Microaerophilic/Capnophilic Bacterial Culture
Materials Required
Principle
Microaerophilic/capnophilic bacterial culture detects viable bacteria that grow only or optimally under reduced oxygen and/or elevated carbon dioxide, with the readout generated by visible colony recovery, colony density, or growth in liquid culture; thermophilic Campylobacter grew under 5-10% O2 with 1-10% CO2, while Helicobacter pylori culture studies describe growth under 5-19% O2 and 5-10% CO2, with CO2 being required for H. pylori and contributing to C. jejuni capnophilic growth through bicarbonate-linked metabolism.
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use an organism-appropriate nonselective or selective culture medium: for Campylobacter jejuni/coli, Preston medium, charcoal-cefoperazone-deoxycholate agar, or charcoal-based selective medium were evaluated for stool or mixed-flora specimens;
• Charcoal-based media support Campylobacter recovery while suppressing fecal flora.
• For H. pylori, use complex culture media, commonly serumor blood-supplemented media, because published methodology reviews describe H. pylori as capnophilic and microaerophilic and typically cultured on complex media.
• Culture confirmation may use organism-specific identification methods, but only culture growth is part of this protocol.
• Use an incubator with a microaerophilic gas system, evacuation-replacement jar, gas-generating system, or automated jar system;
• Comparative studies found that evacuation-replacement produced less variable O2/CO2 concentrations than gas envelopes, and the Anoxomat system gave growth comparable to conventional systems for capnophiles and microaerophiles.
• Charcoal-based media support Campylobacter recovery while suppressing fecal flora.
• For H. pylori, use complex culture media, commonly serumor blood-supplemented media, because published methodology reviews describe H. pylori as capnophilic and microaerophilic and typically cultured on complex media.
• Culture confirmation may use organism-specific identification methods, but only culture growth is part of this protocol.
• Use an incubator with a microaerophilic gas system, evacuation-replacement jar, gas-generating system, or automated jar system;
• Comparative studies found that evacuation-replacement produced less variable O2/CO2 concentrations than gas envelopes, and the Anoxomat system gave growth comparable to conventional systems for capnophiles and microaerophiles.
Experimental Procedure
• Prepare fresh or appropriately transported clinical, food, animal, or environmental samples for direct plating or enrichment according to the organism and specimen type;
• For Campylobacter, studies compared direct plating and enrichment on selective media, and enrichment culture using Preston enrichment broth increased isolation compared with direct plating onto Preston medium in tested specimens.
• Prepare selective agar plates before inoculation and select the medium according to specimen background flora;
• Preston medium showed high isolation/selectivity in one comparative study, while charcoal-based selective media and modified CCDA showed high recovery and suppression of fecal flora in later evaluations.
• Inoculate the sample onto the selected agar medium or into enrichment broth, then incubate plates or broth under a verified microaerophilic/capnophilic atmosphere;
• For thermophilic Campylobacter, reported suitable atmospheres are 5-10% O2 with 1-10% CO2, and 10% O2 was optimal among the concentrations tested in one study.
• For H. pylori, incubate cultures under a CO2-containing atmosphere;
• One comparative study reported that H. pylori is commonly grown at 5-19% O2 and 5-10% CO2, required elevated CO2, and showed similar growth across microaerobic to aerobic O2 tensions only at sufficiently high inoculum densities in CO2-containing conditions.
• Avoid relying on candle jars for Campylobacter culture when reproducible microaerobic conditions are required, because a comparative study found candle jars and modified Butzler medium to be of limited use, and measured gas control was less variable with evacuation-replacement than with gas envelopes.
• Record growth as colony recovery, colony morphology, colony density, or viable count on the selected medium;
• Comparative media studies used isolation rate, colony counts, recovery from simulated positive feces, and suppression of fecal flora to judge culture performance.
• Use a known viable strain as a positive growth control and an uninoculated plate or broth as a sterility control;
• Atmosphere performance should be verified when possible because studies comparing microaerobic systems measured O2 and CO2 concentrations and found that not all jar-envelope combinations produced satisfactory atmospheres.
• For Campylobacter, studies compared direct plating and enrichment on selective media, and enrichment culture using Preston enrichment broth increased isolation compared with direct plating onto Preston medium in tested specimens.
• Prepare selective agar plates before inoculation and select the medium according to specimen background flora;
• Preston medium showed high isolation/selectivity in one comparative study, while charcoal-based selective media and modified CCDA showed high recovery and suppression of fecal flora in later evaluations.
• Inoculate the sample onto the selected agar medium or into enrichment broth, then incubate plates or broth under a verified microaerophilic/capnophilic atmosphere;
• For thermophilic Campylobacter, reported suitable atmospheres are 5-10% O2 with 1-10% CO2, and 10% O2 was optimal among the concentrations tested in one study.
• For H. pylori, incubate cultures under a CO2-containing atmosphere;
• One comparative study reported that H. pylori is commonly grown at 5-19% O2 and 5-10% CO2, required elevated CO2, and showed similar growth across microaerobic to aerobic O2 tensions only at sufficiently high inoculum densities in CO2-containing conditions.
• Avoid relying on candle jars for Campylobacter culture when reproducible microaerobic conditions are required, because a comparative study found candle jars and modified Butzler medium to be of limited use, and measured gas control was less variable with evacuation-replacement than with gas envelopes.
• Record growth as colony recovery, colony morphology, colony density, or viable count on the selected medium;
• Comparative media studies used isolation rate, colony counts, recovery from simulated positive feces, and suppression of fecal flora to judge culture performance.
• Use a known viable strain as a positive growth control and an uninoculated plate or broth as a sterility control;
• Atmosphere performance should be verified when possible because studies comparing microaerobic systems measured O2 and CO2 concentrations and found that not all jar-envelope combinations produced satisfactory atmospheres.
Troubleshooting
Problem: Poor or absent Campylobacter growth.
• Possible Cause: The O2/CO2 atmosphere is outside the supported range.• Literature-supported Solution: Use a controlled microaerophilic/capnophilic atmosphere, because thermophilic Campylobacter grew suitably at 5-10% O2 with 1-10% CO2, and evacuation-replacement produced less variable gas concentrations than envelopes.
Problem: Poor H. Pylori recovery.
• Possible Cause: Insufficient CO2 or low inoculum density.• Literature-supported Solution: Maintain CO2-containing conditions, because H. Pylori and other tested Helicobacter species had an absolute requirement for elevated CO2, and H. Pylori behaved as oxygen-sensitive at low cell density.
Problem: Overgrowth of fecal background flora during Campylobacter isolation.
• Possible Cause: The selected medium does not sufficiently suppress competing flora.• Literature-supported Solution: Use validated selective media such as Preston medium, charcoal-based selective medium, or modified CCDA, which were evaluated for recovery and suppression of fecal flora.
References:
- [1]. Bolton FJ, et al. A study of the oxygen and carbon dioxide requirements of thermophilic campylobacters. J Clin Pathol. 1983;36(7):829-834. [Content Brief]
- [2]. Bury-Moné S, Kaakoush NO, Asencio C, Mégraud F, Thibonnier M, De Reuse H, et al. Is Helicobacter pylori a true microaerophile? Helicobacter. 2006;11(4):296-303. [Content Brief]
- [3]. Al-Haideri H, et al. Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni. Environ Microbiol. 2016;18(2):721-735. [Content Brief]
- [4]. Bolton FJ, et al. Comparison of selective media for isolation of Campylobacter jejuni/coli. J Clin Pathol. 1983;36(1):78-83. [Content Brief]
- [5]. Karmali MA, et al. Evaluation of a blood-free, charcoal-based, selective medium for the isolation of Campylobacter organisms from feces. J Clin Microbiol. 1986;23(3):456-459. [Content Brief]
- [6]. Bolton FJ, et al. Blood-free selective medium for isolation of Campylobacter jejuni from feces. J Clin Microbiol. 1984;19(2):169-171. [Content Brief]
- [7]. Gun-Munro J, et al. Laboratory and clinical evaluation of isolation media for Campylobacter jejuni. J Clin Microbiol. 1987;25(12):2274-2277. [Content Brief]
- [8]. Cover TL. Perspectives on methodology for in vitro culture of Helicobacter pylori. Methods Mol Biol. 2012;921:11-15. [Content Brief]
- [9]. Bolton FJ, et al. A comparison of microaerobic systems for the culture of Campylobacter jejuni and Campylobacter coli. Eur J Clin Microbiol. 1983;2(2):105-110. [Content Brief]
- [10]. Brazier JS, et al. Evaluation of the Anoxomat: a new technique for anaerobic and microaerophilic clinical bacteriology. J Clin Pathol. 1989;42(6):640-644. [Content Brief]
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