Wild-type Bordetella bronchiseptica strain RB50 (B. bronchiseptica ) and a derivative btrS mutant (RB50ΔbtrS) (3 (link)), here called Bbvac (52 ), B. pertussis , and B. parapertussis were grown on plates of Difco Bordet-Gengou agar (BD; catalog no. 248200) supplemented with 20% defibrinated sheep blood from HemoStat and 20 μg/mL of streptomycin as previously described (1 (link)). For liquid cultures, B. pertussis and B. parapertussis were grown in Stainer-Scholte medium (1 (link)). B. bronchiseptica and Bbvac were grown in LB media (1 (link)). Details of the clinical strains included in this study are found in Table S2 in the supplemental material.
Bordet gengou agar
Manufactured by BD
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Bordet-Gengou agar is a microbiological culture medium used for the isolation and identification of Bordetella species, particularly Bordetella pertussis, the causative agent of whooping cough. The medium is designed to support the growth of these fastidious bacteria while inhibiting the growth of other microorganisms.
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Lab products found in correlation
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Sheep blood agar, by BD (2 mentions)
Casamino acid, by Thermo Fisher Scientific (2 mentions)
Dna mini kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Defibrinated horse blood, by bioMérieux (2 mentions)
Lysis buffer, by Roche (2 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Sheep blood, by BD (1 mentions)
Tsb st, by bioMérieux (1 mentions)
Maldi biotyper identification software 3, by Bruker (1 mentions)
Microflex lt, by Bruker (1 mentions)
Polypeptone, by Fujifilm (1 mentions)
Hipolypeptone, by Fujifilm (1 mentions)
Defibrinated horse blood, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Charcoal agar, by Thermo Fisher Scientific (1 mentions)
Api 20ne system, by bioMérieux (1 mentions)
19 protocols using bordet gengou agar
1
Bacterial Culture Protocols for Bordetella spp.
2
Bordetella bronchiseptica Culture Protocol
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Bordetella bronchiseptica RB50 wild-type (Bb) and RB50 mutant strain, herein BbDbtrS [55 (link)], were grown on plates of Difco Bordet-Gengou agar (BD, cat. 248200) supplemented with 10% sheep defibrinated blood and 20 μg/mL concentration of streptomycin, as previously described [55 (link),65 (link)].
3
Bordetella species isolation and DNA extraction
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B. pertussis (ATCC 9797) was obtained from American Type Culture Collection (ATCC, Rockville, MD), and B. holmesii and B. parapertussis were clinical isolates obtained from the Wisconsin Department of Health Services, which were de-identified. B. pertussis was grown on Bordet-Gengou agar (BD, Sparks, MD, USA) supplemented with 10% casamino acid (Fisher Scientific, USA). B. holmesii and B. parapertussis were grown in 5% sheep blood agar (BD, Sparks, MD, USA). The microorganisms were incubated in an aerobic environment with sufficient humidity at 35 °C for 3–4 days. Genomic DNA from B. pertussis, B. holmesii and B. parapertussis was extracted and purified by using the Qiagen DNA Mini kit (Catalog No. 51304, Valencia, CA, USA) following the protocol from the manufacturer. For each organism, bacterial colonies were transferred into a centrifuge tube containing 5 mL of sterile saline adjusting the turbidity to 0.5 McFarland standard (ProLab Diagnostics, Round Rock, TX, USA) to achieve a cell density of ~ 1.5 × 108. Cells were harvested by centrifugation in a Beckman centrifuge, using a C0650 rotor with adapters for 15 mL tubes, at 7500 rpm (5000 × g) for 10 min. Bacterial cell pellets were collected to proceed with the solid-phase extraction of DNA as indicated by the Qiagen protocol. Eluted DNA samples were measured by using Nanodrop (Nanodrop 1000, Thermo Scientific, MA, USA).
4
Bordetella pertussis Strain Isolation and Characterization
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A total of 48 clinical isolates of Bordetella pertussis (Table 1 ) were obtained from 1989 through 1993, and 2005 at the Cincinnati Children’s Hospital Medical Center (Cincinnati, OH) [8 (link),17 (link),18 (link)]. B. pertussis strain Tohama I, which is a widely used vaccine strain, was used as a reference strain [19 (link)]. Strain CVI, an isogenic mutant described previously [8 (link)], and a Japanese clinical isolate KF-1, producing Ptx with PtxA1 and PtxB1 subunit and that with PtxA1 and PtxB2 respectively, were used in this study. Other reference strains are listed in Table 2 [20 (link)–24 (link)]. The amino acid sequences for PtxC-E were identical for these isolates. For routine propagation, B. pertussis was grown on Bordet-Gengou agar (BD Biosciences, Sparks, MD) supplemented with 1% glycerol and 15% defibrinated sheep blood (BG agar) at 36°C for 4 days. The isolates were stored at -80°C.
5
Bordetella bronchiseptica Cultivation and Induction
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Bordetella bronchiseptica strains were streaked from −80°C stocks onto Bordet-Gengou agar (BD Biosciences) supplemented with 15% defibrinated sheep blood (HemoStat Laboratories) and grown at 37°C for 2–3 days. For broth cultures, colonies were picked from these plates and cultured overnight in Stainer-Scholte broth (SS) (15 (link)). For experiments that monitored production and processing of nascent FhaB and ACT across time (such as in Fig. 8 ), bacteria were instead first grown overnight in BvgAS non-inducing media (SS supplemented with 50 mM MgSO4) to prevent the production of Bvg-induced virulence factors, then washed with Dulbecco’s phosphate-buffered saline (PBS; Thermo Fisher), and sub-cultured in fresh BvgAS inducing media (standard SS) for times listed. Escherichia coli strains were grown at 37°C in lysogeny broth or on lysogeny broth agar. Where appropriate, media was supplemented with streptomycin (20 µg/mL), kanamycin (50 µg/mL), ampicillin (100 µg/mL), MgSO4 (50 mM), and CaCl2 (1.8 mM).
6
Genomic DNA Extraction from Bordetella Species
7
Whole-Genome Sequencing of Bordetella Isolates
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We sequenced a set of 55 isolates (Technical Appendix 1 Table 1). Of these, 24 isolates corresponded to 11 related groups of isolates: 8 isolates originated from 4 different pairs of intrafamilial transmission cases and 16 isolates corresponded to multiple isolates collected from 7 patients (6 pairs and 1 quadruplet); 30 corresponded to a random selection of temporally cocirculating isolates. We used as reference the Tohama isolate (GenBank accession no. NC_002929).
We grew isolates at 36°C for 72 hours on Bordet-Gengou agar (Becton Dickinson, Le Pont de Claix, France) supplemented with 15% defibrinated horse blood (BioMérieux, Marcy l’Étoile, France) and subcultured them in the same medium for 24 hours. We suspended the bacteria in physiologic salt to reach an optical density at 650 nm of 1, and pelleted 400 μL. We suspended the pellets in 100 μL of 1× phosphate-buffered saline, 100 μL of lysis buffer (Roche), and 40 μL of proteinase K; heated them at 65°C for 10 minutes and then at 95°C for 10 minutes; and used them for DNA extraction.
We grew isolates at 36°C for 72 hours on Bordet-Gengou agar (Becton Dickinson, Le Pont de Claix, France) supplemented with 15% defibrinated horse blood (BioMérieux, Marcy l’Étoile, France) and subcultured them in the same medium for 24 hours. We suspended the bacteria in physiologic salt to reach an optical density at 650 nm of 1, and pelleted 400 μL. We suspended the pellets in 100 μL of 1× phosphate-buffered saline, 100 μL of lysis buffer (Roche), and 40 μL of proteinase K; heated them at 65°C for 10 minutes and then at 95°C for 10 minutes; and used them for DNA extraction.
8
Canine-Origin Bb Strain Challenge
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A virulent Bb strain of canine origin was used for challenge. This challenge strain was grown in TSB-ST (BioMérieux SA, Marcy l’étoile, France) at 37 °C, with shaking at 180 rpm, for 6 h. The bacterial culture was collected when an OD600 of 1 was reached (approximately equivalent to 1 × 109 colony-forming units (CFU)/mL). On D35, all the study animals were anesthetized with isoflurane. Rats were inoculated with 25 µL of the challenge strain in each nostril, resulting in a total dose of 50 µL of the strain in PBS, or a titer of 1 × 103 CFU/animal. The diluted bacterial cultures were back-titrated to confirm the titers used (CFU/animal). Each suspension was serially diluted and 10 µL drops were dispensed onto Bordet Gengou agar containing 15% sheep blood (Becton Dickinson, Heidelberg, Germany).
9
Cultivation of Bordetella pertussis Strains
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B. pertussis strains 18323 (85 (link)) and Tohama (86 (link)) were maintained in our laboratory. The B. pertussis clinical strains BP140, BP141, BP142, and BP144 were provided by K. Kamachi (National Institute of Infectious Diseases). B. pertussis was grown on Bordet-Gengou agar (Becton, Dickinson) plates containing 1% Hipolypepton (Nihon Pharmaceutical), 1% glycerol, 15% defibrinated horse blood, and 10 μg/mL ceftibuten (BG plate). The bacteria recovered from colonies on BG plates were suspended in Stainer-Scholte (SS) medium (87 (link)) to yield an optical density at 650 nm (OD650) value of 0.2 and incubated at 37°C for 12 to 14 h with shaking. The obtained bacteria were used as Bvg+-phase bacteria. Unless otherwise specified, B. pertussis in the Bvg− phase was obtained by cultivation in the presence of 40 mM MgSO4. The number of CFU was estimated from the OD650 values of fresh cultures according to the following equation: 1 OD650 unit = 3.3 × 109 CFU/mL. Escherichia coli was grown with Luria-Bertani (LB) agar or broth. E. coli strains DH5α λpir and HB101 harboring pRK2013 (88 (link)) were provided by K. Minamisawa (Tohoku University). The growth media were supplemented with antibiotics when necessary at the following concentrations: ampicillin, 50 μg/mL; gentamicin 10 μg/mL; kanamycin 50 μg/mL.
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10
Bacterial Growth Evaluation Protocol
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The samples were applied to culture media using standard techniques. Culture media included Bordetella Agar (DifcoTM Bordet Gengou Agar Ref#248200, Becton Dickinson, Le Pont de Claix, France (BD) with 15% sheep blood), BBLTM Columbia Agar with 5% sheep blood (Ref# 211124, BD) and BBLTM Columbia colistin nalidixic acid (CNA) agar with 5% sheep blood (Ref 212104, BD) in order to provide appropriate media for a variety of aerobic bacterial species.
The inoculated agar plates were incubated at 36–38 °C and examined for bacterial growth after 24, 48, and 72 h. Bacterial growth was semiquantitatively assessed. Degree of contamination was defined as negligible (<10 colony forming units (CFU)), moderate (10–50 CFU), or severe (>50 CFU). If bacterial growth was identified, bacterial species were further classified using matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF, Microflex LT and MALDI Biotyper Identification-Software 3.1, Bruker Daltonik GmbH, Bremen, Germany; Library: Bruker Taxonomy Tree (8599 Spectra)).
The inoculated agar plates were incubated at 36–38 °C and examined for bacterial growth after 24, 48, and 72 h. Bacterial growth was semiquantitatively assessed. Degree of contamination was defined as negligible (<10 colony forming units (CFU)), moderate (10–50 CFU), or severe (>50 CFU). If bacterial growth was identified, bacterial species were further classified using matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF, Microflex LT and MALDI Biotyper Identification-Software 3.1, Bruker Daltonik GmbH, Bremen, Germany; Library: Bruker Taxonomy Tree (8599 Spectra)).
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