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  • 99
    Thermo Fisher brain heart infusion bhi broth
    Detection of E. faecalis EbpC on the cell surface by immunoelectron microscopy. Anti-EbpC MAb-probed <t>OG1RF</t> cells grown in <t>BHI</t> were immunogold labeled, negatively stained with 1% uranyl acetate, and viewed by transmission electron microscopy.
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    Millipore bhi broth
    FTL_1753 is required for F. tularensis subsp. holarctica to survive hypoosmotic shock. (A and B) Survival data from downshock experiments performed in <t>BHI</t> broth. Bacteria were grown in BHI broth plus 300 mM <t>NaCl,</t> and then diluted in BHI broth plus 300, 200, 100, or 0 mM NaCl, producing hypoosmotic shocks of 0, 100, 200, and 300 mM, respectively. (C) Survival data for PBS-equilibrated bacteria diluted in water retrieved from a freshwater lake or double-distilled H 2 O. Statistical differences were determined by one-way analyses of variance (ANOVAs) with Dunnett's posttests. *, P
    Bhi Broth, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco brain heart infusion bhi broth
    Protease activation in the extracellular protein fraction from P. <t>gingivalis</t> FLL93. P. gingivalis was grown to stationary phase (OD 600 of 1.3) in 1 liter of <t>BHI</t> broth supplemented with hemin and vitamin K. Acetone (37.5%)-precipitated proteins were mixed with 8 M urea and were incubated at 4°C for 1 h. The urea was slowly removed from the mixture by centrifugation (10,000 × g ) in a Millipore filtration unit with the addition of increasing volumes of 100 mM Tris-HCl buffer (pH 7.4). Activities for Rgp and Kgp were tested. Error bars indicate the standard errors of the means of three independent trials.
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    Becton Dickinson brain heart infusion bhi broth
    Prevention of infectious abortion by HO-1 and Bcl-XL expression. (A) Pregnant mice were infected with 10 5 cells of L. <t>monocytogenes</t> in 0.1 ml of saline at day 13.5 of pregnancy with or without Co-PP treatment (5 mg/kg). At day 16.5, the placentas, fetuses, and livers were removed. HO-1 and Bcl-XL expression in the placenta was analyzed by immunoblotting. A representative immunoblot of three independent experiments is shown. (B) Survival rates were determined by the presence or absence of a heartbeat in the fetuses. (C) Livers were homogenized in saline and diluted with PBS. CFU was determined by plating the diluted samples on <t>BHI</t> agar plate.
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    Difco brain heart infusion broth
    Prevention of infectious abortion by HO-1 and Bcl-XL expression. (A) Pregnant mice were infected with 10 5 cells of L. <t>monocytogenes</t> in 0.1 ml of saline at day 13.5 of pregnancy with or without Co-PP treatment (5 mg/kg). At day 16.5, the placentas, fetuses, and livers were removed. HO-1 and Bcl-XL expression in the placenta was analyzed by immunoblotting. A representative immunoblot of three independent experiments is shown. (B) Survival rates were determined by the presence or absence of a heartbeat in the fetuses. (C) Livers were homogenized in saline and diluted with PBS. CFU was determined by plating the diluted samples on <t>BHI</t> agar plate.
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    Difco brain heart infusion bhi
    Growth patterns of E. <t>faecalis</t> strains in <t>BHI</t> and BHIS. Data from BHI grown cultures were marked with solid squares, circles and triangles and BHIS were marked with open squares, circles and triangles. Time points at which adherence was tested are marked
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    92
    Difco brain heart infusion bhi medium
    Role of the c-di-GMP-induced EPS in biofilm formation, cell aggregation, and tolerance of L. <t>monocytogenes</t> to disinfectants and desiccation. A : Biofilm formation of L. monocytogenes in 96-well polystyrene plates (measured using a Crystal violet dye-binding assay). Cultures were grown for 6 days at 30°C in LB (top panel) or LB supplemented with 3% glycerol (bottom panel). Shown are average results from two biological replicates, where each strain was grown in six wells in a replicate (i.e., six technical replicates). Black circle, wild type; red square, Δ pdeB/C/D ; green triangle, Δ pdeB/C/D Δ pssC ; blue cross, Δ pdeB/C/D Δ pssE . B : EPS-dependent L. monocytogenes cell aggregation (clumping) in HTM medium. Overnight cultures grown in <t>BHI</t> were inoculated into HTM liquid medium at A 600 of 0.01 and incubated at 30°C with gentle shaking (rotary shaker, 125 rpm) for 48 h. 1, ΔpdeB/C/D ; 2, wild type; 3, ΔpdeB/C/D ΔpssC ; 4, ΔpdeB/C/D ΔpssE ; C : Protective role of the c-di-GMP-inducible EPS in disinfection. Aliquots of the HTM-grown cultures were mixed with disinfectant solutions for 10 min at room temperature. Disinfection was stopped by adding a D/E neutralizing broth (Difco); the cultures were vortexed vigorously (5 min) with glass beads to break clumps and plated on BHI agar. Colonies were enumerated after a 48-h growth at 37°C. SH, sodium hydrochloride (1600 ppm); HP, hydrogen peroxide (200 mM); BC, benzalkonium chloride (100 ppm). White background, EGD-e; black, ΔpdeB/C/D ; grey, ΔpdeB/C/D ΔpssC . SH, sodium hypochlorite; HP, hydrogen peroxide; BC, benzalkonium chloride. The absence of the bar for the EGD-e strain treated with SH indicates the lack of survivors. D : Protective role of the c-di-GMP-inducible EPS in desiccation. Aliquots of overnight cultures grown in HTM at 37°C were spun down, the supernatants were removed, and cell pellets were stored in desiccators at room temperature for the indicated periods. The pellets were rehydrated, vortexed with glass beads for better suspension and plated on BHI agar. The numbers of surviving colonies after incubation at 37°C for 24 h are plotted. In panels C and D, bars denote mean values for data from three biological replicates. *, significantly different (p
    Brain Heart Infusion Bhi Medium, supplied by Difco, used in various techniques. Bioz Stars score: 92/100, based on 431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco bhi agar
    Expression and autolytic activity of Cwp19 in <t>BHI</t> medium. (A) qRT-PCR analysis of cwp19 expression during cellular growth in BHI medium. Total RNAs were extracted from C. difficile 630Δ <t>erm</t> grown in BHI medium at different times of growth. cDNAs were prepared and qRT-PCR was performed using gene-specific primers as outlined in Materials and Methods. Cells incubated for 5 h served as the reference condition. *, P ≤ 0.05 (Student’s t test). (B) Schematic representation of the genetic organization of the cwp19 chromosomal region of C. difficile strain 630Δ erm . Large arrows represent the genes, and their orientation shows the transcriptional direction. The nucleotide sequence of the cwp19 promoter region is shown. The transcriptional initiation nucleotide (+1) identified by 5′ RACE, the putative ribosome binding site sequence, the −10 and −35 motifs, and the translational initiation codon are underlined. (C) Triton X-100-induced autolysis of the C. difficile wild type (●), cwp19 mutant (■), and complemented cwp19 mutant (▲) grown in BHI medium. The strains were harvested in the exponential-growth phase and transferred in 50 mM potassium phosphate buffer (pH 7.0) containing 0.01% Triton X-100. Autolysis was monitored by measuring the decrease of the bacterial suspension at OD 600 and is expressed as a percentage of the initial OD 600 value. Error bars indicate standard deviations. Values are given as means ± standard deviations ( n = 3).
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    Becton Dickinson brain heart infusion bhi
    Detection of E. faecalis mixed colonies by fluorescence imaging. Colonies were obtained by subculturing from a <t>BHI-S</t> broth culture of the gfp -tagged strain (SD234; <t>OG1RF</t> : : gfp ) grown with a non- gfp tagged strain (OG1SSp). Plates
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    Becton Dickinson brain heart infusion bhi medium
    S. <t>oralis</t> biofilms growing for 24 or 48 h on polystyrene. Biofilms of wild-type (So34), gtfR mutant (So ∆gtfR) or complemented (So pgtfR) strains were grown in RPMI, 10%FBS, 10% <t>BHI</t> media supplemented with 1% sucrose or 1% glucose. a X – Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. S. oralis (blue) was visualized after fluorescence in situ hybridization with a Streptococcus -specific probe conjugated to Alexa 405. Alexa Fluor 647-labeled dextran conjugate probe (red) was used to stain biofilm matrix (glucans). Scale bars, 50 µm ( X – Y isosurfaces) and 70 µm (three-dimensional reconstructions). b Average total biovolumes (in µm 3 ) for 24 and 48 h biofilms exposed to glucose (white bars) or sucrose (black bars) shown in ( a ) above. Biovolumes were measured in two different confocal laser scanning microscopy image stacks from two independent experiments. c Average biofilm thickness (in µm) in biofilms growing with 1%sucrose for 24 and 48 h. d Average S. oralis colony-forming units (CFU) in logarithmic scale for each 24 and 48 h biofilm. e Average matrix (α-glucan) biovolumes (in µm 3 ) for 24 and 48 h biofilms. * p
    Brain Heart Infusion Bhi Medium, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher brain heart infusion bhi agar
    Maximum specific growth rates of Listeria monocytogenes LO28 WT and 24 HHP-resistant variants at <t>30°C</t> in <t>BHI</t> under aerobic conditions (a); at 30°C in BHI under anaerobic conditions (b); or at 7°C in BHI under aerobic conditions
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    Becton Dickinson bhi agar
    Maximum specific growth rates of Listeria monocytogenes LO28 WT and 24 HHP-resistant variants at <t>30°C</t> in <t>BHI</t> under aerobic conditions (a); at 30°C in BHI under anaerobic conditions (b); or at 7°C in BHI under aerobic conditions
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    Becton Dickinson brain heart infusion broth
    Maximum specific growth rates of Listeria monocytogenes LO28 WT and 24 HHP-resistant variants at <t>30°C</t> in <t>BHI</t> under aerobic conditions (a); at 30°C in BHI under anaerobic conditions (b); or at 7°C in BHI under aerobic conditions
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    Thermo Fisher bhi agar
    Stress resistance of late-exponential phase cells of L. monocytogenes LO28 WT and acid resistant variants in <t>BHI</t> broth . Late-exponential phase cells were exposed to pH 2.5 for 3.5 min at 37∘C (adapted from Metselaar et al., 2013 ) (A), 55∘C for 6 min (B), 420 mM hydrogen peroxide for 9 min at <t>30∘C</t> (C), and 20 mg/L benzalkonium chloride (BAC) for 5 min at 30∘C (D) . Results are expressed as reduction in log 10 cfu/ml after exposure compared to log 10 cfu/ml at t = 0. Errors bars represent the SD and significant differences from the WT are indicated with ∗( p
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    Millipore bhi agar
    Effect of the hfq mutation on the sensitivity of M. catarrhalis to methyl <t>viologen.</t> Bacterial cells of each strain were suspended to the same OD 600 and then serially diluted and spotted onto <t>BHI</t> agar (A) and BHI agar containing 40 μM methyl viologen (B). Four strains were tested: wild-type O35E strain (WT), the O35EΔ hfq mutant (Δ hfq ), the O35EΔ hfq mutant containing the pAA200 plasmid with the wild-type O35E hfq gene [Δ hfq (pAA200)], and this same mutant containing only the plasmid vector [Δ hfq (pWW115)]. The plates were dried and incubated overnight, and the resultant growth was photographed.
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    Difco brain heart infusion
    Effect of the hfq mutation on the sensitivity of M. catarrhalis to methyl <t>viologen.</t> Bacterial cells of each strain were suspended to the same OD 600 and then serially diluted and spotted onto <t>BHI</t> agar (A) and BHI agar containing 40 μM methyl viologen (B). Four strains were tested: wild-type O35E strain (WT), the O35EΔ hfq mutant (Δ hfq ), the O35EΔ hfq mutant containing the pAA200 plasmid with the wild-type O35E hfq gene [Δ hfq (pAA200)], and this same mutant containing only the plasmid vector [Δ hfq (pWW115)]. The plates were dried and incubated overnight, and the resultant growth was photographed.
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    Becton Dickinson brain heart infusion
    Effect of the hfq mutation on the sensitivity of M. catarrhalis to methyl <t>viologen.</t> Bacterial cells of each strain were suspended to the same OD 600 and then serially diluted and spotted onto <t>BHI</t> agar (A) and BHI agar containing 40 μM methyl viologen (B). Four strains were tested: wild-type O35E strain (WT), the O35EΔ hfq mutant (Δ hfq ), the O35EΔ hfq mutant containing the pAA200 plasmid with the wild-type O35E hfq gene [Δ hfq (pAA200)], and this same mutant containing only the plasmid vector [Δ hfq (pWW115)]. The plates were dried and incubated overnight, and the resultant growth was photographed.
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    Thermo Fisher brain heart infusion
    Effect of the hfq mutation on the sensitivity of M. catarrhalis to methyl <t>viologen.</t> Bacterial cells of each strain were suspended to the same OD 600 and then serially diluted and spotted onto <t>BHI</t> agar (A) and BHI agar containing 40 μM methyl viologen (B). Four strains were tested: wild-type O35E strain (WT), the O35EΔ hfq mutant (Δ hfq ), the O35EΔ hfq mutant containing the pAA200 plasmid with the wild-type O35E hfq gene [Δ hfq (pAA200)], and this same mutant containing only the plasmid vector [Δ hfq (pWW115)]. The plates were dried and incubated overnight, and the resultant growth was photographed.
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    Image Search Results


    Detection of E. faecalis EbpC on the cell surface by immunoelectron microscopy. Anti-EbpC MAb-probed OG1RF cells grown in BHI were immunogold labeled, negatively stained with 1% uranyl acetate, and viewed by transmission electron microscopy.

    Journal: Infection and Immunity

    Article Title: Targeting Pili in Enterococcal Pathogenesis

    doi: 10.1128/IAI.01403-13

    Figure Lengend Snippet: Detection of E. faecalis EbpC on the cell surface by immunoelectron microscopy. Anti-EbpC MAb-probed OG1RF cells grown in BHI were immunogold labeled, negatively stained with 1% uranyl acetate, and viewed by transmission electron microscopy.

    Article Snippet: Briefly, overnight-cultured OG1RF was inoculated into 20 ml brain heart infusion (BHI) broth (Gibco) at a starting optical density at 600 nm (OD600 ) of 0.05 and harvested at mid-log phase.

    Techniques: Immuno-Electron Microscopy, Labeling, Staining, Transmission Assay, Electron Microscopy

    Oxacillin susceptibility and autolysis phenotypes of strains USA300 and HoR34. (A) Oxacillin MICs of strains USA300, HoR34, and HoR34 carrying plasmids pLI50 (control), p gdpP , p guaA , and p camS determined using Etests. (B) Autolytic activity in USA300 and HoR34. Strains USA300, HoR34, and HoR34 carrying plasmids pLI50 (control), p gdpP , p guaA , and p camS , and a USA300 JE2 atl mutant (negative control) were grown to early exponential phase in BHI at 37°C, washed in phosphate-buffered saline (PBS), and adjusted to an A 600 of 1.0 in 0.01% Triton X-100. The A 600 was measured initially and at 15-min intervals thereafter with shaking incubation at 37°C. Autolytic activity is expressed as a percentage of the initial A 600 . Average results from three independent experiments are shown.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Tandem Amplification of the Staphylococcal Cassette Chromosome mec Element Can Drive High-Level Methicillin Resistance in Methicillin-Resistant Staphylococcus aureus

    doi: 10.1128/AAC.00869-17

    Figure Lengend Snippet: Oxacillin susceptibility and autolysis phenotypes of strains USA300 and HoR34. (A) Oxacillin MICs of strains USA300, HoR34, and HoR34 carrying plasmids pLI50 (control), p gdpP , p guaA , and p camS determined using Etests. (B) Autolytic activity in USA300 and HoR34. Strains USA300, HoR34, and HoR34 carrying plasmids pLI50 (control), p gdpP , p guaA , and p camS , and a USA300 JE2 atl mutant (negative control) were grown to early exponential phase in BHI at 37°C, washed in phosphate-buffered saline (PBS), and adjusted to an A 600 of 1.0 in 0.01% Triton X-100. The A 600 was measured initially and at 15-min intervals thereafter with shaking incubation at 37°C. Autolytic activity is expressed as a percentage of the initial A 600 . Average results from three independent experiments are shown.

    Article Snippet: Bacterial strains used in this study are listed in and were grown at 37°C in LB (Sigma), brain heart infusion (BHI) (Oxoid), Mueller-Hinton (Oxoid), or nutrient (Oxoid) broth supplemented with ampicillin (50 μg/ml), oxacillin (0.5, 64, 100, or 130 μg/ml), chloramphenicol (10 μg/ml), or erythromycin (10 μg/ml) as indicated.

    Techniques: Activity Assay, Mutagenesis, Negative Control, Incubation

    ESR determination of hydroxyl radicals formed in cultures of S. aureus treated with hydrogen peroxide or antibiotics. Hydroxyl-radical signals were monitored in BHI (A) or TSB (B) medium (filled diamonds) or in cultures of S. aureus grown in the corresponding

    Journal: Applied and Environmental Microbiology

    Article Title: Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

    doi: 10.1128/AEM.01199-15

    Figure Lengend Snippet: ESR determination of hydroxyl radicals formed in cultures of S. aureus treated with hydrogen peroxide or antibiotics. Hydroxyl-radical signals were monitored in BHI (A) or TSB (B) medium (filled diamonds) or in cultures of S. aureus grown in the corresponding

    Article Snippet: Staphylococcus aureus Newman (NTCT 8178) was cultivated in 25 ml of tryptic soy broth (TSB) or brain heart infusion broth (BHI) (Oxoid, Denmark) in a 250-ml narrow-neck Erlenmeyer flask at 37°C and 200 rpm.

    Techniques: Electron Paramagnetic Resonance

    FTL_1753 is required for F. tularensis subsp. holarctica to survive hypoosmotic shock. (A and B) Survival data from downshock experiments performed in BHI broth. Bacteria were grown in BHI broth plus 300 mM NaCl, and then diluted in BHI broth plus 300, 200, 100, or 0 mM NaCl, producing hypoosmotic shocks of 0, 100, 200, and 300 mM, respectively. (C) Survival data for PBS-equilibrated bacteria diluted in water retrieved from a freshwater lake or double-distilled H 2 O. Statistical differences were determined by one-way analyses of variance (ANOVAs) with Dunnett's posttests. *, P

    Journal: Applied and Environmental Microbiology

    Article Title: A Single Mechanosensitive Channel Protects Francisella tularensis subsp. holarctica from Hypoosmotic Shock and Promotes Survival in the Aquatic Environment

    doi: 10.1128/AEM.02203-17

    Figure Lengend Snippet: FTL_1753 is required for F. tularensis subsp. holarctica to survive hypoosmotic shock. (A and B) Survival data from downshock experiments performed in BHI broth. Bacteria were grown in BHI broth plus 300 mM NaCl, and then diluted in BHI broth plus 300, 200, 100, or 0 mM NaCl, producing hypoosmotic shocks of 0, 100, 200, and 300 mM, respectively. (C) Survival data for PBS-equilibrated bacteria diluted in water retrieved from a freshwater lake or double-distilled H 2 O. Statistical differences were determined by one-way analyses of variance (ANOVAs) with Dunnett's posttests. *, P

    Article Snippet: For downshock experiments conducted in medium, single colonies of each strain were picked from MH chocolate agar plates and grown in BHI broth supplemented with 300 mM NaCl (EMD Chemicals).

    Techniques:

    Expression of ace/ Ace by E. faecalis OG1RF and its Δers derivative. (A) Transcriptional changes by strains grown in BHI with serum at 37°C and in BHI at 46°C. Northern blot analysis was performed with total RNA (gel, 10 µg/lane)

    Journal: FEMS microbiology letters

    Article Title: Expression of the Collagen Adhesin ace by Enterococcus faecalis Strain OG1RF is not Repressed by Ers but Requires the Ers box

    doi: 10.1111/1574-6968.12146

    Figure Lengend Snippet: Expression of ace/ Ace by E. faecalis OG1RF and its Δers derivative. (A) Transcriptional changes by strains grown in BHI with serum at 37°C and in BHI at 46°C. Northern blot analysis was performed with total RNA (gel, 10 µg/lane)

    Article Snippet: Bacteria were grown at 37°C in Luria-Bertani (LB) broth, Brain Heart Infusion (BHI) broth, M17 medium supplemented with 0.5% glucose (M17-G) or MM9YEG with 10 mM p -chloro-phenylalanine (p-Cl-Phe, Sigma-Aldrich Co.) ( ).

    Techniques: Expressing, Northern Blot

    Protease activation in the extracellular protein fraction from P. gingivalis FLL93. P. gingivalis was grown to stationary phase (OD 600 of 1.3) in 1 liter of BHI broth supplemented with hemin and vitamin K. Acetone (37.5%)-precipitated proteins were mixed with 8 M urea and were incubated at 4°C for 1 h. The urea was slowly removed from the mixture by centrifugation (10,000 × g ) in a Millipore filtration unit with the addition of increasing volumes of 100 mM Tris-HCl buffer (pH 7.4). Activities for Rgp and Kgp were tested. Error bars indicate the standard errors of the means of three independent trials.

    Journal: Infection and Immunity

    Article Title: The vimE Gene Downstream of vimA Is Independently Expressed and Is Involved in Modulating Proteolytic Activity in Porphyromonas gingivalis W83

    doi: 10.1128/IAI.72.10.5555-5564.2004

    Figure Lengend Snippet: Protease activation in the extracellular protein fraction from P. gingivalis FLL93. P. gingivalis was grown to stationary phase (OD 600 of 1.3) in 1 liter of BHI broth supplemented with hemin and vitamin K. Acetone (37.5%)-precipitated proteins were mixed with 8 M urea and were incubated at 4°C for 1 h. The urea was slowly removed from the mixture by centrifugation (10,000 × g ) in a Millipore filtration unit with the addition of increasing volumes of 100 mM Tris-HCl buffer (pH 7.4). Activities for Rgp and Kgp were tested. Error bars indicate the standard errors of the means of three independent trials.

    Article Snippet: P. gingivalis strains were grown in brain heart infusion (BHI) broth supplemented with 0.5% yeast extract (both from Difco Laboratories, Detroit, Mich.), hemin (5 μg/ml), vitamin K (0.5 μg/ml), and cysteine (0.1%) (all from Sigma-Aldrich, St. Louis, Mo.).

    Techniques: Activation Assay, Incubation, Centrifugation, Filtration

    Inactivation of vimE resulted in decreased proteolytic activity of P. gingivalis. P. gingivalis was grown to late log phase (OD 600 of 0.7 to 0.8) or stationary phase (OD 600 of 1.4 to 1.5) in BHI broth supplemented with yeast extract, hemin, and vitamin K. Activities against Rgp (A) and Kgp (B) were tested in whole-cell culture. The results shown are representative of three independent experiments in triplicate. E, exponential phase; L, stationary phase. Error bars indicate the standard errors of the means of three independent trials.

    Journal: Infection and Immunity

    Article Title: The vimE Gene Downstream of vimA Is Independently Expressed and Is Involved in Modulating Proteolytic Activity in Porphyromonas gingivalis W83

    doi: 10.1128/IAI.72.10.5555-5564.2004

    Figure Lengend Snippet: Inactivation of vimE resulted in decreased proteolytic activity of P. gingivalis. P. gingivalis was grown to late log phase (OD 600 of 0.7 to 0.8) or stationary phase (OD 600 of 1.4 to 1.5) in BHI broth supplemented with yeast extract, hemin, and vitamin K. Activities against Rgp (A) and Kgp (B) were tested in whole-cell culture. The results shown are representative of three independent experiments in triplicate. E, exponential phase; L, stationary phase. Error bars indicate the standard errors of the means of three independent trials.

    Article Snippet: P. gingivalis strains were grown in brain heart infusion (BHI) broth supplemented with 0.5% yeast extract (both from Difco Laboratories, Detroit, Mich.), hemin (5 μg/ml), vitamin K (0.5 μg/ml), and cysteine (0.1%) (all from Sigma-Aldrich, St. Louis, Mo.).

    Techniques: Activity Assay, Cell Culture

    Immunoreactivity of the RgpB proenzyme in P. gingivalis FLL93. Western blot analysis using specific RgpB proenzyme antibodies as probes was done on the extracellular fractions from P. gingivalis W83 (lane 1), FLL92 (lane 2), and FLL93 (lane 3). All lanes contained 20 μg of acetone (37.5%)-precipitated proteins from the supernatant fractions of cultures in BHI medium at exponential growth phase.

    Journal: Infection and Immunity

    Article Title: The vimE Gene Downstream of vimA Is Independently Expressed and Is Involved in Modulating Proteolytic Activity in Porphyromonas gingivalis W83

    doi: 10.1128/IAI.72.10.5555-5564.2004

    Figure Lengend Snippet: Immunoreactivity of the RgpB proenzyme in P. gingivalis FLL93. Western blot analysis using specific RgpB proenzyme antibodies as probes was done on the extracellular fractions from P. gingivalis W83 (lane 1), FLL92 (lane 2), and FLL93 (lane 3). All lanes contained 20 μg of acetone (37.5%)-precipitated proteins from the supernatant fractions of cultures in BHI medium at exponential growth phase.

    Article Snippet: P. gingivalis strains were grown in brain heart infusion (BHI) broth supplemented with 0.5% yeast extract (both from Difco Laboratories, Detroit, Mich.), hemin (5 μg/ml), vitamin K (0.5 μg/ml), and cysteine (0.1%) (all from Sigma-Aldrich, St. Louis, Mo.).

    Techniques: Western Blot

    Prevention of infectious abortion by HO-1 and Bcl-XL expression. (A) Pregnant mice were infected with 10 5 cells of L. monocytogenes in 0.1 ml of saline at day 13.5 of pregnancy with or without Co-PP treatment (5 mg/kg). At day 16.5, the placentas, fetuses, and livers were removed. HO-1 and Bcl-XL expression in the placenta was analyzed by immunoblotting. A representative immunoblot of three independent experiments is shown. (B) Survival rates were determined by the presence or absence of a heartbeat in the fetuses. (C) Livers were homogenized in saline and diluted with PBS. CFU was determined by plating the diluted samples on BHI agar plate.

    Journal: PLoS ONE

    Article Title: Protective Role of Heme Oxygenase-1 in Listeria monocytogenes-Induced Abortion

    doi: 10.1371/journal.pone.0025046

    Figure Lengend Snippet: Prevention of infectious abortion by HO-1 and Bcl-XL expression. (A) Pregnant mice were infected with 10 5 cells of L. monocytogenes in 0.1 ml of saline at day 13.5 of pregnancy with or without Co-PP treatment (5 mg/kg). At day 16.5, the placentas, fetuses, and livers were removed. HO-1 and Bcl-XL expression in the placenta was analyzed by immunoblotting. A representative immunoblot of three independent experiments is shown. (B) Survival rates were determined by the presence or absence of a heartbeat in the fetuses. (C) Livers were homogenized in saline and diluted with PBS. CFU was determined by plating the diluted samples on BHI agar plate.

    Article Snippet: Bacterial strains L. monocytogenes EGD was maintained as a frozen glycerol stock and cultured in brain heart infusion (BHI) broth (Becton Dickinson) or on BHI broth containing 1.5% agar.

    Techniques: Expressing, Mouse Assay, Infection

    Decreased HO-1 and Bcl-XL expression in TG cells infected with L. monocytogenes . (A) TG cells were first treated with Co-PP and then infected with L. monocytogenes . The infected cells were cultured in 50 µg/ml of gentamicin. After 6 h, HO-1 and Bcl-XL expression was analyzed by immunoblotting. A representative immunoblot of three independent experiments is shown. (B) TG cells were treated with Co-PP and then infected with L. monocytogenes . The infected cells were cultured in 50 µg/ml of gentamicin. After 0.5, 2, and 6 h incubation, the infected cells were washed with PBS and lysed with cold distilled water. CFU were determined by serial dilution on BHI agar plates. (C) L. monocytogenes was deposited on TG cells by centrifugation at 150×g for 10 min at room temperature, incubated for 6 h, fixed, and stained. The figure shows FITC-labeled bacteria (green) and Alexa Fluor 594-labeled actin filaments (red) merged images. The left-hand panel shows untreated cells, the center panel Co-PP (9 µg/ml)-treated cells, and the right-hand panel, cytochalasin D-treated cells.

    Journal: PLoS ONE

    Article Title: Protective Role of Heme Oxygenase-1 in Listeria monocytogenes-Induced Abortion

    doi: 10.1371/journal.pone.0025046

    Figure Lengend Snippet: Decreased HO-1 and Bcl-XL expression in TG cells infected with L. monocytogenes . (A) TG cells were first treated with Co-PP and then infected with L. monocytogenes . The infected cells were cultured in 50 µg/ml of gentamicin. After 6 h, HO-1 and Bcl-XL expression was analyzed by immunoblotting. A representative immunoblot of three independent experiments is shown. (B) TG cells were treated with Co-PP and then infected with L. monocytogenes . The infected cells were cultured in 50 µg/ml of gentamicin. After 0.5, 2, and 6 h incubation, the infected cells were washed with PBS and lysed with cold distilled water. CFU were determined by serial dilution on BHI agar plates. (C) L. monocytogenes was deposited on TG cells by centrifugation at 150×g for 10 min at room temperature, incubated for 6 h, fixed, and stained. The figure shows FITC-labeled bacteria (green) and Alexa Fluor 594-labeled actin filaments (red) merged images. The left-hand panel shows untreated cells, the center panel Co-PP (9 µg/ml)-treated cells, and the right-hand panel, cytochalasin D-treated cells.

    Article Snippet: Bacterial strains L. monocytogenes EGD was maintained as a frozen glycerol stock and cultured in brain heart infusion (BHI) broth (Becton Dickinson) or on BHI broth containing 1.5% agar.

    Techniques: Expressing, Infection, Cell Culture, Incubation, Serial Dilution, Centrifugation, Staining, Labeling

    Prevention of cell death by HO-1 and Bcl-XL expression. (A) TG cells were treated for 48 h with either siRNA targeting HO-1, Bcl-XL, or control siRNA (QIAGEN AllStars Negative Control). Bcl-XL overexpression was achieved by transfecting the cells with pcDNA4/TO-Bcl-XL. HO-1 and Bcl-XL expression was monitored by immunoblotting. β-actin was used as an internal control. A representative immunoblot of three independent experiments is shown. (B) TG cells were infected with L. monocytogenes . The infected cells were cultured with media containing 50 µg/ml gentamicin for 2, 6, and 12 h. The cells were then washed with PBS and lysed with cold distilled water. CFU was determined by serial dilution on BHI agar plates. All values represent the average and the standard deviation of three identical experiments. (C) Cell death was determined using the JC-1 Mitochondrial Membrane Potential Assay Kit. One hundred TG cells per coverslip were examined to determine the total number of live or dead cells. All values represent the average and the standard deviation of three identical experiments. Statistically significant differences compared with the control are indicated by asterisks (*, P

    Journal: PLoS ONE

    Article Title: Protective Role of Heme Oxygenase-1 in Listeria monocytogenes-Induced Abortion

    doi: 10.1371/journal.pone.0025046

    Figure Lengend Snippet: Prevention of cell death by HO-1 and Bcl-XL expression. (A) TG cells were treated for 48 h with either siRNA targeting HO-1, Bcl-XL, or control siRNA (QIAGEN AllStars Negative Control). Bcl-XL overexpression was achieved by transfecting the cells with pcDNA4/TO-Bcl-XL. HO-1 and Bcl-XL expression was monitored by immunoblotting. β-actin was used as an internal control. A representative immunoblot of three independent experiments is shown. (B) TG cells were infected with L. monocytogenes . The infected cells were cultured with media containing 50 µg/ml gentamicin for 2, 6, and 12 h. The cells were then washed with PBS and lysed with cold distilled water. CFU was determined by serial dilution on BHI agar plates. All values represent the average and the standard deviation of three identical experiments. (C) Cell death was determined using the JC-1 Mitochondrial Membrane Potential Assay Kit. One hundred TG cells per coverslip were examined to determine the total number of live or dead cells. All values represent the average and the standard deviation of three identical experiments. Statistically significant differences compared with the control are indicated by asterisks (*, P

    Article Snippet: Bacterial strains L. monocytogenes EGD was maintained as a frozen glycerol stock and cultured in brain heart infusion (BHI) broth (Becton Dickinson) or on BHI broth containing 1.5% agar.

    Techniques: Expressing, Negative Control, Over Expression, Infection, Cell Culture, Serial Dilution, Standard Deviation

    Growth patterns of E. faecalis strains in BHI and BHIS. Data from BHI grown cultures were marked with solid squares, circles and triangles and BHIS were marked with open squares, circles and triangles. Time points at which adherence was tested are marked

    Journal:

    Article Title: Role of Serum, a Biological Cue, in Adherence of Enterococcus faecalis to Extracellular Matrix Proteins, Collagen, Fibrinogen, and Fibronectin

    doi: 10.1086/588143

    Figure Lengend Snippet: Growth patterns of E. faecalis strains in BHI and BHIS. Data from BHI grown cultures were marked with solid squares, circles and triangles and BHIS were marked with open squares, circles and triangles. Time points at which adherence was tested are marked

    Article Snippet: E. faecalis cells were grown in Brain Heart Infusion (BHI) (Difco) at 37°C for routine purposes.

    Techniques:

    Growth of mutant H. influenzae Rd strains. The strains Rd, REI1012 (Δ hel :: kan ), and GK04 ( nadN :: cat Δ hel :: kan ) were grown on BHI agar plates supplemented with hemin (20 μg/ml) and different nicotinamide nucleotide concentrations and sources. Growth phenotype is shown with 1.5 and 35 μM NAD, with 1.5 and 15 μM NMN, and with 1.5 and 15 μM NR, as indicated.

    Journal: Journal of Bacteriology

    Article Title: NadN and e (P4) Are Essential for Utilization of NAD and Nicotinamide Mononucleotide but Not Nicotinamide Riboside in Haemophilus influenzae

    doi: 10.1128/JB.183.13.3974-3981.2001

    Figure Lengend Snippet: Growth of mutant H. influenzae Rd strains. The strains Rd, REI1012 (Δ hel :: kan ), and GK04 ( nadN :: cat Δ hel :: kan ) were grown on BHI agar plates supplemented with hemin (20 μg/ml) and different nicotinamide nucleotide concentrations and sources. Growth phenotype is shown with 1.5 and 35 μM NAD, with 1.5 and 15 μM NMN, and with 1.5 and 15 μM NR, as indicated.

    Article Snippet: H. influenzae was grown at 37°C under aerobic conditions on 3.8% brain heart infusion (BHI) agar (Difco Laboratories, Detroit, Mich.) supplemented with NAD (15 to 30 μM) and hemin-chloride (20 μg/ml) (Sigma, Deisenhofen, Germany).

    Techniques: Mutagenesis

    Role of the c-di-GMP-induced EPS in biofilm formation, cell aggregation, and tolerance of L. monocytogenes to disinfectants and desiccation. A : Biofilm formation of L. monocytogenes in 96-well polystyrene plates (measured using a Crystal violet dye-binding assay). Cultures were grown for 6 days at 30°C in LB (top panel) or LB supplemented with 3% glycerol (bottom panel). Shown are average results from two biological replicates, where each strain was grown in six wells in a replicate (i.e., six technical replicates). Black circle, wild type; red square, Δ pdeB/C/D ; green triangle, Δ pdeB/C/D Δ pssC ; blue cross, Δ pdeB/C/D Δ pssE . B : EPS-dependent L. monocytogenes cell aggregation (clumping) in HTM medium. Overnight cultures grown in BHI were inoculated into HTM liquid medium at A 600 of 0.01 and incubated at 30°C with gentle shaking (rotary shaker, 125 rpm) for 48 h. 1, ΔpdeB/C/D ; 2, wild type; 3, ΔpdeB/C/D ΔpssC ; 4, ΔpdeB/C/D ΔpssE ; C : Protective role of the c-di-GMP-inducible EPS in disinfection. Aliquots of the HTM-grown cultures were mixed with disinfectant solutions for 10 min at room temperature. Disinfection was stopped by adding a D/E neutralizing broth (Difco); the cultures were vortexed vigorously (5 min) with glass beads to break clumps and plated on BHI agar. Colonies were enumerated after a 48-h growth at 37°C. SH, sodium hydrochloride (1600 ppm); HP, hydrogen peroxide (200 mM); BC, benzalkonium chloride (100 ppm). White background, EGD-e; black, ΔpdeB/C/D ; grey, ΔpdeB/C/D ΔpssC . SH, sodium hypochlorite; HP, hydrogen peroxide; BC, benzalkonium chloride. The absence of the bar for the EGD-e strain treated with SH indicates the lack of survivors. D : Protective role of the c-di-GMP-inducible EPS in desiccation. Aliquots of overnight cultures grown in HTM at 37°C were spun down, the supernatants were removed, and cell pellets were stored in desiccators at room temperature for the indicated periods. The pellets were rehydrated, vortexed with glass beads for better suspension and plated on BHI agar. The numbers of surviving colonies after incubation at 37°C for 24 h are plotted. In panels C and D, bars denote mean values for data from three biological replicates. *, significantly different (p

    Journal: PLoS Pathogens

    Article Title: Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes

    doi: 10.1371/journal.ppat.1004301

    Figure Lengend Snippet: Role of the c-di-GMP-induced EPS in biofilm formation, cell aggregation, and tolerance of L. monocytogenes to disinfectants and desiccation. A : Biofilm formation of L. monocytogenes in 96-well polystyrene plates (measured using a Crystal violet dye-binding assay). Cultures were grown for 6 days at 30°C in LB (top panel) or LB supplemented with 3% glycerol (bottom panel). Shown are average results from two biological replicates, where each strain was grown in six wells in a replicate (i.e., six technical replicates). Black circle, wild type; red square, Δ pdeB/C/D ; green triangle, Δ pdeB/C/D Δ pssC ; blue cross, Δ pdeB/C/D Δ pssE . B : EPS-dependent L. monocytogenes cell aggregation (clumping) in HTM medium. Overnight cultures grown in BHI were inoculated into HTM liquid medium at A 600 of 0.01 and incubated at 30°C with gentle shaking (rotary shaker, 125 rpm) for 48 h. 1, ΔpdeB/C/D ; 2, wild type; 3, ΔpdeB/C/D ΔpssC ; 4, ΔpdeB/C/D ΔpssE ; C : Protective role of the c-di-GMP-inducible EPS in disinfection. Aliquots of the HTM-grown cultures were mixed with disinfectant solutions for 10 min at room temperature. Disinfection was stopped by adding a D/E neutralizing broth (Difco); the cultures were vortexed vigorously (5 min) with glass beads to break clumps and plated on BHI agar. Colonies were enumerated after a 48-h growth at 37°C. SH, sodium hydrochloride (1600 ppm); HP, hydrogen peroxide (200 mM); BC, benzalkonium chloride (100 ppm). White background, EGD-e; black, ΔpdeB/C/D ; grey, ΔpdeB/C/D ΔpssC . SH, sodium hypochlorite; HP, hydrogen peroxide; BC, benzalkonium chloride. The absence of the bar for the EGD-e strain treated with SH indicates the lack of survivors. D : Protective role of the c-di-GMP-inducible EPS in desiccation. Aliquots of overnight cultures grown in HTM at 37°C were spun down, the supernatants were removed, and cell pellets were stored in desiccators at room temperature for the indicated periods. The pellets were rehydrated, vortexed with glass beads for better suspension and plated on BHI agar. The numbers of surviving colonies after incubation at 37°C for 24 h are plotted. In panels C and D, bars denote mean values for data from three biological replicates. *, significantly different (p

    Article Snippet: L. monocytogenes was grown in Brain Heart Infusion (BHI) medium (Difco), HTM (minimal medium containing 3% glucose) or LB, supplemented with appropriate antibiotics at 25, 30, 37 or 42°C, as indicated.

    Techniques: Binding Assay, Incubation

    Expression and autolytic activity of Cwp19 in BHI medium. (A) qRT-PCR analysis of cwp19 expression during cellular growth in BHI medium. Total RNAs were extracted from C. difficile 630Δ erm grown in BHI medium at different times of growth. cDNAs were prepared and qRT-PCR was performed using gene-specific primers as outlined in Materials and Methods. Cells incubated for 5 h served as the reference condition. *, P ≤ 0.05 (Student’s t test). (B) Schematic representation of the genetic organization of the cwp19 chromosomal region of C. difficile strain 630Δ erm . Large arrows represent the genes, and their orientation shows the transcriptional direction. The nucleotide sequence of the cwp19 promoter region is shown. The transcriptional initiation nucleotide (+1) identified by 5′ RACE, the putative ribosome binding site sequence, the −10 and −35 motifs, and the translational initiation codon are underlined. (C) Triton X-100-induced autolysis of the C. difficile wild type (●), cwp19 mutant (■), and complemented cwp19 mutant (▲) grown in BHI medium. The strains were harvested in the exponential-growth phase and transferred in 50 mM potassium phosphate buffer (pH 7.0) containing 0.01% Triton X-100. Autolysis was monitored by measuring the decrease of the bacterial suspension at OD 600 and is expressed as a percentage of the initial OD 600 value. Error bars indicate standard deviations. Values are given as means ± standard deviations ( n = 3).

    Journal: mBio

    Article Title: Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile

    doi: 10.1128/mBio.00648-18

    Figure Lengend Snippet: Expression and autolytic activity of Cwp19 in BHI medium. (A) qRT-PCR analysis of cwp19 expression during cellular growth in BHI medium. Total RNAs were extracted from C. difficile 630Δ erm grown in BHI medium at different times of growth. cDNAs were prepared and qRT-PCR was performed using gene-specific primers as outlined in Materials and Methods. Cells incubated for 5 h served as the reference condition. *, P ≤ 0.05 (Student’s t test). (B) Schematic representation of the genetic organization of the cwp19 chromosomal region of C. difficile strain 630Δ erm . Large arrows represent the genes, and their orientation shows the transcriptional direction. The nucleotide sequence of the cwp19 promoter region is shown. The transcriptional initiation nucleotide (+1) identified by 5′ RACE, the putative ribosome binding site sequence, the −10 and −35 motifs, and the translational initiation codon are underlined. (C) Triton X-100-induced autolysis of the C. difficile wild type (●), cwp19 mutant (■), and complemented cwp19 mutant (▲) grown in BHI medium. The strains were harvested in the exponential-growth phase and transferred in 50 mM potassium phosphate buffer (pH 7.0) containing 0.01% Triton X-100. Autolysis was monitored by measuring the decrease of the bacterial suspension at OD 600 and is expressed as a percentage of the initial OD 600 value. Error bars indicate standard deviations. Values are given as means ± standard deviations ( n = 3).

    Article Snippet: Transconjugants were selected by subculturing on BHI agar containing Cfx, Ccs, and Tm and then plated on BHI agar containing Erm.

    Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Incubation, Sequencing, Binding Assay, Mutagenesis

    Cwp19 is a PG-degrading enzyme. (A) Detection of bacteriolytic activities in surface protein extracts of C. difficile 630 Δ erm (WT), cwp19 mutant (mut.), and complemented cwp19 mutant (comp) strains by zymogram. The gel contained 0.2% (wt/vol) M. lysodeikticus cells as a substrate. Surface proteins were extracted from cells grown to the exponential phase in BHI medium. Arrows indicate the positions of the bacteriolytic bands. (B) Schematic representation of Cwp19. The relevant characteristics are a signal peptide (SP), a putative catalytic domain (GHL10), and three copies of a cell wall binding motif termed CWB2. (C) Analysis of purified His 6 -tagged Cwp19 by SDS-PAGE (left panel), Western blotting performed with anti-His antibody (middle panel), and zymogram assay (right panel).

    Journal: mBio

    Article Title: Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile

    doi: 10.1128/mBio.00648-18

    Figure Lengend Snippet: Cwp19 is a PG-degrading enzyme. (A) Detection of bacteriolytic activities in surface protein extracts of C. difficile 630 Δ erm (WT), cwp19 mutant (mut.), and complemented cwp19 mutant (comp) strains by zymogram. The gel contained 0.2% (wt/vol) M. lysodeikticus cells as a substrate. Surface proteins were extracted from cells grown to the exponential phase in BHI medium. Arrows indicate the positions of the bacteriolytic bands. (B) Schematic representation of Cwp19. The relevant characteristics are a signal peptide (SP), a putative catalytic domain (GHL10), and three copies of a cell wall binding motif termed CWB2. (C) Analysis of purified His 6 -tagged Cwp19 by SDS-PAGE (left panel), Western blotting performed with anti-His antibody (middle panel), and zymogram assay (right panel).

    Article Snippet: Transconjugants were selected by subculturing on BHI agar containing Cfx, Ccs, and Tm and then plated on BHI agar containing Erm.

    Techniques: Mutagenesis, Binding Assay, Purification, SDS Page, Western Blot

    Role of Cwp19 in cell autolysis and toxin release of C. difficile cultivated in BHI medium. (A) Growth and autolysis of the wild-type 630Δ erm (●), cwp19 mutant (■), and complemented cwp19 mutant (▲) strains in BHI medium. Values are given as means ± standard deviations ( n = 3). (B) LDH activity released in the supernatant fractions of wild-type 630Δ erm (black bars), cwp19 mutant (gray bars), and complemented cwp19 mutant (white bars) strains in BHI medium at different time points. LDH activity was determined using Promega CytoTox 96. The signal from the test was recorded as absorbance at 490 nm. Values are given as means ± standard deviations ( n = 3). *, P ≤ 0.05 (Student’s t test). (C) TEM micrographs of C. difficile wild-type 630Δ erm , cwp19 mutant ( cwp19 mut.), and complemented cwp19 mutant (comp.) strains incubated for 33 h in BHI medium. The percentage of damaged cells for each strain is indicated. (D and E) Toxin titers in the cytosol (D) and in the culture supernatant (E) of wild-type 630Δ erm (black bars), cwp19 mutant (gray bars), and complemented cwp19 mutant (white bars) strains grown in BHI medium. Toxins were quantified by ELISA. The signal from the test was recorded as absorbance at 450 nm. Values are given as means ± standard deviations ( n = 3). *, P ≤ 0.05 (Student’s t test). ND, not detectable.

    Journal: mBio

    Article Title: Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile

    doi: 10.1128/mBio.00648-18

    Figure Lengend Snippet: Role of Cwp19 in cell autolysis and toxin release of C. difficile cultivated in BHI medium. (A) Growth and autolysis of the wild-type 630Δ erm (●), cwp19 mutant (■), and complemented cwp19 mutant (▲) strains in BHI medium. Values are given as means ± standard deviations ( n = 3). (B) LDH activity released in the supernatant fractions of wild-type 630Δ erm (black bars), cwp19 mutant (gray bars), and complemented cwp19 mutant (white bars) strains in BHI medium at different time points. LDH activity was determined using Promega CytoTox 96. The signal from the test was recorded as absorbance at 490 nm. Values are given as means ± standard deviations ( n = 3). *, P ≤ 0.05 (Student’s t test). (C) TEM micrographs of C. difficile wild-type 630Δ erm , cwp19 mutant ( cwp19 mut.), and complemented cwp19 mutant (comp.) strains incubated for 33 h in BHI medium. The percentage of damaged cells for each strain is indicated. (D and E) Toxin titers in the cytosol (D) and in the culture supernatant (E) of wild-type 630Δ erm (black bars), cwp19 mutant (gray bars), and complemented cwp19 mutant (white bars) strains grown in BHI medium. Toxins were quantified by ELISA. The signal from the test was recorded as absorbance at 450 nm. Values are given as means ± standard deviations ( n = 3). *, P ≤ 0.05 (Student’s t test). ND, not detectable.

    Article Snippet: Transconjugants were selected by subculturing on BHI agar containing Cfx, Ccs, and Tm and then plated on BHI agar containing Erm.

    Techniques: Mutagenesis, Activity Assay, Transmission Electron Microscopy, Incubation, Enzyme-linked Immunosorbent Assay

    Detection of E. faecalis mixed colonies by fluorescence imaging. Colonies were obtained by subculturing from a BHI-S broth culture of the gfp -tagged strain (SD234; OG1RF : : gfp ) grown with a non- gfp tagged strain (OG1SSp). Plates

    Journal: Microbiology

    Article Title: Enterococcus faecalis Ebp pili are important for cell-cell aggregation and intraspecies gene transfer

    doi: 10.1099/mic.0.000276

    Figure Lengend Snippet: Detection of E. faecalis mixed colonies by fluorescence imaging. Colonies were obtained by subculturing from a BHI-S broth culture of the gfp -tagged strain (SD234; OG1RF : : gfp ) grown with a non- gfp tagged strain (OG1SSp). Plates

    Article Snippet: Unless otherwise specified, E. faecalis strain OG1RF (Ebp+ ) ( ) and its derivative were cultivated in brain heart infusion (BHI) (Becton Dickinson) broth supplemented, when appropriate, with 25 mg fusidic acid l− 1 (Sigma-Aldrich).

    Techniques: Fluorescence, Imaging, Subculturing Assay

    Levels of PsaE and PsaF are impacted by temperature and pH. (A) Diagram of the psaEF locus showing the location of primers and the predicted PCR product used to analyze psaEF cotranscription via RT-PCR. Templates were as follows: RT+, cDNA; RT−, no reverse transcriptase (negative control); gDNA, YP6 gDNA; dH 2 O, no template (negative control). (B) WT containing a psaEF transcriptional reporter plasmid (pJC126, psaEF-gfp ) was grown at 37°C and 26°C in buffered BHI broth, and the RFU/OD 600 was determined under each condition, as described in Materials and Methods. The number depicted over each bar indicates fold change in psaEF-gfp expression in the WT compared to the expression from the vector control under the given condition. Each bar represents the mean RFU/OD 600 , and error bars represent standard deviations. Each sample was assayed in biological triplicates, and at least three independent experiments were performed. ***, P

    Journal: Journal of Bacteriology

    Article Title: Temperature Control of psaA Expression by PsaE and PsaF in Yersinia pestis

    doi: 10.1128/JB.00217-19

    Figure Lengend Snippet: Levels of PsaE and PsaF are impacted by temperature and pH. (A) Diagram of the psaEF locus showing the location of primers and the predicted PCR product used to analyze psaEF cotranscription via RT-PCR. Templates were as follows: RT+, cDNA; RT−, no reverse transcriptase (negative control); gDNA, YP6 gDNA; dH 2 O, no template (negative control). (B) WT containing a psaEF transcriptional reporter plasmid (pJC126, psaEF-gfp ) was grown at 37°C and 26°C in buffered BHI broth, and the RFU/OD 600 was determined under each condition, as described in Materials and Methods. The number depicted over each bar indicates fold change in psaEF-gfp expression in the WT compared to the expression from the vector control under the given condition. Each bar represents the mean RFU/OD 600 , and error bars represent standard deviations. Each sample was assayed in biological triplicates, and at least three independent experiments were performed. ***, P

    Article Snippet: Y. pestis CO92/pCD1− (YP6) was cultivated on brain heart infusion (BHI) agar (BD Biosciences, Bedford, MA) at 26°C for 48 h and in BHI broth cultures grown with aeration at 26°C or 37°C.

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control, Plasmid Preparation, Expressing

    Expression of psaA and enhanced PsaE stability correspond with PsaF. (A) The psaA-gfp reporter was introduced into derivatives of the Δ psaEF mutant expressing psaEF from the psaE native 5′ UTR (YPA260, native), the psaE 5G 5′ UTR (YPA361, 5G), and the psaE 5C 5′ UTR (YPA360, 5C); these strains were grown at 37°C and 26°C in pH 6.3 buffered BHI broth, and reporter expression was determined as described in Materials and Methods. Each bar represents the mean RFU/OD 600 , and error bars represent standard deviations. Each sample was assayed in biological triplicate. ***, P

    Journal: Journal of Bacteriology

    Article Title: Temperature Control of psaA Expression by PsaE and PsaF in Yersinia pestis

    doi: 10.1128/JB.00217-19

    Figure Lengend Snippet: Expression of psaA and enhanced PsaE stability correspond with PsaF. (A) The psaA-gfp reporter was introduced into derivatives of the Δ psaEF mutant expressing psaEF from the psaE native 5′ UTR (YPA260, native), the psaE 5G 5′ UTR (YPA361, 5G), and the psaE 5C 5′ UTR (YPA360, 5C); these strains were grown at 37°C and 26°C in pH 6.3 buffered BHI broth, and reporter expression was determined as described in Materials and Methods. Each bar represents the mean RFU/OD 600 , and error bars represent standard deviations. Each sample was assayed in biological triplicate. ***, P

    Article Snippet: Y. pestis CO92/pCD1− (YP6) was cultivated on brain heart infusion (BHI) agar (BD Biosciences, Bedford, MA) at 26°C for 48 h and in BHI broth cultures grown with aeration at 26°C or 37°C.

    Techniques: Expressing, Mutagenesis

    Expression of a psaA transcriptional reporter and production of PsaA require high temperature and low pH. WT Y. pestis (YP6) carrying a psaA transcriptional reporter (pEW102, psaA-gfp ) was grown at 26°C and 37°C, and psaA transcription (A, C, and D), medium pH (B), and PsaA (E) were analyzed as described in Materials and Methods. (A and B) psaA expression (A) and growth medium pH (B) were determined following growth at 26°C and 37°C in unbuffered BHI broth over time. (C) WT carrying psaA-gfp was grown at 37°C in BHI broth buffered to pH 6.3, 6.7, and 7.3, and reporter expression was determined. (D) WT carrying psaA-gfp was grown for 8 h at 37°C and 26°C in buffered BHI broth, and reporter expression was determined. Each bar represents the mean RFU/OD 600 , and error bars represent standard deviations. For reporter experiments, each sample was assayed in biological triplicates. ***, P

    Journal: Journal of Bacteriology

    Article Title: Temperature Control of psaA Expression by PsaE and PsaF in Yersinia pestis

    doi: 10.1128/JB.00217-19

    Figure Lengend Snippet: Expression of a psaA transcriptional reporter and production of PsaA require high temperature and low pH. WT Y. pestis (YP6) carrying a psaA transcriptional reporter (pEW102, psaA-gfp ) was grown at 26°C and 37°C, and psaA transcription (A, C, and D), medium pH (B), and PsaA (E) were analyzed as described in Materials and Methods. (A and B) psaA expression (A) and growth medium pH (B) were determined following growth at 26°C and 37°C in unbuffered BHI broth over time. (C) WT carrying psaA-gfp was grown at 37°C in BHI broth buffered to pH 6.3, 6.7, and 7.3, and reporter expression was determined. (D) WT carrying psaA-gfp was grown for 8 h at 37°C and 26°C in buffered BHI broth, and reporter expression was determined. Each bar represents the mean RFU/OD 600 , and error bars represent standard deviations. For reporter experiments, each sample was assayed in biological triplicates. ***, P

    Article Snippet: Y. pestis CO92/pCD1− (YP6) was cultivated on brain heart infusion (BHI) agar (BD Biosciences, Bedford, MA) at 26°C for 48 h and in BHI broth cultures grown with aeration at 26°C or 37°C.

    Techniques: Expressing

    psaF translation requires high temperature and is regulated independently of the psaE 5′ UTR. The psaEF coding sequence was cloned into a tet -inducible expression vector (pPsaEF). (A) The Δ psaEF mutant carrying pPsaEF was grown at 37°C and 26°C in buffered BHI broth in the presence or absence of 50 ng/ml ATc, and whole-cell lysates were prepared and used to analyze PsaE and PsaF via Western blotting, as indicated in Materials and Methods. P λ , phage lambda promoter. (B) Diagram of sequence used to construct the psaF translational reporter (pJQ043, psaF up - lacZ ); the psaEF promoter was ligated directly to sequences upstream of psaF . The fragments outlined in red were excluded from the reporter. (C) The psaF up - lacZ reporter was introduced at the Tn 7 site in the Y. pestis Δ lacZ mutant (YPA87), and this strain was grown at 37°C or 26°C in buffered BHI broth. β-Galactosidase activity was analyzed as indicated in Materials and Methods. Bar graphs represent mean values of Miller units, and error bars represent standard deviations. ***, P

    Journal: Journal of Bacteriology

    Article Title: Temperature Control of psaA Expression by PsaE and PsaF in Yersinia pestis

    doi: 10.1128/JB.00217-19

    Figure Lengend Snippet: psaF translation requires high temperature and is regulated independently of the psaE 5′ UTR. The psaEF coding sequence was cloned into a tet -inducible expression vector (pPsaEF). (A) The Δ psaEF mutant carrying pPsaEF was grown at 37°C and 26°C in buffered BHI broth in the presence or absence of 50 ng/ml ATc, and whole-cell lysates were prepared and used to analyze PsaE and PsaF via Western blotting, as indicated in Materials and Methods. P λ , phage lambda promoter. (B) Diagram of sequence used to construct the psaF translational reporter (pJQ043, psaF up - lacZ ); the psaEF promoter was ligated directly to sequences upstream of psaF . The fragments outlined in red were excluded from the reporter. (C) The psaF up - lacZ reporter was introduced at the Tn 7 site in the Y. pestis Δ lacZ mutant (YPA87), and this strain was grown at 37°C or 26°C in buffered BHI broth. β-Galactosidase activity was analyzed as indicated in Materials and Methods. Bar graphs represent mean values of Miller units, and error bars represent standard deviations. ***, P

    Article Snippet: Y. pestis CO92/pCD1− (YP6) was cultivated on brain heart infusion (BHI) agar (BD Biosciences, Bedford, MA) at 26°C for 48 h and in BHI broth cultures grown with aeration at 26°C or 37°C.

    Techniques: Sequencing, Clone Assay, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Construct, Activity Assay

    S. oralis biofilms growing for 24 or 48 h on polystyrene. Biofilms of wild-type (So34), gtfR mutant (So ∆gtfR) or complemented (So pgtfR) strains were grown in RPMI, 10%FBS, 10% BHI media supplemented with 1% sucrose or 1% glucose. a X – Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. S. oralis (blue) was visualized after fluorescence in situ hybridization with a Streptococcus -specific probe conjugated to Alexa 405. Alexa Fluor 647-labeled dextran conjugate probe (red) was used to stain biofilm matrix (glucans). Scale bars, 50 µm ( X – Y isosurfaces) and 70 µm (three-dimensional reconstructions). b Average total biovolumes (in µm 3 ) for 24 and 48 h biofilms exposed to glucose (white bars) or sucrose (black bars) shown in ( a ) above. Biovolumes were measured in two different confocal laser scanning microscopy image stacks from two independent experiments. c Average biofilm thickness (in µm) in biofilms growing with 1%sucrose for 24 and 48 h. d Average S. oralis colony-forming units (CFU) in logarithmic scale for each 24 and 48 h biofilm. e Average matrix (α-glucan) biovolumes (in µm 3 ) for 24 and 48 h biofilms. * p

    Journal: The ISME Journal

    Article Title: Role of glucosyltransferase R in biofilm interactions between Streptococcus oralis and Candida albicans

    doi: 10.1038/s41396-020-0608-4

    Figure Lengend Snippet: S. oralis biofilms growing for 24 or 48 h on polystyrene. Biofilms of wild-type (So34), gtfR mutant (So ∆gtfR) or complemented (So pgtfR) strains were grown in RPMI, 10%FBS, 10% BHI media supplemented with 1% sucrose or 1% glucose. a X – Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. S. oralis (blue) was visualized after fluorescence in situ hybridization with a Streptococcus -specific probe conjugated to Alexa 405. Alexa Fluor 647-labeled dextran conjugate probe (red) was used to stain biofilm matrix (glucans). Scale bars, 50 µm ( X – Y isosurfaces) and 70 µm (three-dimensional reconstructions). b Average total biovolumes (in µm 3 ) for 24 and 48 h biofilms exposed to glucose (white bars) or sucrose (black bars) shown in ( a ) above. Biovolumes were measured in two different confocal laser scanning microscopy image stacks from two independent experiments. c Average biofilm thickness (in µm) in biofilms growing with 1%sucrose for 24 and 48 h. d Average S. oralis colony-forming units (CFU) in logarithmic scale for each 24 and 48 h biofilm. e Average matrix (α-glucan) biovolumes (in µm 3 ) for 24 and 48 h biofilms. * p

    Article Snippet: S. oralis strains were reactivated from glycerol stocks by overnight growth in brain–heart infusion (BHI) medium (Becton, Dickinson and Company, Sparks, MD, USA) supplemented with antibiotics (Spectinomycin, 250 μg/ml, Erythromycin, 5 μg/ml) as needed, under static conditions at 37 °C, in a 5% CO2 incubator.

    Techniques: Mutagenesis, Confocal Laser Scanning Microscopy, Fluorescence, In Situ Hybridization, Labeling, Staining

    Twenty-four and forty-eight hour biofilms of C. albicans (Ca) alone or with wild-type S. oralis (So34), gtfR mutant (So ∆gtfR) or complemented (So pgtfR) strains. Biofilms were grown on polystyrene surfaces in RPMI, 10% FBS, 10% BHI media supplemented with 1% sucrose. a X – Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. Please note overlap of green and red signals shown in yellow, suggesting close physical proximity between the two organisms. Scale bars 50 μm. b Average total biofilm biovolume (in µm 3 ) for 24 and 48 h biofilms. c S. oralis CFU counts expressed as fold of mixed over single biofilms. d Average matrix biovolumes (in µm 3 ). e Relative expression of gtfR gene levels assessed by RT-qPCR. Results represent mean fold change gene expression in C. albicans with S. oralis (CaSo, black bars) over S. oralis (So, white bars) biofilms alone, in three independent experiments. f C. albicans CFU counts expressed as fold of mixed biofilms with each of the three S. oralis strains (CaSo) over single biofilms (Ca). * p

    Journal: The ISME Journal

    Article Title: Role of glucosyltransferase R in biofilm interactions between Streptococcus oralis and Candida albicans

    doi: 10.1038/s41396-020-0608-4

    Figure Lengend Snippet: Twenty-four and forty-eight hour biofilms of C. albicans (Ca) alone or with wild-type S. oralis (So34), gtfR mutant (So ∆gtfR) or complemented (So pgtfR) strains. Biofilms were grown on polystyrene surfaces in RPMI, 10% FBS, 10% BHI media supplemented with 1% sucrose. a X – Y isosurfaces (top panel) and three-dimensional reconstructions (bottom panel) of representative confocal laser scanning microscopy images of biofilms. Please note overlap of green and red signals shown in yellow, suggesting close physical proximity between the two organisms. Scale bars 50 μm. b Average total biofilm biovolume (in µm 3 ) for 24 and 48 h biofilms. c S. oralis CFU counts expressed as fold of mixed over single biofilms. d Average matrix biovolumes (in µm 3 ). e Relative expression of gtfR gene levels assessed by RT-qPCR. Results represent mean fold change gene expression in C. albicans with S. oralis (CaSo, black bars) over S. oralis (So, white bars) biofilms alone, in three independent experiments. f C. albicans CFU counts expressed as fold of mixed biofilms with each of the three S. oralis strains (CaSo) over single biofilms (Ca). * p

    Article Snippet: S. oralis strains were reactivated from glycerol stocks by overnight growth in brain–heart infusion (BHI) medium (Becton, Dickinson and Company, Sparks, MD, USA) supplemented with antibiotics (Spectinomycin, 250 μg/ml, Erythromycin, 5 μg/ml) as needed, under static conditions at 37 °C, in a 5% CO2 incubator.

    Techniques: Mutagenesis, Confocal Laser Scanning Microscopy, Expressing, Quantitative RT-PCR

    Maximum specific growth rates of Listeria monocytogenes LO28 WT and 24 HHP-resistant variants at 30°C in BHI under aerobic conditions (a); at 30°C in BHI under anaerobic conditions (b); or at 7°C in BHI under aerobic conditions

    Journal: Applied and Environmental Microbiology

    Article Title: Population Diversity of Listeria monocytogenes LO28: Phenotypic and Genotypic Characterization of Variants Resistant to High Hydrostatic Pressure ▿

    doi: 10.1128/AEM.02434-09

    Figure Lengend Snippet: Maximum specific growth rates of Listeria monocytogenes LO28 WT and 24 HHP-resistant variants at 30°C in BHI under aerobic conditions (a); at 30°C in BHI under anaerobic conditions (b); or at 7°C in BHI under aerobic conditions

    Article Snippet: Stock cultures of all strains were kept in 15% (vol/vol) glycerol (Fluka, Buchs, Switzerland) at −80°C, and before the experiments, cells from stock were grown for 2 days at 30°C on brain heart infusion (BHI) agar (Oxoid, Hampshire, England).

    Techniques:

    Responses or roles of aguA1 and aguA2 under acidic stresses. A , relative quantification of aguA1 and aguA2 mRNA levels in L. monocytogenes 10403S exposed to BHI medium at pH 5.0 and 7.0. Values are expressed as means ± S.D. B , survival of L. monocytogenes

    Journal: The Journal of Biological Chemistry

    Article Title: Listeria monocytogenes aguA1, but Not aguA2, Encodes a Functional Agmatine Deiminase

    doi: 10.1074/jbc.M113.477380

    Figure Lengend Snippet: Responses or roles of aguA1 and aguA2 under acidic stresses. A , relative quantification of aguA1 and aguA2 mRNA levels in L. monocytogenes 10403S exposed to BHI medium at pH 5.0 and 7.0. Values are expressed as means ± S.D. B , survival of L. monocytogenes

    Article Snippet: L. monocytogenes was cultured in brain-heart infusion (BHI) medium (Oxoid, Hampshire, England).

    Techniques:

    Stress resistance of late-exponential phase cells of L. monocytogenes LO28 WT and acid resistant variants in BHI broth . Late-exponential phase cells were exposed to pH 2.5 for 3.5 min at 37∘C (adapted from Metselaar et al., 2013 ) (A), 55∘C for 6 min (B), 420 mM hydrogen peroxide for 9 min at 30∘C (C), and 20 mg/L benzalkonium chloride (BAC) for 5 min at 30∘C (D) . Results are expressed as reduction in log 10 cfu/ml after exposure compared to log 10 cfu/ml at t = 0. Errors bars represent the SD and significant differences from the WT are indicated with ∗( p

    Journal: Frontiers in Microbiology

    Article Title: Diversity of acid stress resistant variants of Listeria monocytogenes and the potential role of ribosomal protein S21 encoded by rpsU

    doi: 10.3389/fmicb.2015.00422

    Figure Lengend Snippet: Stress resistance of late-exponential phase cells of L. monocytogenes LO28 WT and acid resistant variants in BHI broth . Late-exponential phase cells were exposed to pH 2.5 for 3.5 min at 37∘C (adapted from Metselaar et al., 2013 ) (A), 55∘C for 6 min (B), 420 mM hydrogen peroxide for 9 min at 30∘C (C), and 20 mg/L benzalkonium chloride (BAC) for 5 min at 30∘C (D) . Results are expressed as reduction in log 10 cfu/ml after exposure compared to log 10 cfu/ml at t = 0. Errors bars represent the SD and significant differences from the WT are indicated with ∗( p

    Article Snippet: The stock culture was kept in 15% (v/v) glycerol (Fluka, Buchs) at -80∘C, and before the experiments cells from stock were grown for 2 days at 30∘C on BHI agar (Oxoid, Hampshire).

    Techniques: BAC Assay

    Effect of the hfq mutation on the sensitivity of M. catarrhalis to methyl viologen. Bacterial cells of each strain were suspended to the same OD 600 and then serially diluted and spotted onto BHI agar (A) and BHI agar containing 40 μM methyl viologen (B). Four strains were tested: wild-type O35E strain (WT), the O35EΔ hfq mutant (Δ hfq ), the O35EΔ hfq mutant containing the pAA200 plasmid with the wild-type O35E hfq gene [Δ hfq (pAA200)], and this same mutant containing only the plasmid vector [Δ hfq (pWW115)]. The plates were dried and incubated overnight, and the resultant growth was photographed.

    Journal: Infection and Immunity

    Article Title: Moraxella catarrhalis Expresses an Unusual Hfq Protein

    doi: 10.1128/IAI.01652-07

    Figure Lengend Snippet: Effect of the hfq mutation on the sensitivity of M. catarrhalis to methyl viologen. Bacterial cells of each strain were suspended to the same OD 600 and then serially diluted and spotted onto BHI agar (A) and BHI agar containing 40 μM methyl viologen (B). Four strains were tested: wild-type O35E strain (WT), the O35EΔ hfq mutant (Δ hfq ), the O35EΔ hfq mutant containing the pAA200 plasmid with the wild-type O35E hfq gene [Δ hfq (pAA200)], and this same mutant containing only the plasmid vector [Δ hfq (pWW115)]. The plates were dried and incubated overnight, and the resultant growth was photographed.

    Article Snippet: A 10-μl aliquot of each dilution was spotted onto BHI agar with or without 40 μM methyl viologen (Sigma, St. Louis, MO).

    Techniques: Mutagenesis, Plasmid Preparation, Incubation

    Complementation of an E. coli hfq mutant with the M. catarrhalis Hfq protein. (A) Growth curves of wild-type, mutant, and recombinant E. coli strains. The wild-type strain MC4100 (⋄), the hfq mutant GS081 (▪), the hfq mutant with the M. catarrhalis hfq gene provided in trans [GS081(pAA105)] (▴), and the hfq mutant containing the negative control plasmid [GS081(pAA105B)] (○) were suspended in LB broth to an OD 600 of 1.0 and then diluted 1:20. These cultures were allowed to grow with aeration at 37°C, and the growth was monitored by measuring the absorbance at 600 nm every hour. (B and C) Cells of the wild-type, mutant, and recombinant E. coli strains were suspended to the same OD 600 and then serially diluted and spotted onto BHI agar (B) and onto BHI agar containing 40 μM methyl viologen (C). The plates were dried and incubated overnight at 37°C.

    Journal: Infection and Immunity

    Article Title: Moraxella catarrhalis Expresses an Unusual Hfq Protein

    doi: 10.1128/IAI.01652-07

    Figure Lengend Snippet: Complementation of an E. coli hfq mutant with the M. catarrhalis Hfq protein. (A) Growth curves of wild-type, mutant, and recombinant E. coli strains. The wild-type strain MC4100 (⋄), the hfq mutant GS081 (▪), the hfq mutant with the M. catarrhalis hfq gene provided in trans [GS081(pAA105)] (▴), and the hfq mutant containing the negative control plasmid [GS081(pAA105B)] (○) were suspended in LB broth to an OD 600 of 1.0 and then diluted 1:20. These cultures were allowed to grow with aeration at 37°C, and the growth was monitored by measuring the absorbance at 600 nm every hour. (B and C) Cells of the wild-type, mutant, and recombinant E. coli strains were suspended to the same OD 600 and then serially diluted and spotted onto BHI agar (B) and onto BHI agar containing 40 μM methyl viologen (C). The plates were dried and incubated overnight at 37°C.

    Article Snippet: A 10-μl aliquot of each dilution was spotted onto BHI agar with or without 40 μM methyl viologen (Sigma, St. Louis, MO).

    Techniques: Mutagenesis, Recombinant, Negative Control, Plasmid Preparation, Incubation