For selective pre-enrichment, 25 g of each sample (chicken meat, liver, or gizzard), were added to 225 mL of a Brucella broth supplemented with 5% sheep defibrinated blood, and DENT selective supplement (Oxoid, Hampshire, UK), and the mixture was homogenized in Stomacher® 400 (Seward, Worthing, UK). The mixture was then divided into two 250 mL flasks and incubated at 37 °C for 48 h under a microaerophilic condition using BBL GasPak™ jars (Becton Dickinson, Franklin Lakes, NJ, USA), supplemented with CampyGen bags (Oxoid, Hampshire, UK). After incubation, 100 µL of the mixture were inoculated onto Columbia blood agar base (Oxoid, Hampshire, UK), supplemented with 5% sheep blood and DENT selective supplements. The plates were incubated for up to 7 days at 37 °C under a microaerophilic condition, as previously described. Suspected colonies were further identified using Gram’s staining (Gram-negative for Helicobacter spp.), oxidase (positive for Helicobacter spp.), urease (positive for H. pylori), and nitrate reduction (positive for H. pullorum) tests.
Dent selective supplement
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Dent selective supplement is a laboratory product designed to support the growth and isolation of Dent bacteria in culture media. It provides the necessary nutrients and selective agents to enhance the recovery of Dent organisms from various samples.
Automatically generated - may contain errors
Lab products found in correlation
Columbia blood agar, by Thermo Fisher Scientific (5 mentions)
Columbia blood agar base, by Thermo Fisher Scientific (5 mentions)
Bbl gaspak jars, by BD (4 mentions)
Campygen bags, by Thermo Fisher Scientific (4 mentions)
Sheep blood, by Thermo Fisher Scientific (3 mentions)
Horse serum, by Thermo Fisher Scientific (3 mentions)
Trypticase soy broth (tsb), by Thermo Fisher Scientific (3 mentions)
Campygen atmosphere generating system, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Kanamycine, by Merck Group (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Stomacher 400, by Seward Medical (2 mentions)
Bhi medium, by BD (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Yeast extract, by BD (1 mentions)
Brain heart infusion medium, by BD (1 mentions)
Atcc 43504, by American Type Culture Collection (1 mentions)
Brain heart infusion medium, by Thermo Fisher Scientific (1 mentions)
Horse blood, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
14 protocols using dent selective supplement
1
Selective Enrichment and Identification of Helicobacter
2
Isolation and Identification of H. pylori
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For the isolation of H. pylori, 25 g of each sample was suspended in 225 mL of Brucella broth supplemented with 5% sheep defibrinated blood and DENT selective supplement (Oxoid, Hampshire, UK). After incubation at 37 °C for 48 h under a microaerophilic condition using BBL GasPak™ jars (Becton Dickinson, Franklin Lakes, NJ, USA), supplemented with CampyGen bags (Oxoid, Hampshire, UK) 100 µL of the mixture was inoculated onto Columbia blood agar base (Oxoid, Hampshire, UK), supplemented with 5% sheep blood and DENT selective supplements, and then incubated for 5–7 days under a microaerophilic condition. Suspected colonies were further identified using Gram staining (Gram-negative for Helicobacter spp.), oxidase (positive for Helicobacter spp.), urease (positive for H. pylori), and nitrate reduction (positive for H. pullorum) tests.
3
H. pylori ATCC 43504 Cultivation Conditions
4
Construction and Characterization of H. pylori Mutants
5
Isolation and Characterization of H. pylori Strains
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Twelve H. pylori clinical isolates and one reference strain of H. pylori (NCTC 11916). The clinical strains were isolated from gastric biopsy samples obtained by a gastroenterologist of the Jordan University Hospital during a routine endoscopy. The gastric biopsy material was processed according to the standard methodology [37 ]. Briefly, each biopsy for culture was homogenized using a tissue homogenizer (IKA, Staufen, Germany). Aliquots of 100 μL of the homogenate were primarily cultured on Columbia blood agar (Oxoid, Hampshire, UK) supplemented with 7% (v/v) horse blood and Dent selective supplement (Oxoid, Hampshire, UK). Subcultures of the bacteria were performed using the same plates but without the Dent supplement. All of the plates were incubated at 37 °C under microaerophilic conditions using CampyGen atmosphere generating system (Oxoid, Hampshire, UK) in anaerobic jars for 5–7 days. Growth of H. pylori was confirmed according to colony morphology, Gram staining, microaerophilic growth (at 37 °C), biochemical tests (positive for oxidase, catalase and urease), and subsequently by standard PCR of 16S ribosomal DNA test [38 (link)]. H. pylori cultures were stored at −70 °C in Trypticase soy broth (Oxoid, Hampshire, UK) containing 10% (v/v) fetal calf serum (PAA, Pasching, Austria) and 15% glycerol.
6
Isolation and Characterization of H. pylori Strains
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Twelve H. pylori clinical isolates and one reference strain of H. pylori (NCTC 11916) were used in this study. The clinical strains were isolated from gastric biopsy samples obtained by a gastroenterologist at the Jordan University Hospital during a routine endoscopy. Gastric biopsies were processed according to standard methodology [8 ]. Briefly, each biopsy was homogenized using a tissue homogenizer (IKA, Staufen, Germany). Aliquots of 100 μL of the homogenate were primarily cultured on Columbia blood agar (Oxoid, Hampshire, UK) supplemented with 7% (v/v) horse blood and Dent selective supplement (Oxoid, Hampshire, UK). The same bacterial plates were used for subcultures but without the Dent supplement. All plates were incubated at 37 °C under microaerophilic conditions using the CampyGen atmosphere generating system (Oxoid, Hampshire, UK) in anaerobic jars for 5-7 days. The growth of H. pylori was confirmed according to biochemical tests (positive for oxidase, catalase, and urease). H. pylori cultures were stored at −70°C in trypticase soy broth (Oxoid, Hampshire, UK) containing 10% (v/v) fetal calf serum (PAA, Pasching, Austria) and 15% glycerol [1 ,8 ]. All experiments were performed in triplicates to ensure consistency of the results.
7
H. pylori Strain G27 Growth
8
Cultivation and Genetic Manipulation of Helicobacter pylori
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The H. pylori strains used in this study (Supplementary Table S1 ) were 26 695 (11 (link)) B128 (12 ,13 ) and X47-2AL (14 (link)). Strains were grown on Columbia agar plates supplemented with 7% horse blood and Dent selective supplement (Oxoid, Basingstoke, UK) for 24–48 h depending on the strain. Liquid cultures were performed in brain-heart infusion medium (Oxoid) supplemented with 10% fetal bovine serum and Dent. H. pylori plates and liquid cultures were incubated at 37°C under microaerobic conditions (10% CO2, 6% O2, 84% N2) using an Anoxomat (MART microbiology) atmosphere generator. For liquid cultures, bacteria harvested from plates were inoculated at an optical density at 600 nm of 0.05 (OD600 = 0.05) into 5 ml (tubes, shaking at 175 rpm) brain-heart infusion medium supplemented with 10% fetal bovine serum and Dent supplement. After 12–24 h, pre-cultures were diluted to an OD600 of 0.05 into 25 ml (flasks, shaking at 125 rpm). Plasmids used for cloning were amplified in Escherichia coli strain JM109, which was grown in Luria–Bertani medium, supplemented either with kanamycin (50 μg.ml−1) or chloramphenicol (30 μg.ml−1). For H. pylori mutant selection and culture, antibiotics were used at the following final concentrations 20 μg.ml−1 kanamycine (Sigma) and 8 μg.ml−1 chloramphenicol (Sigma).
9
Isolation and Characterization of H. pylori
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Twelve H. pylori clinical isolates and one reference strain of H. pylori (NCTC 11916) were used in this study. The clinical strains were isolated from gastric biopsy samples obtained by a gastroenterologist of the Jordan University Hospital during a routine endoscopy. The gastric biopsy material was processed according to the standard methodology. Briefly, each biopsy for culture was homogenized using a tissue homogenizer (IKA, Staufen, Germany). Aliquots of 100 µL of the homogenate were primarily cultured on Columbia blood agar (Oxoid, Hampshire, UK) supplemented with 7% (v/v) horse blood and Dent selective supplement (Oxoid, Hampshire, UK). Subcultures of the bacteria were performed using the same plates but without the Dent supplement. All of the plates were incubated at 37°C under microaerophilic conditions using CampyGen atmosphere generating system (Oxoid, Hampshire, UK) in anaerobic jars for 5–7 days. Growth of H. pylori was confirmed according to colony morphology, Gram staining, microaerophilic growth (at 37 °C), biochemical tests (positive for oxidase, catalase, and urease), and subsequently by standard PCR of 16S ribosomal DNA test [21 (link)]. H. pylori cultures were stored at −70 °C in Trypticase soy broth (Oxoid, Hampshire, UK) containing 10% (v/v) fetal calf serum (PAA, Pasching, Austria) and 15% glycerol.
10
Helicobacter Spp. Protocol for Wastewater
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One liter of each wastewater sample was centrifuged at 3,220 g for 20 min. The supernatant was removed and centrifuged again at 8,000 g for 8 min at 4ºC to ensure the elimination of all residual water. The resulted pellet was suspended in 10 mL of phosphate-buffered saline (PBS 1X: 130 mM sodium chloride, 10 mM sodium phosphate, pH 7.2). From this, 200 L were taken for Helicobacter spp. culture, 1 mL for qPCR analysis, and another 1 mL for DVC-FISH analysis ("D" samples). An additional aliquot of 5 mL from each sample was incubated in 10 mL of Brucella broth enrichment media (BBL TM (Becton Dickinson, USA), 10% (v/v) fetal bovine serum (Fisher, USA) and Dent selective supplement (Oxoid, UK)) under microaerophilic conditions at 37ºC for 24 hours. After the incubation period, the presence of Helicobacter spp. was also analyzed by culture, qPCR and FISH ("E" samples).
For Illumina-based NGS analysis, 1 mL of the 8 samples from tertiary effluent (after disinfection), both directly and after the enrichment step, were processed as described below.
For Illumina-based NGS analysis, 1 mL of the 8 samples from tertiary effluent (after disinfection), both directly and after the enrichment step, were processed as described below.
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