Stool specimens were collected from patients with healthcare-associated diarrhea admitted to Hanyang University Guri Hospital in Guri, South Korea, from September 2008 to January 2010. After alcohol shock treatment, specimens were inoculated on Clostridium difficile Selective Agar (Oxoid Ltd., Cambridge, UK) supplemented with cycloserine, cefoxitin, and 7% horse blood. Specimens were cultured anaerobically for 72 hr. Colonies of C. difficile were identified with an API RapidID 32A system (bioMérieux SA, Lyon, France), and toxigenic isolates were stored in a -70℃ deep freezer. A total of 171 C. difficile isolates were collected and used for the study. This study was approved by the institutional review board of Hanyang University Guri Hospital (HYUGH IRB 2013-24).
Api rapid id 32a system
Manufactured by bioMérieux
Sourced in France
The API Rapid ID 32A system is a rapid identification system for anaerobic bacteria. It provides a standardized and automated method for the identification of anaerobic microorganisms from clinical specimens.
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Lab products found in correlation
4 protocols using api rapid id 32a system
1
Isolation and Identification of Clostridium difficile
2
Detecting C. difficile from Stool Samples
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The stool samples were pre-treated with 99% ethanol for 30 min at room temperature. The samples were inoculated on selective media with antibiotics (Clostridium difficile Moxalactam Norfloxacin), and cultured anaerobically for 24-48 h at 36℃. Suspected C. difficile colonies were identified with an API Rapid ID 32A system (bioMérieux SA, Lyon, France). C. difficile toxin A and B were detected by VIDAS C. difficile Toxin A&B (bioMérieux SA) kits.
3
Clostridium difficile Identification Protocol
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Stool specimens were cultured anaerobically on cycloserine–cefoxitin–taurocholate agar (Oxoid Ltd., Cambridge, UK) supplemented with 7% horse blood, after alcohol shock treatment. Colonies of C. difficile were identified with an API® Rapid ID 32A system (bioMérieux SA, Lyon, France). Multiplex PCR and agar gel electrophoresis were performed with DNA from the cultured C. difficile isolates, as described elsewhere with minor modifications [8 (link), 10 (link)]. PCR-ribotyping was performed as described elsewhere [8 (link), 10 (link)], and banding patterns were checked by eye. Each unique pattern was assigned its own ribotype code and was matched with the PCR-ribotypes of reference strains ribotype 027 (BI/NAP1/027), ribotype 017 (ATCC 43598) and standard strains from the ECDC-Brazier collection.
4
Isolation and Identification of C. difficile
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Samples were processed immediately after arrival at the Lab or refrigerated for no more than 72 h before being processed. Stool samples were initially tested for free C. difficle A/B toxins using one or two enzyme immunoassays (EI) ImmunoCard toxin A&B (Meridian Bioscience, Cincinnati, OH) or MiniVidas C. difficile Toxin A/B assay (bioMerieux, Marcy I´Etoile, France). All samples were inoculated on cycloserine-cefoxitin-fructose-taurocholate agar (TCCFA) [25 (link)], and incubated at 37°C for 48 h in an anaerobic chamber (90% N2, 5%CO2, 5%H2). From C. difficile positive TCCFA, a single colony was subcultured on blood agar and then identified on the basis of colony morphology, fluorescence under UV light, L-proline aminopeptidase production and confirmed using the API Rapid ID 32A system (BioMérieux Inc., Durhan, NC, USA). Isolates not clearly confirmed as C. difficile were subjected to matrix- assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). C. difficile isolates were stored at -70°C in brain-heart infusion (BHI) broth (Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 20% glycerol, for further analysis.
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