Ccda selective supplement

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

CCDA selective supplement is a bacterial growth medium used in microbiological laboratories. It is designed to facilitate the selective isolation and identification of Campylobacter species from clinical and food samples.

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10 protocols using ccda selective supplement

Antimicrobial Effects of Citrox and Chitosan on Campylobacter

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This study evaluated the effects of using 1% or 2% Citrox alone or in combination with 1% chitosan on C. jejuni in Campylobacter blood-free selective medium (modified CCDA-Preston) (Oxoid CM 0739, Basingstoke, UK) supplemented with CCDA Selective Supplement (Oxoid SR 0155, Basingstoke, UK) at 42 °C for 24 to 48 h.
One hundred microliters of C. jejuni ATCC 33291 culture (10⁶ cfu/mL) was spread using a cotton swab on the surface of Campylobacter blood-free selective medium (modified CCDA-Preston) (Oxoid CM 0739, Basingstoke, UK) supplemented with CCDA Selective Supplement (Oxoid SR 0155, Basingstoke, UK). Using the agar diffusion method, a hole with a diameter of 8 mm was punched aseptically using a sterile cork borer, and 100 µL of 1% or 2% Citrox solution was introduced into each well. To compare its effects on C. jejuni, 1% chitosan was mixed with the same volume of 1% or 2% Citrox. The plates were then incubated at 42 °C for 48 to 72 h under a microaerophilic atmosphere in the presence of CO₂. The diameter (mm) of the zone of inhibition was determined.

Yehia H.M., Al-Masoud A.H., Elkhadragy M.F., Korany S.M., Nada H.M., Albaridi N.A., Alzahrani A.A, & AL-Dagal M.M. (2021). Improving the Quality and Safety of Fresh Camel Meat Contaminated with Campylobacter jejuni Using Citrox, Chitosan, and Vacuum Packaging to Extend Shelf Life. Animals : an Open Access Journal from MDPI, 11(4), 1152.

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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) .

Wieczorek K, & Osek J. (2015). Poultry flocks as a source of Campylobacter contamination of broiler carcasses. Polish journal of veterinary sciences, 18(1).

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Cultivation and Maintenance of Bacterial Strains

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E. coli BL21 (DE3) Star (Invitrogen) was grown at 37°C on Luria-Bertani (LB) plates or in LB broth (Biotrading) supplemented with 100 μg x ml^-1 of ampicillin. C. jejuni strain 81116 [23 (link)] and C. jejuni 81116 ΔflaAB [22 (link)] were routinely grown on agar plates with 5% saponin-lysed horse blood or in heart infusion (HI) broth (Biotrading) at 37°C or 42°C under microaerobic conditions (10% CO₂, 5% O₂, 85% N₂). The presence of Campylobacter in cloacal swabs was tested using CCDA (charcoal cefoperazone deoxycholate agar) plates containing Campylobacter blood free selective agar base (Oxoid) and CCDA selective supplement (Oxoid) according to the manufacturer’s instructions. HeLa57A cell line stably transfected with a NF-κB luciferase reporter construct [24 (link)], was generously provided by Dr. R. T. Hay (Institute of Biomolecular Sciences, University of St. Andrews, St. Andrews, Scotland, U.K.). HeLa57A cells were propagated in Dulbecco modified Eagle medium (DMEM, Invitrogen) supplemented with 5% fetal calf serum (FCS) at 37°C under 10% CO₂.

Radomska K.A., Vaezirad M.M., Verstappen K.M., Wösten M.M., Wagenaar J.A, & van Putten J.P. (2016). Chicken Immune Response after In Ovo Immunization with Chimeric TLR5 Activating Flagellin of Campylobacter jejuni. PLoS ONE, 11(10), e0164837.

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Isolation of Campylobacter spp. from Samples

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The preparation of the samples and isolation of Campylobacter spp. from the examined samples was done according to FDA [14 ]. The pH of the samples was adjusted to 7.5±0.2, and then centrifugation of 50 g portion at 20,000 ×g for 40 min was attained. Supernatant was discarded and pellets were dissolved in 10 ml Bolton broth (supplemented with Bolton broth Selective Supplement and Laked Horse Blood, Oxoid) and then was transmitted to 90 ml enrichment broth and incubated at 42°C for 48 h in an anaerobic jar containing a gas generating Kit (Oxoid). The Campylobacter blood free selective agar (mCCDA-Preston, Oxoid) which was supplemented with CCDA selective supplement (Oxoid), were then streaked with a loopful of each enrichment broth, and subsequently, incubated at 42°C for 48 h under microaerobic condition. From 2 to 3 presumptive Campylobacter colonies were purified on Columbia blood agar (containing 7% defibrinated sheep blood)without supplement. About 100 Campylobacter isolates were submitted to Gram-stain, oxidase, catalase, inability to grow aerobically at 25°C, hippurate hydrolysis and resistance to naladixic acid and cephalothin to exclude Campylobacter spp. except C. jejuni and C. coli.

El-Zamkan M.A, & Hameed K.G. (2016). Prevalence of Campylobacter jejuni and Campylobacter coli in raw milk and some dairy products. Veterinary World, 9(10), 1147-1151.

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Isolation and Identification of C. jejuni

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One gram of each sample was homogenized with 9 mL of Bolton broth containing 5% hemolyzed horse blood and a Bolton broth selective supplement (Oxoid, United Kingdom). The samples were incubated at 42°C for 48 h microaerobically. On the following day, one loop of broth was streaked onto modified charcoal cefoperazone deoxycholate agar (mCCDA) containing CCDA selective supplement (Oxoid), and then the samples were incubated at 42°C for 48 h microaerobically. Next, two to six suspected colonies from the mCCDA plates were picked, subcultured in blood agar, and incubated microaerobically at 42°C for 48 h. To identify C. jejuni isolates, multiplex PCR targeting the 16S rRNA gene and cj0414 and singleplex PCR targeting hipO were performed using the DNA templates, according to methods described previously (Yamazaki-Matsune et al., 2007 (link); Supplementary Table S2).

An J.U., Ho H., Kim J., Kim W.H., Kim J., Lee S., Mun S.H., Guk J.H., Hong S, & Cho S. (2018). Dairy Cattle, a Potential Reservoir of Human Campylobacteriosis: Epidemiological and Molecular Characterization of Campylobacter jejuni From Cattle Farms. Frontiers in Microbiology, 9, 3136.

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Isolation and Identification of Campylobacter

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Isolation of Campylobacter spp. was achieved according to the International Standards Organization (ISO) guidelines [40 ]. Briefly, the enrichment broth containing samples was incubated for 48 h at 42 °C in darkness under microaerophilic conditions (5% O₂, 10% CO₂ and 85% N₂) using CampyGen sachets (Oxoid, Cheshire, UK) and anaerobic jar (Oxoid, Cheshire, UK). After that, 10 µL of the enrichment broth was streaked onto the surface of the selective modified charcoal cefoperazone deoxycholate agar (mCCDA) plates with CCDA selective supplement (Oxoid, Cheshire, UK), and the plates were incubated for 48 h at 42 °C in darkness under microaerophilic conditions. For more purification, suspicious colonies were cultivated onto blood agar (Oxoid, Cheshire, UK) supplemented with 5% sterile defibrinated sheep blood, and the plates were incubated for 48 h under microaerophilic conditions. The Campylobacter isolates were presumptively confirmed via cultural characteristics on mCCDA, Gram’s staining, motility, some biochemical tests such as catalase, oxidase and indoxyl acetate and sodium hippurate hydrolysis tests and finally susceptibility to nalidixic and cephalothin.

Ammar A.M., Abd El-Hamid M.I., El-Malt R.M., Azab D.S., Albogami S., Al-Sanea M.M., Soliman W.E., Ghoneim M.M, & Bendary M.M. (2021). Molecular Detection of Fluoroquinolone Resistance among Multidrug-, Extensively Drug-, and Pan-Drug-Resistant Campylobacter Species in Egypt. Antibiotics, 10(11), 1342.

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Campylobacter Isolation and Identification

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For Campylobacter spp. isolation, the collected samples in Campy-Thio broth were incubated for 24–48 h at 42 °C with less than 1 cm of headspace left in the culture vessels, which were kept with tightly-capped lids in darkness under microaerophilic conditions (85% N₂, 10% CO₂ and 5% O₂) using CampyGen sachets (Oxoid, Cambridge, UK) and the anaerobic jar (Sigma-Aldrich, St. Louis, MI, USA). Following the enrichment step, 0.1 mL of the broth was inoculated onto the surface of modified charcoal cefoperazone deoxycholate agar (mCCDA) with CCDA selective supplement (Oxoid, Cambridge, UK) and the plates were incubated under microaerophilic conditions at 42 °C in darkness for 48 h. Additionally, three to four presumptive campylobacter colonies that had similar colonial morphology were further inoculated onto 5% sheep blood agar plates for 24–48 h at 42 °C under microaerophilic conditions in darkness. After incubation, suspected colonies were identified via Gram’s staining, motility test and biochemical identification using catalase, oxidase and rapid hippurate hydrolysis tests [25 (link),26 ].

Ammar A.M., El-Naenaeey E.S., El-Malt R.M., El-Gedawy A.A., Khalifa E., Elnahriry S.S, & Abd El-Hamid M.I. (2020). Prevalence, Antimicrobial Susceptibility, Virulence and Genotyping of Campylobacter jejuni with a Special Reference to the Anti-Virulence Potential of Eugenol and Beta-Resorcylic Acid on Some Multi-Drug Resistant Isolates in Egypt. Animals : an Open Access Journal from MDPI, 11(1), 3.

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Campylobacter jejuni ATCC 33291 Activation

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An inoculum of C. jejuni ATCC 33291 (~10⁴ colony-forming units (cfu/mL)) was used. Two types of media were used to activate the strain, Campylobacter blood-free selective medium (modified CCDA-Preston) (Oxoid CM 0739, Basingstoke, UK) supplemented with CCDA Selective Supplement (SR 0155) and Bolton Selective Enrichment Broth (Oxoid CM0983, Basingstoke, UK), and then cooled to 45 to 50 °C, and 25 mL of laked horse blood (SR0048) and one vial of Bolton Broth Selective Supplement (Oxoid SR0183 Basingstoke, UK), were aseptically added. The plates were incubated at 42 °C for 24 to 72 h.

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Culturing and Storing Lactobacillus and Campylobacter

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L. johnsonii strain FI10058 is derived from strain FI9785 and contains plasmid pFI2431, produced by the insertion of a chloramphenicol resistance gene into the highly stable native small plasmid p9785 (Horn et al., 2005) . It was routinely grown at 37°C on de Man, Rogosa, Sharpe agar or broth (MRS, Oxoid) supplemented with 10 µg/ml neomycin and 7.5 µg/ml chloramphenicol. Overnight cultures used for inoculation were harvested by centrifugation at 3000 g for 15 min at 20°C, washed twice and resuspended in phosphate-buffered saline (PBS) at approximately 1×10 9 colony forming units (cfu)/ml. C. jejuni strain 81-176 was isolated from an outbreak associated with unpasteurised milk (Korlath et al., 1985) . It is known that this strain readily colonises chickens (Guccione et al., 2008) . It was routinely cultured on sheep blood agar (Oxoid) under standard microaerobic conditions (10% O 2 , 5%
CO 2 and 85% N 2 ) at 37 o C in a MACS-VA500 incubator (Don Whitley Scientific). Cultures used for inoculation were incubated for 24 h in 10 ml Mueller-Hinton broth (MH, Oxoid).
Long-term storage of Campylobacter was at -80°C in Microbank vials (Prolab Diagnostics).
Campylobacter blood-free selective agar (CBF) plates were prepared according to the manufacturer's instructions from Campylobacter blood-free selective agar (CCDA, Oxoid)
and CCDA selective supplement (Oxoid).

Mañes-Lázaro R., Van Diemen P.M., Pin C., Mayer M.J., Stevens M.P, & Narbad A. (2017). Administration of Lactobacillus johnsonii FI9785 to chickens affects colonisation by Campylobacter jejuni and the intestinal microbiota. British poultry science, 58(4).

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Establishing Campylobacter-Colonized Chicken Model

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To establish an initial gut flora, day-of-hatch specific-pathogen-free (SPF) Light Sussex chicks were fed with 0.1 ml of Campylobacter-free adult gut flora preparations. These preparations were generated by taking 1 g of caecal contents from a 50-week old SPF chicken and using it to inoculate 10 ml of LB broth, which was incubated in static culture for 24 h at 37˚C. Birds were fed a vegetable based diet (Special Diet Services). After two weeks, five chickens housed in a single cage, were orally infected with 0.1 ml of a MH broth culture containing 1x10 9 , 1x10 9 , 1x10 10 CFU ml -1 of the M1 wild type, M1 ΔCJM1_0368b and M1 CJM1_0368*, respectively. Seven days post inoculation (p.i.), chickens were sacrificed and caecal contents were serially diluted in PBS and plated onto blood-free Campylobacter selective agar containing CCDA-selective supplement (Oxoid). Plates were incubated in microaerophilic conditions for 48 h and CFU enumerated. The number of Campylobacter CFU per gram of caecal content was calculated.

Kanji A., Jones M.A., Maskell D.J, & Grant A.J. (2015). Campylobacter jejuni PflB is required for motility and colonisation of the chicken gastrointestinal tract. Microbial pathogenesis, 89.

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