H. pylori strain 7.13 was routinely cultured on GC agar (Oxoid), supplemented with 10% (v/v) horse serum (Invitrogen), 1x (v/v) vitamin mix, vancomycin (10 μg ml−1), and nystatin (10 μg ml−1) as described previously (Kwok, Backert, Schwarz, Berger, & Meyer,
Gc agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Germany, Australia
GC agar is a culture medium used for the growth and isolation of Neisseria species, particularly Neisseria gonorrhoeae. It is a complex agar medium that provides the necessary nutrients and growth factors for the cultivation of these fastidious microorganisms. The medium supports the growth of Neisseria species while inhibiting the growth of other bacteria that may be present in clinical samples.
Automatically generated - may contain errors
Lab products found in correlation
Horse serum, by Thermo Fisher Scientific (4 mentions)
Vancomycin, by Thermo Fisher Scientific (2 mentions)
Campygen system, by Thermo Fisher Scientific (2 mentions)
Vancomycin, by Merck Group (2 mentions)
Vitox, by Thermo Fisher Scientific (2 mentions)
Isovitalex, by BD (2 mentions)
Campygen sachet, by Thermo Fisher Scientific (1 mentions)
Protease peptone, by Thermo Fisher Scientific (1 mentions)
Donor horse serum, by Biowest (1 mentions)
2.5 l anaerobic jar, by Thermo Fisher Scientific (1 mentions)
Bhi medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Heracell 150i incubator, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Bhi medium, by BD (1 mentions)
Nitrocefin test, by Thermo Fisher Scientific (1 mentions)
Etest, by bioMérieux (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
23 protocols using gc agar
1
Culturing and Harvesting H. pylori
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H. pylori strain 7.13 was routinely cultured on GC agar (Oxoid), supplemented with 10% (v/v) horse serum (Invitrogen), 1x (v/v) vitamin mix, vancomycin (10 μg ml−1), and nystatin (10 μg ml−1) as described previously (Kwok, Backert, Schwarz, Berger, & Meyer,2002 ). H. pylori culture plates were incubated under microaerobic conditions in an anaerobic jar with CampyGen sachet (Oxoid) at 37°C. Bacteria were harvested from 48‐hr postinoculated plates with a sterile swab. The cells were gently resuspended in brain heart infusion (BHI) media and washed once with the same media prior to subcellular fractionation.
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H. pylori strain 7.13 was routinely cultured on GC agar (Oxoid), supplemented with 10% (v/v) horse serum (Invitrogen), 1x (v/v) vitamin mix, vancomycin (10 μg ml−1), and nystatin (10 μg ml−1) as described previously (Kwok, Backert, Schwarz, Berger, & Meyer,
2
Culturing and Stress Testing H. pylori
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After revival from frozen stocks, the H. pylori wt strains and the N6 derivatives were cultured for two days at 37 °C under microaerobic conditions produced by sachets (CampyGen) in 2.5 L anaerobic jars (Oxoid, Wesel, Germany) on the GC agar (Oxoid, Wesel, Germany) supplemented with 10% donor horse serum (Biowest, Nuaillé, France), protease peptone (Oxoid, Wesel, Germany), vitamin mix (1%), and antibiotics: vancomycin (10 µg/mL), trimethoprim (5 µg/mL), amphotericin (4 µg/mL) and colistin (10 µg/mL), as described [12 (link), 26 (link)]. To allow for selection, the media were supplemented additionally with chloramphenicol (8 µg/mL). The cells were suspended in the BHI medium (Oxoid, Germany), and the number of bacteria was evaluated by measurement of the optical density at 600 nm (OD600). To investigate the survival of H. pylori the number of colony-forming units (CFU) was determined by plating serial dilutions of bacterial suspensions as described [12 (link)]. The various temperatures (39 °C and 41 °C) and pH (pH5.2) stress conditions were generated as described [12 (link), 31 (link)]. All experiments were performed in triplicate.
3
Meningococcal Strain Characterization Study
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A total of 201 meningococcal strains isolated from disease cases were used in this study– 131 from VIC and 70 from WA (Fig 2 ). Isolates were passaged fewer than 5 times and were cultured under aerobic conditions with 5% CO2 at 37°C on GC agar (Oxoid) supplemented with 0.4% glucose, 0.01% glutamine, 0.2 mg/L of cocarboxylase and 5 mg/L of iron(III) nitrate. Serogroup-specific antisera were used to determine the serogroup of each isolate using slide agglutination.
4
Cultivation of IMD-related Meningococci
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Two hundred and seventy-eight IMD-related meningococci isolated in WA from 2000 to 2014 were collected from the PathWest reference laboratory. Isolates were passaged fewer than 5 times and were cultured under aerobic conditions with 5% CO2 at 37°C on GC agar (Oxoid) supplemented with 0.4% glucose, 0.01% glutamine, 0.2 mg/L of cocarboxylase and 5 mg/L of iron(III) nitrate.
5
Cultivation and Manipulation of H. pylori
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Construction of the various isogenic mutants of H. pylori strain P12 has been described in detail previously (Gorrell et al., 2013 (link)). H. pylori strain 7.13 and its isogenic ΔcagA mutant have also been described elsewhere (Franco et al., 2005 (link)). H. pylori strains were routinely cultured on GC agar (Oxoid) supplemented with 10% (v/v) horse serum (Invitrogen), vitamin mix, vancomycin, and nystatin as described previously (Kwok et al., 2002 (link)). For infection experiments, GC agar-cultured H. pylori was used to inoculate Heart Infusion (HI) broth (Oxoid) supplemented with 10% (v/v) FBS, 1% (v/v) vitamin mix and 10 μg/ml vancomycin (Sigma-Aldrich). Kanamycin sulfate (15 μg/ml) or chloramphenicol (4 μg/ml) was added as required for the culture of H. pylori mutant strains. All H. pylori cultures were grown at 37°C under microaerophilic conditions (CampyGen™ system; Oxoid); liquid cultures were incubated with gentle agitation at 120 rpm to an O.D550nm of 0.8–1.2 prior to use in infections.
6
Culturing and Characterizing H. pylori Strains
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H. pylori strains were routinely grown from glycerol stocks for 2 days on GC agar (Oxoid, Basingstoke, UK) plates supplemented with 10% (v/v) horse serum (Invitrogen Corp, Carlsbad, CA), vitamin mix and antibiotics (nystatin, 20 μg/ml; trimethoprim, 2.5 μg/ml; vancomycin, 10 μg/ml) in a microaerobic atmosphere as described previously11 (link). Plates for cultivation of mutant strains were further supplemented with chloramphenicol (4 μg/ml for routine culture, 10 μg/ml for selection of transformants). H. pylori strains used in this study were P12 wild-type (also designated modH5 ON; in-frame G10 tract; phenotype modH ON), P12∆modH::cat (also designated ∆modH5; replacement of modH DRD with cat cassette; phenotype modH OFF)11 (link), P12 modHOFF::cat (also designated modH5 OFF; in-frame G10-tract substituted to out-of-frame G6AG4, and region after premature stop codon replaced with a cat cassette; phenotype modH OFF)11 (link), and 7.13 wild-type showing heterogeneous modH G-tract lengths.
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H. pylori strains were routinely grown from glycerol stocks for 2 days on GC agar (Oxoid, Basingstoke, UK) plates supplemented with 10% (v/v) horse serum (Invitrogen Corp, Carlsbad, CA), vitamin mix and antibiotics (nystatin, 20 μg/ml; trimethoprim, 2.5 μg/ml; vancomycin, 10 μg/ml) in a microaerobic atmosphere as described previously11 (link). Plates for cultivation of mutant strains were further supplemented with chloramphenicol (4 μg/ml for routine culture, 10 μg/ml for selection of transformants). H. pylori strains used in this study were P12 wild-type (also designated modH5 ON; in-frame G10 tract; phenotype modH ON), P12∆modH::cat (also designated ∆modH5; replacement of modH DRD with cat cassette; phenotype modH OFF)11 (link), P12 modHOFF::cat (also designated modH5 OFF; in-frame G10-tract substituted to out-of-frame G6AG4, and region after premature stop codon replaced with a cat cassette; phenotype modH OFF)11 (link), and 7.13 wild-type showing heterogeneous modH G-tract lengths.
7
Preparation of GC Agar with Blood
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GC agar (Oxoid, UK) supplemented with Vitox (Oxoid, UK) and 5 % sheep blood were prepared following the manufacturer’s instructions. Briefly, 18 grams of GC agar base was weighed and dissolved in 500 ml of distilled water. The mixture was then sterilized in an autoclave for 15 min at 121 °C and allowed to cool to ~50 °C. Defibrinated sheep blood and Vitox supplement prepared following the manufacturer’s instructions were used to supplement the media.
8
Cultivation of Neisseria Strains
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N. gonorrhoeae 1291 (30 (link)), 20 clinical isolates from mucosal and disseminated gonococcal infections (31 (link)) and N. meningitidis MC58¢3 (32 ) strains were grown on GC agar (Oxoid) with 1% IsoVitaleX (Becton Dickinson) or Brain Heart Infusion (BHI, Oxoid) 1% agar with 10% Levinthal's Base medium at 37°C with 5% CO2, respectively, with either kanamycin (kan) (100 μg/ml) or tetracycline (5 μg/ml) as required.
9
Cultivation of E. coli and H. pylori
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E. coli strains were grown in Luria Bertani (LB) medium supplemented with 100 µg/ml ampicillin, 20 µg/ml chloramphenicol, 20 µg/ml kanamycin, and/or 10 µg/ml gentamicin if applicable. H. pylori strains were grown on GC-agar (Oxoid) plates supplemented with 10% horse serum (DHS, Biochrom AG), 1% vitamin mix, 10 µg/ml vancomycin, 5 µg/ml trimethoprim, and 1 µg/ml nystatin. For transformant selection and growth of mutant strains, 20 µg/ml kanamycin, 20 µg/ml chloramphenicol, 10 µg/ml gentamicin or 10 µg/ml erythromycin were added. For liquid cultures, 15 or 50 ml Brain Heart Infusion medium (BHI, Roth) supplemented with 10% FBS (Biochrom AG) and 10 µg/ml vancomycin, 5 µg/ml trimethoprim, and 1 µg/ml nystatin were inoculated with H. pylori strains from plates to a final OD600 of 0.02–0.04 and grown under agitation at 140 rpm in 25 cm3 or 75 cm3 cell culture flasks. Bacteria were grown at 37 °C in a HERAcell 150i incubator (Thermo Scientific) in a microaerobic environment (10% CO2, 5% O2, and 85% N2).
10
Isolation and Culture of Neisseria gonorrhoeae
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Neisseria gonorrhoeae strain ExNg63 was isolated from cerebrospinal fluid of patient with meningitis in Australia in 2015. Isolates were stored in GC broth with 20% glycerol at −80 °C, were cultured under aerobic conditions with 5% CO2 at 37 °C on GC agar (Oxoid, Australia), and supplemented with 0.4% glucose, 0.01% glutamine, 0.2 mg/l of cocarboxylase, and 5 mg/l of iron (III) nitrate.
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Neisseria gonorrhoeae strain ExNg63 was isolated from cerebrospinal fluid of patient with meningitis in Australia in 2015. Isolates were stored in GC broth with 20% glycerol at −80 °C, were cultured under aerobic conditions with 5% CO2 at 37 °C on GC agar (Oxoid, Australia), and supplemented with 0.4% glucose, 0.01% glutamine, 0.2 mg/l of cocarboxylase, and 5 mg/l of iron (III) nitrate.
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