Helicobacter pylori strains 26695 (ATCC® 700392, cagA+, vacA s1/m1), 60190 (ATCC® 49503, cagA+, vacA s1/m1) and Tx30a (ATCC® 51932; cagA–, vacA s2/m2) were routinely cultured in TrypticaseTM Soy Agar (TSA) supplemented with 5% Sheep Blood (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) and incubated in a sealed jar with a microaerophilic atmosphere (GENBox microaer; bioMérieux S.A., Marcy l’Etoile, France) at 37°C for 48 h. Bacteria were sub-cultured for a maximum of 12 passages.
Genbox microaer
Manufactured by bioMérieux
Sourced in France
The GENbox microaer is a laboratory equipment designed for the cultivation and incubation of microorganisms. It provides a controlled environment with reduced oxygen levels, suitable for the growth of anaerobic and microaerophilic bacteria. The GENbox microaer maintains the desired atmospheric conditions during the incubation process, facilitating the cultivation and analysis of these types of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Sheep blood, by BD (3 mentions)
Genbox anaer, by bioMérieux (2 mentions)
Trypticasetm soy agar with 5 sheep blood tsaii, by BD (2 mentions)
Genbox jar, by bioMérieux (2 mentions)
Hicrome uti agar, by HiMedia (1 mentions)
Cm0739, by Thermo Fisher Scientific (1 mentions)
Amicon, by Merck Group (1 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Columbia blood agar cba, by Thermo Fisher Scientific (1 mentions)
Dmem with glutamax, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin sulfate, by Thermo Fisher Scientific (1 mentions)
Columbia agar plate, by bioMérieux (1 mentions)
Columbia blood agar plates, by bioMérieux (1 mentions)
Api campy, by bioMérieux (1 mentions)
Campylobacter blood, by Thermo Fisher Scientific (1 mentions)
Trypticase soy agar, by BD (1 mentions)
Kanamycin, by Thermo Fisher Scientific (1 mentions)
Campyfood agar, by bioMérieux (1 mentions)
Columbia agar, by bioMérieux (1 mentions)
Lysed horse blood, by Thermo Fisher Scientific (1 mentions)
20 protocols using genbox microaer
1
Culturing Diverse H. pylori Strains
2
Campylobacter Detection Using mCCDA
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Charcoal cefoperazone deoxycholate modified agar base media (mCCDA) was prepared according to the manufacturer’s instructions (HiMedia, Mumbai, India). Briefly, 22.4 g of powder was suspended in sterile distilled water and brought to boiling temperature to dissolve. The mixture was then sterilized in the autoclave and kept to cool down to 50°C. One vial of the Campylobacter CCDA selective supplement containing two antibiotics, cefoperazone and amphoterricin B, was added and carefully mixed in sterile Petri dishes. Using the direct plating technique, each sample was swabbed on a dried Petri dish containing the mCCDA media using the 4 quadrants method. The dishes were then incubated at 37°C for 48 h in microaerophilic conditions using atmospheric generators (GENbox Microaer, bioMérieux, France).
3
Extended Culturomics Protocol for Urine Samples
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The extended culturomics protocol included inoculation of 0.1 mL of urine onto a large plate surface (140-mm diameter) of Columbia agar with 5% sheep blood (blood agar plates [BAPs], Biogerm, Portugal) and HiCrome UTI agar (chromogenic agar plates [CAPs], HiMedia, India) supplemented as previously described (45 (link), 46 (link)). BAPs and CAPs were incubated under aerobic and microaerophilic conditions (GENbox Microaer, bioMérieux) at 37°C for 48 h. Additionally, BAPs were incubated under anaerobic conditions (GENbox Anaer, bioMérieux) at 37°C for 48 h. In the case of a suspected high bacterial load based on microscopic observation, 10-fold serial dilutions (up to 0.001) were performed using sterile saline solution (0.9% NaCl) to obtain a countable range of CFU/mL. Each morphologically distinct colony type was counted, and 1 to 5 colonies of each morphology were further identified. The plate presenting the higher CFU count was considered the representative count of each isolate in a sample. When more than one species was identified within the same colony morphotype, the CFU count was split proportionally between the identified species. Relative abundance (RA [%]) was calculated by generating the percentage of total CFU/sample.
4
Cultivation and Quantification of C. jejuni
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C. jejuni reference strain NCTC 12744 was cultivated at 41.5°C for 48 h under microaerophilic conditions (Genbox microaer, BioMerieux, Vienna, Austria) on Campylobacter Selective Agar (CASA, BioMerieux), which was also used to determine bacterial load in different organs. Modified charcoal–cefaprazone–deoxycholate agar (CM0739, OXOID, Hampshire, UK) was used to determine fecal shedding based on cloacal swabs.
5
Characterizing C. albicans Phenotypes
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The strains used are described in Table 2 . Cells were grown at 37°C in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) unless otherwise stated. Two independent WOR1OE clones (c1 and c2) were generated which gave similar expression levels and in vitro phenotypes (not shown) and only one (c1) was used for in vivo studies. The susceptibility/resistance to different compounds was performed through drop test as follows. Cultures grown at 37°C from either stationary or exponential phase (O.D. = 1) were adjusted to 2 × 107 cells/mL, serially 10-fold diluted and deposited (5 μL) onto solid YPD plates supplemented (or not) with the indicated compounds. Plates were incubated at 37°C for 24 and 48 h before scanned. Microaerophilia was achieved using an anaerobic chamber and a commercial system that ensures the adequate percentages of O2 and CO2 (GENBox Microaer, BioMérieux, ≈15% CO2 and ≈6% O2). For the observation of white-opaque switching, C. albicans strains were grown on YPD plates supplemented with phloxine B (10 μg/mL) at 37 and 21°C. When necessary, doxycycline was added to either liquid or solid media at 10 or 20 μg/mL respectively.
6
Culturing H. pylori and H. acinonychis
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H. pylori 26695, obtained from the American Type Culture Collection (ATCC 700392, VA, USA) and H. acinonychis strain 90-119, obtained from the Health Protection Agency Culture Collections (HPA Culture Collections 12686, Salisbury, UK), were subcultured every 48 h in trypticase soy agar (TSA) supplemented with 5 % (v/v) sheep blood (Becton Dickinson GmbH, Germany) and incubated at 37 °C under microaerobic conditions using a GENbox microaer (bioMérieux, Marcy l′Étoile, France). Cell concentration was obtained by optical density (O. D.), and each initial culture was diluted in saline buffer in order to obtain a final concentration of 10 6 total cells/mL.
7
Helicobacter pylori Strain Culture and Conditioned Media Production
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H. pylori strain 26695 (ATCC 700392, cagPAI+, vacA s1/m1) was obtained from ATCC (LGC Standards, United Kingdom), and H. pylori strain 60190 (ATCC 49503 cagPAI+, vacA s1/m1) and its respective 60190CagA-, 60190CagE-, and 60190VacA- mutants were a kind gift from Professor John Atherton (University of Nottingham). Strains were cultured in TrypticaseTM Soy Agar with 5% Sheep Blood (TSAII; Becton, Dickinson and Company) at 37°C under microaerophilic atmosphere (GENbox microaer; bioMérieux S.A.) for 48 h, as previously described (Costa et al., 2016 (link)). For the production of conditioned media, bacteria were grown in F12 medium (Gibco) supplemented with 1x cholesterol (Gibco) under microaerophilic conditions at 37°C with constant rotation (150 rpm) over 24 h. Subsequently, bacterial suspensions were centrifuged at 15,000 × g, for 15 min, and the supernatants were filtered through a 0.2-μm sterile filter, and concentrated by ultrafiltration using a 10 kDa pore size Amicon (EMD Millipore). The resulting concentrated bacterial conditioned media was used as media for gastric cell cultures in comparison with F12 medium plus 1x cholesterol alone without bacteria, as control.
8
Campylobacter coli Suspension Quantification
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The test strain Campylobacter coli ATCC® 43478™ (LGC Standards GmbH) was recovered from storage and purifi ed in selective media according to a previously described preparation protocol [15] . For the generation of a pure C. coli suspension, fi ve overnight and well-isolated colonies on Columbia blood agar (CBA; Oxoid) were inoculated in 200 mL of Cation-Adjusted Mueller Hinton Broth (CAMHB; BBL™ BD) and cultivated at 37°C for 22 ± 2 hours under microaerophilic conditions (GENbox microaer and GENbox jar; BioMérieux). Then, two aliquots of 100 μL each (quantifi cation controls) from the pure bacterial suspension (Figure 1 -point A) were subjected to DNA extraction followed by real-time PCR (qPCR) (Figure 1 points B & C) in order to determine the corresponding average count (cell equivalents mL -1 ; one genome copy = one cell equivalent) of the initial C. coli population (100 μL) from the same pure bacterial suspension that was inoculated on each sampling material and lamb meat sample (Figure 1 -point D).
9
Soft-Agar Overlay Method for Enterococcus faecalis Phage Propagation
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For E. faecalis, the soft-agar overlay methods was used for phage propagation and plaque phenotype determination. BHI-fc soft agar (4% agar, 37 g/L BHI, 4 g/L glycine, 50 mM Tris) supplemented with 3.2 mM L-cysteine HCl was used as top agar layer and BHI-fc agar (1% agar, 37 g/L BHI, 4 g/L glycine, 50 mM Tris, 5 ng/ml choline chloride) served as bottom agar. A total of 200 μl log-phase host bacteria were mixed with 10 μl phage dilution and poured on top of the bottom agar layer. EfS3 and EfS7 were propagated under microaerophilic conditions (Genbox Microaer, biomerieux) at 30 °C. Progeny virions were extracted using 5 ml SM buffer per plate (100 mM NaCl, 8 mM MgSO4, and 50 mM Tris, pH 7.4) and filter-sterilized (0.2 μm) to obtain crude lysates.
10
Optimizing Probe Hybridization for Helicobacter Detection
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All bacterial cultures were grown in trypticase soy agar (TSA) supplemented with 5% (v/v) sheep blood (Becton Dickinson GmbH, Germany) and incubated for 48 hours at 37°C under microaerobic conditions using a GENbox microaer (bioMérieux, Marcy l’Étoile, France). Bacteria were collected from TSA plates using water or saline (microscopy or cytometry analysis, respectively). The bacterial density was determined by the dilution of initial culture in water or saline and the absorbance was measured at 600 nm. H. pylori 26695 was used for the optimization of probe hybridization conditions, whereas other bacteria, either Helicobacter spp. or the non-Helicobacter spp., were used for the analysis of probe specificity and sensitivity. All bacteria used in this study are listed in Table 2 .
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