long-term storage, pick Campylobacter colonies grown on Brucella agar plates, and inoculate the Bolton broth. Place the samples inside a sealed container that maintains a gas mixture of 85%nitrogen, 10%carbon dioxide, and 5%oxygen.
Incubate the samples at 42 degrees Celsius overnight. Then add 900 microliters of the overnight grown culture of Campylobacter to a two milliliter cryo vial containing 100 microliters of DMSO. Rapidly cool in a dry ice ethanol bath for 10 minutes, and transfer to a minus 80 degree Celsius freezer.
Comparison of different pore size filter membranes revealed that more cells were able to pass through the 0.65 micrometer filter compared to the 0.45 micrometer filter. To begin, take the Brucella agar plates with Campylobacter colonies, disperse purified Campylobacter colonies in a 96 well plate with 100 microliters of extraction solution per well. Lyse samples at 99 degrees Celsius for 10 minutes, followed by cooling at 20 degrees Celsius for 2 minutes in a thermo cycler.
Then centrifuge the plate at 8, 000 G for 10 minutes at room temperature. Prepare a 20 microliter reaction mixture consisting of a master mix, DNA sample, internal amplification control template, primer, and probe. Load all samples in triplicates in a 96 well optical plate covered with an optical film, and place them in a real-time PCR system.
