A stool sample was obtained from a 2-year-old Nigerian boy with kwashiorkor, a severe form of acute malnutrition, who was admitted to the emergency room in the national hospital in Niamey, the capital city of Niger, in October 2013 [2] (link). The study was approved by the local ethics committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France, under agreement 09-022. The fecal specimen was preserved at 4°C after collection. Then the stool was sent to Marseille, where it was kept at −80°C until laboratory culture isolation. Strain SIT1T was isolated in March 2014 by cultivation on 5% sheep's blood–enriched Columbia agar (bioMérieux, Marcy l’Étoile, France) in an anaerobic atmosphere at 37°C after 10 days of stool specimen incubation in a culture bottle containing a blood-enriched Columbia agar liquid medium (bioMérieux). Growth of the strain was tested under anaerobic conditions using GENbag anaer system (bioMérieux), and under aerobic conditions, with or without 5% CO2. Different growth temperatures (25, 30, 37, 45, 55°C) were also tested.
Columbia agar
Manufactured by bioMérieux
Sourced in France, Poland, United Kingdom, Germany, Spain
Columbia agar is a general-purpose microbiological growth medium used for the cultivation of a wide range of aerobic and facultative anaerobic bacteria. It provides the necessary nutrients and growth factors to support the growth of a diverse range of bacterial species.
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Lab products found in correlation
Sheep blood, by bioMérieux (27 mentions)
Chocolate agar, by bioMérieux (9 mentions)
Macconkey agar, by bioMérieux (8 mentions)
Genbag anaer, by bioMérieux (7 mentions)
Sheep s blood, by bioMérieux (6 mentions)
Mannitol salt agar, by bioMérieux (5 mentions)
Schaedler agar, by bioMérieux (5 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (5 mentions)
Sabouraud agar, by bioMérieux (4 mentions)
Genbag anaer system, by bioMérieux (4 mentions)
Microflex spectrometer, by Bruker (4 mentions)
Campygen, by Thermo Fisher Scientific (4 mentions)
Genbag microaer system, by bioMérieux (4 mentions)
Horse blood, by bioMérieux (3 mentions)
Polyvitex, by bioMérieux (3 mentions)
Vitek ms, by bioMérieux (3 mentions)
Clarus 500 sq 8 s, by PerkinElmer (3 mentions)
Api candida, by bioMérieux (3 mentions)
Vitek 2 automated system, by bioMérieux (2 mentions)
Sabouraud gentamicin chloramphenicol 2 agar, by bioMérieux (2 mentions)
138 protocols using columbia agar
1
Isolation of Severe Malnutrition Bacterium
2
Comprehensive Microbial Cultivation Protocol
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Standard microbiological cultures were performed on all specimen included into the study by plating on Columbia agar supplemented with 5% sheep blood (bioMérieux) under aerobic conditions, on Chocolate agar (bioMérieux) under atmosphere enriched with 9% C02 for 48h and, under anaerobic conditions on Columbia agar supplemented with 5% sheep blood (bioMérieux) for 48 h at 37°C. Reference identifications from culture plates were performed using the same MALDI Biotyper system as below, using standard procedures.
3
Comprehensive Characterization of Strain Marseille-P2645
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Different growth conditions were tested on a 5% sheep's blood–enriched Columbia agar (bioMérieux) for strain Marseille-P2645T. Five temperatures (room temperature, 28, 37, 45 and 55°C) and three atmospheres—anaerobic (anaeroGen Compact; Oxoid), microaerophilic (campyGen Compact; Oxoid) and aerobic (in a plastic pouch to maintain a humid atmosphere)—were evaluated. Tolerance of this strain to salt was tested using 5%, 7.5%, 10%, 15% and 20% of NaCl, and the pH tolerance (5, 5.5, 6, 6.5, 7, 7.5 and 8) was also tested. Individual cells of strain Marseille-P2645T were visualized using a Tecnai G20 electron microscope (FEI Company, Limeil-Brevannes, France). Gram staining was performed and observed using a photonic microscope Leica DM2500 (Leica, Wetzlar, Germany) with a 100× oil-immersion objective. Motility testing was performed by observation of a fresh colony between the blades and slats using a DM1000 photonic microscope (Leica) at 40×. To check the ability to sporulate, strain Marseille-P2645T was grown on 5% sheep's blood–enriched Columbia agar (bioMérieux) for 2 days, and then a heat-shock test (20 minutes at 80°C) was performed.
4
Enterobacteriaceae Detection in Broiler Faeces
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The presence of Enterobacteriaceae and their count in broiler faeces samples were demonstrated by the ISO 21528-2:2017 method [46 ]. Pooled faeces samples were tested weekly. Faeces decimal dilutions (from 10−1 to 10−5) previously prepared according to the ISO 6887-6:2013 and ISO 6887-1:2017 methods [47 ,48 ] from the three experimental groups and the control group were inoculated onto blood agar (Columbia agar + 10% sheep blood), Columbia agar (bioMérieux, Craponne, France), Tryptone Bile X-glucuronic chromogenic agar (TBX agar, bioMérieux, France), and Plate Count Agar (PCA, bioMérieux, Craponne, France). The identification of E. coli was performed on a Bruker Microflex LT MALDI TOF mass spectrometer (Bruker Daltonics, Bremen, Germany), whereas the presence of the Salmonella genus was demonstrated by use of the ISO 6579-1:2017/A1:2020 method [49 ].
5
Comparative Evaluation of S. aureus Strains
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Three reference strains were used in this study: S. aureus ATCC 29213 (methicillin-sensitive S. aureus – MSSA), S. aureus ATCC 43300 (MRSA) and S. aureus ATCC 6538 (MSSA) as a reference biofilm forming strain [14 (link)]. The bacteria were cultivated for 18 h at 37°C on Columbia agar with 5% sheep blood (bioMérieux, Warsaw, Poland).
6
Duplex real-time PCR for pneumococcal detection
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A duplex real-time PCR targeting the lytA gene and RNase P (single-copy gene of the human genome used as an amplification control) was used for pneumococcal DNA detection/quantification in invasive samples and NPAs [31 ]. Primers and probes were those recommended by the Centers for Disease Control and Prevention (CDC). Blood agar plates (Columbia agar supplemented with 5% sheep blood; bioMérieux) were utilized for the isolation of pneumococcal strains directly from invasive samples or from positive blood cultures. Standard microbiological tests were performed for S. pneumoniae S. pneumoniae https://doi.org/10.6084/m9.figshare.13280435.v3 ).
7
Enterococcal UTI Strain Identification
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The collection of 58 enterococcal clinical strains isolated from urinary tract infections (UTIs) was initially grown on Columbia agar (BioMérieux, Madrid, Spain) added with 5% lamb blood and grown at 37 °C for 24 h. Phenotypic identification continued with Gram staining, catalase activity, and hydrolysis of esculin during growth on Bile Esculin Agar (Biomedics, Madrid, Spain). Several vancomycin-resistant strains and reference strains from the lab collection were included: E. gallinarum C86 (vanC1+) [44 (link)], E. faecium C135 (vanB+) [44 (link)], E. faecium AR1 (vanA+) [44 (link)], E. casseliflavus C85 (vanC2+) [45 (link)], E. durans AR23 (vanA+) [45 (link)], E. faecalis G8 (vanB+), E. faecium G74 (vanA+), E. faecium LMG16003 (unknown van+ resistance gene), E. faecium LMG11423 (van−), E. faecalis LMG16216 (vanB+), E. faecalis LMG8222 (van−), and E. faecalis FI9190 (multiple virulence factors) [46 (link)]. MIC tests were performed in Mueller-Hinton Broth (Scharlau, Barcelona, Spain), according to internationally accepted protocols [47 (link)].
8
Isolation and Characterization of Anti-Staphylococcus capitis Phages
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The Staphylococcus capitis strains used in this study were obtained from the collection of the French National Reference Centre for Staphylococci (Lyon, France). These strains were conserved in cryotubes containing glycerol at -20°C. They were grown on Columbia agar + 5% sheep blood (BioMérieux, Marcy l’Etoile, France) for 24 hours at 37°C. A collection of 31 strains was included in this study to isolate, produce and assess the host range of anti-S. capitis bacteriophages. These strains were selected to reflect the phylogenetic diversity of the S. capitis NRCS-A clone composed of three subgroups successively appeared: Proto-outbreak I, Proto-outbreak II and the most recent and specialized in NICU infections Outbreak (Table 1
9
Anthrax Strain Recultivation Protocol
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Isolates were recultured from the Public Health England anthrax strain collection glycerol stocks. A 10 μl loop of glycerol suspension was streaked onto Columbia agar plus 5 % horse blood (bioMérieux) and incubated aerobically for 16–24 h at 37 °C.
10
Isolation and Characterization of Pseudomonas aeruginosa
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Pure bacterial cultures were isolated on solid media plates—Columbia Agar (bioMerieux, France) was prepared according to the following recipe: 400 mL agar solution with manufacturer’s recommended concentration was sterilized by autoclaving, and 20 mL of sheep blood (Biomed Lublin, Lublin, Poland) was added. After 24 h of culture, the bacteria were again sieved to the same medium, and after 24 h, pure culture suspensions were prepared in Dulbecco’s PBS with Ca2+ and Mg2+ (PAA, Pasching, Austria). A density of 1 McFarland (McF, about 3 × 108 CFU/mL) was measured by using a Densimat photometer (BioMerieux, Marcy l’Etoile, France) to determine the concentration of the solution by measuring the intensity of light scattering.
Based on the type of growth of Pseudomonas aeruginosa colonies on solid media, the mucoid and non-mucoid types were differentiated. Mucus type growth was observed in strains 2, 3, 5, 7, 9, 11 and 14.
Based on the type of growth of Pseudomonas aeruginosa colonies on solid media, the mucoid and non-mucoid types were differentiated. Mucus type growth was observed in strains 2, 3, 5, 7, 9, 11 and 14.
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