Campygen gas pack

Manufactured by Thermo Fisher Scientific

Sourced in Germany, United Kingdom

CampyGen gas packs are a laboratory product designed to create an anaerobic atmosphere for the cultivation of Campylobacter species. The gas packs generate a controlled mixture of gases, including hydrogen and carbon dioxide, to support the growth of these microaerophilic bacteria.

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50 protocols using campygen gas pack

Culturing and Passaging Streptococcus pneumoniae

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Viable bacteria were recovered from the storage medium by inoculation onto 5% Columbia Blood Agar plates (E & O Laboratories Ltd, Bonnybridge, Scotland), and incubated at 37°C for 2–3 days in an anaerobic jar containing a CampyGen^™ gas pack (Oxoid Ltd, Basingstoke, UK) for the generation of a microaerophilic atmosphere (5% Oxygen, 10% Carbon dioxide, and 85% Nitrogen). Between 20 and 30 S. pneumoniae colonies were passaged initially into 10 mL Brain Heart Infusion (BHI) broth (Oxoid, UK) supplemented with 5% (v/v) Foetal Calf Serum (FCS). The inoculated BHI-FCS broth medium was incubated stationary at 37°C for 15 h in an anaerobic jar containing a CampyGen^™ gas pack. In order to generate a sufficient number of bacterial cells for the analysis of S. pneumoniae cellular proteins, a subculture in BHI-FCS broth from the first bacterial passage was performed. Briefly, 3 mL of bacterial suspension from the 10 mL broth culture (OD650 of 1.2) was added into 20 mL fresh BHI-FCS broth to give a starting OD650 of 0.1 and incubated stationary at 37°C under a microaerophilic atmosphere until an OD650 of 1.0 was reached.

Bittaye M., Cash P, & Forbes K. (2017). Proteomic variation and diversity in clinical Streptococcus pneumoniae isolates from invasive and non-invasive sites. PLoS ONE, 12(6), e0179075.

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Antibacterial Susceptibility of Campylobacter

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Culture media (Columbia Agar, Brain Heart Infusion (BHI), lacked horse blood, Horse Serum, Vitox Supplement) and CampyGen gas pack were obtained from Oxoid, Basingstoke, England. Doxycycline (doxycycline 200 mg, Combitic Global Caplet, India), metronidazole (metronidazole Tablets BP 500 mg, Strides Arcolab, India), and ciprofloxacin (zoflox, ciprofloxacin 750 mg, Odypharm) were used as reference antibiotics for the determination of the MIC and MBC and for the in vivo test were obtained from local pharmacy. P-Iodonitrotetrazolium chloride (INT, Sigma-Aldrich) was used to indicate microbial growth [27 (link)].

Kouitcheu Mabeku L.B., Eyoum Bille B, & Nguepi E. (2016). In Vitro and In Vivo Anti-Helicobacter Activities of Eryngium foetidum (Apiaceae), Bidens pilosa (Asteraceae), and Galinsoga ciliata (Asteraceae) against Helicobacter pylori. BioMed Research International, 2016, 2171032.

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Isolation and Identification of Helicobacter pylori

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Colombia blood agar base (Oxoid Ltd., Basingstoke, Hampshire, England) was sterilized by autoclaving (121°C for 15 min), then enriched with 7% (v/v) laked horse blood (Oxoid Ltd., Basingstoke, Hampshire, England), and supplied with H. pylori selective supplement (Dent) (Oxoid Ltd., Basingstoke, Hampshire, England). Our standard strain (NCTCC 11916) of H. pylori was used for testing.
After spreading the bacteria, plates were incubated in Candle jar 2.5L (Oxoid ltd., Basingstoke, Hampshire, England) with 98% humidity at 37°C, this environment achieved by using CampyGen Gas Pack (Oxoid Ltd., Basingstoke, Hampshire, England), and the incubation period was 7 days.[13 (link)]
The presence of H. pylori growth on plates was confirmed by typical spiral morphology and flagella presence of the colonies, Gram staining, and conventional biochemical tests, including urease, catalase, and oxidase reactions (positive for urease, catalase, and oxidase reactions).[14 ]

Al Tawalbeh D., Aburjai T., Al Balas Q, & Al Samydai A. (2022). In Silico and In Vitro Investigation of Anti Helicobacter Activity of Selected Phytochemicals. Journal of Pharmacy & Bioallied Sciences, 14(3), 132-139.

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Quantification of Pathogenic Loads

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The pathogenic loads were surveyed in fecal samples at defined time points p.i., and upon necropsy in luminal samples derived from the colon, ileum, duodenum and stomach and additionally, in ex vivo biopsies taken from mesenteric lymph nodes (MLN), spleen, lungs, liver, and kidneys by culture as reported earlier [36 (link),38 (link)]. Briefly, by using sterile pestles respective samples were homogenized in sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA). Serial dilutions were then streaked onto karmali agar and Columbia agar (supplemented with 5% sheep blood; both from Oxoid, Wesel, Germany) and incubated in a jar containing CampyGen gas packs (Oxoid, Wesel, Germany) under microaerophilic conditions at 37 °C for at least 48 h. In order to survey systemic translocation, cardiac blood (0.1 mL) was directly plated onto Columbia agar (supplemented with 5% sheep blood) and incubated likewise. The detection limit of viable pathogens was 100 CFU per g.

Heimesaat M.M., Mousavi S., Weschka D, & Bereswill S. (2021). Garlic Essential Oil as Promising Option for the Treatment of Acute Campylobacteriosis—Results from a Preclinical Placebo-Controlled Intervention Study. Microorganisms, 9(6), 1140.

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Quantifying C. jejuni Colonization

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Viable C. jejuni were detected in feces over time p.i. or in luminal colonic samples at time of necropsy (i.e. day 8 p.i.), dissolved in sterile PBS and serial dilutions cultured on Karmali- and Columbia-Agar supplemented with 5% sheep blood (Oxoid) for two days at 37°C under microaerobic conditions using CampyGen gas packs (Oxoid). To quantify bacterial translocation, ex vivo biopsies derived from MLNs, spleen, liver and kidney were homogenized in 1 mL sterile PBS, whereas cardiac blood (≈100 μL) was directly streaked onto Karmali-Agar and Columbia-Agar supplemented with 5% sheep blood and cultivated accordingly. The respective weights of fecal or tissue samples were determined by the difference of the sample weights before and after asservation. The detection limit of viable pathogens was ≈100 colony forming units (CFU) per gram (g) as assessed by direct plating.

Bereswill S., Alutis M.E., Grundmann U., Fischer A., Göbel U.B, & Heimesaat M.M. (2016). Interleukin-18 Mediates Immune Responses to Campylobacter jejuni Infection in Gnotobiotic Mice. PLoS ONE, 11(6), e0158020.

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Characterization of Campylobacter jejuni Strains

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C. jejuni 81-176 is a well characterized sequenced strain used widely in infection studies, C. jejuni NCTC11168 is the type strain of C. jejuni. Strain C19 was isolated from a chicken and was acquired from the laboratory of Professor Seamus Fanning. NCTC11168 cadF- and flpA- were acquired from Professor Steffen Backert and Dr Nick Dorrell respectively. 81-176 flgI-, 81-176 flgE- and 81-176 fliD- as well as the pRY107 plasmid were acquired from Professor Patricia Guerry. All C. jejuni strains were cultured on Mueller Hinton (MH) agar (Oxoid) at 37°C under microaerophilic conditions created using Campygen gas packs (Oxoid). C. jejuni stock cultures were maintained using MH broth (Oxoid) supplemented with 20% glycerol and stored at −80°C. To revive stocks of C. jejuni strains, bacteria were streaked with a single use sterile inoculation loop (Sarstedt) on MH agar and incubated under microaerophilic conditions for 48 h at 37°C. For liquid cultures, C. jejuni strains were equalized to specific optical densities in MH broth and incubated under microaerophilic conditions at 37°C, shaking at 200 rpm. NCTC11168 cadF-, NCTC11168 flpA- or strains transformed with plasmid pRY107 were cultured with the addition of 50 μg/ml kanamycin. 81-176 flgI-, 81-176 flgE- and 81-176 fliD- were cultured with the addition of 10 μg/ml chloramphenicol.

Scanlan E., Ardill L., Whelan M.V., Shortt C., Nally J.E., Bourke B, & Ó Cróinín T. (2017). Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni. Molecular microbiology, 104(1), 92-104.

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Detecting Campylobacter in Food Samples

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Identification was performed following the method given by ISO 10272:1995(E) with certain modifications. Samples from caecal material were directly cultured on modified Charcoal Cefoperazone Deoxycholate agar (mCCDA) plates. Twenty five grams from the pooled neck skins or retail meat samples were enriched in 225 mL of Preston enrichment broth, incubated at 42 °C for 48 h. After enrichment, a loopful taken from a wire loop was plated on mCCDA plates. The mCCDA plates were incubated at 42 °C for 24 to 48 h at a microaerobic atmosphere which was created using a Campy-gen gas pack. All the media and Campy-gen gas packs utilized were from Oxoid.
Culture plates were observed after 24 to 48 h for the presence of typical Campylobacter colonies. Suspected colonies were selected and cultured on blood agar plates. Thereafter, Gram staining, catalase test, oxidase test, aerobic growth at 42 °C, anaerobic growth at 25 °C and reactions in Triple Sugar Iron (TSI) agar slants were utilized for genus identification of Campylobacter.

Kottawatta K.S., Van Bergen M.A., Abeynayake P., Wagenaar J.A., Veldman K.T, & Kalupahana R.S. (2017). Campylobacter in Broiler Chicken and Broiler Meat in Sri Lanka: Influence of Semi-Automated vs. Wet Market Processing on Campylobacter Contamination of Broiler Neck Skin Samples. Foods, 6(12), 105.

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Comparative Study of H. pylori and C. jejuni

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H. pylori strains P12 [21 (link)], 26695 [22 (link)], J99 [23 (link)] and G27 [24 (link)] were used in this study. Campylobacter jejuni strains used were the invasive strain, 81-176 [25 (link),26 (link)] and the less invasive strain, NCTC11168 [27 (link)]. H. pylori strains were cultured on Columbia blood agar base containing 7% (v/v) horse defibrinated blood. C. jejuni strains were cultured at 37 °C on Mueller-Hinton (MH) agar. Microaerophilic conditions were generated using CampyGen gas packs (Oxoid).

Dunne C., Naughton J., Duggan G., Loughrey C., Kilcoyne M., Joshi L., Carrington S., Earley H., Backert S., Robbe Masselot C., May F.E, & Clyne M. (2018). Binding of Helicobacter pylori to Human Gastric Mucins Correlates with Binding of TFF1. Microorganisms, 6(2), 44.

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Cultivation and Harvesting of Campylobacter Strains

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Campylobacter hepaticus HV10^T (NCTC 13823) isolated from a SLD-affected flock in Australia, Campylobacter jejuni 354, Campylobacter coli 54/2, Campylobacter fetus 54/3, Campylobacter lari 54/6, Campylobacter upsaliensis 54/7 were grown on Brucella agar (Amyl Media) with 5% horse blood (HBA) plates and incubated at 37 °C under microaerophilic conditions (85% N2, 10% CO2 and 5% O2) using CampyGen gas packs (Oxoid) for 48 h. All Campylobacter strains but C. hepaticus HV10^T are human clinical isolates obtained from RMIT University’s collection. Plates were harvested by flooding with ice-cold phosphate buffered saline (PBS) and gently resuspending cells using a spreader bar. Escherichia coli JM109 and Salmonella Typhimurium STM1^{48 (link)} were grown in Luria broth (tryptone (1%) (Sigma), yeast extract (0.05%) (Oxoid), and sodium chloride (1%). Cell suspensions were spun down at 8000 g for 5 min and the supernatant removed, cell pellets were washed twice with ice-cold PBS and stored at − 20 °C.

McDonald J.B., Scott N.E., Underwood G.J., Andrews D.M., Van T.T, & Moore R.J. (2023). Characterisation of N-linked protein glycosylation in the bacterial pathogen Campylobacter hepaticus. Scientific Reports, 13, 227.

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Culturing and Maintaining Campylobacter jejuni NCTC11168

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C. jejuni NCTC11168 is the type strain of C. jejuni and a commonly used laboratory strain. C. jejuni were cultured on Mueller Hinton (MH) agar (Oxoid) at 37 °C under microaerobic conditions generated using Campygen gas packs (Oxoid). For liquid cultures, C. jejuni strains were equalised to specific optical densities in MH broth and incubated under microaerobic conditions at 37 °C, shaking at 200 rpm. C. jejuni stock cultures were maintained using MH broth (Oxoid) supplemented with 20% glycerol and stored at -80 °C. To relax DNA supercoiling levels and induce biofilm formation, strains were grown in the presence of 10 μg/ml novobiocin as previously described [27 (link), 30 (link)].

Whelan M.V., Simpson J.C, & Ó Cróinín T. (2021). A novel high-content screening approach for the elucidation of C. jejuni biofilm composition and integrity. BMC Microbiology, 21, 2.

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