Structured Review

Difco brain heart infusion bhi medium
Inducible expression of LLO and PC-PLC in L. <t>monocytogenes.</t> (A) Hemolytic activity assay. Hemolytic activities were determined from culture supernatants of L. monocytogenes as described in Materials and Methods. LLO was expressed from the inducible SPAC/ lac Oid promoter in the presence of 1 mM IPTG or from the native PrfA- or PrfA*-regulated hly promoter in strains 10403S and SLCC-5764, respectively. (B) PC-PLC activity assay. PC-PLC was expressed under the control of the inducible SPAC/ lac Oid promoter (i- plcB ) or the native PrfA-regulated actA-plcB promoter in the L. monocytogenes 10403S (PrfA) and SLCC-5764 (PrfA*) backgrounds. Overnight cultures were diluted 1:10 in <t>BHI</t> medium and grown for 5 h at 37°C in the presence or absence of 1 mM IPTG. Culture supernatants were TCA precipitated, and an equivalent of 8 or 0.32 ml of culture supernatant was separated by SDS-PAGE. PC-PLC activities were detected as described in Materials and Methods. Lane 1, DH-L377 (SLCC-5764 Δ hly ); lane 2, DH-L419 (SLCC-5764 Δ hly Δ plcB ); lane 3, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 4, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; lane 5, DH-L727 (10403S Δ hly , pPL2); lane 6, DP-L726 (10403S Δ hly Δ plcB , pPL2); lane 7, DH-L718 (10403S Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 8, DH-L718 (10403S Δ hly Δ plcB i- plcB ) without IPTG. (C) Intracellular growth in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. Symbols: •, DH-L377 (SLCC-5764 Δ hly ); ○, DH-L419 (SLCC-5764 Δ hly Δ plcB ); ▵, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; ▴, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 10 mM IPTG. The data points in growth curves represent the means ± standard deviations of three coverslips from one of two experiments.
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1) Product Images from "Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells"

Article Title: Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.21.6295-6307.2003

Inducible expression of LLO and PC-PLC in L. monocytogenes. (A) Hemolytic activity assay. Hemolytic activities were determined from culture supernatants of L. monocytogenes as described in Materials and Methods. LLO was expressed from the inducible SPAC/ lac Oid promoter in the presence of 1 mM IPTG or from the native PrfA- or PrfA*-regulated hly promoter in strains 10403S and SLCC-5764, respectively. (B) PC-PLC activity assay. PC-PLC was expressed under the control of the inducible SPAC/ lac Oid promoter (i- plcB ) or the native PrfA-regulated actA-plcB promoter in the L. monocytogenes 10403S (PrfA) and SLCC-5764 (PrfA*) backgrounds. Overnight cultures were diluted 1:10 in BHI medium and grown for 5 h at 37°C in the presence or absence of 1 mM IPTG. Culture supernatants were TCA precipitated, and an equivalent of 8 or 0.32 ml of culture supernatant was separated by SDS-PAGE. PC-PLC activities were detected as described in Materials and Methods. Lane 1, DH-L377 (SLCC-5764 Δ hly ); lane 2, DH-L419 (SLCC-5764 Δ hly Δ plcB ); lane 3, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 4, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; lane 5, DH-L727 (10403S Δ hly , pPL2); lane 6, DP-L726 (10403S Δ hly Δ plcB , pPL2); lane 7, DH-L718 (10403S Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 8, DH-L718 (10403S Δ hly Δ plcB i- plcB ) without IPTG. (C) Intracellular growth in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. Symbols: •, DH-L377 (SLCC-5764 Δ hly ); ○, DH-L419 (SLCC-5764 Δ hly Δ plcB ); ▵, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; ▴, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 10 mM IPTG. The data points in growth curves represent the means ± standard deviations of three coverslips from one of two experiments.
Figure Legend Snippet: Inducible expression of LLO and PC-PLC in L. monocytogenes. (A) Hemolytic activity assay. Hemolytic activities were determined from culture supernatants of L. monocytogenes as described in Materials and Methods. LLO was expressed from the inducible SPAC/ lac Oid promoter in the presence of 1 mM IPTG or from the native PrfA- or PrfA*-regulated hly promoter in strains 10403S and SLCC-5764, respectively. (B) PC-PLC activity assay. PC-PLC was expressed under the control of the inducible SPAC/ lac Oid promoter (i- plcB ) or the native PrfA-regulated actA-plcB promoter in the L. monocytogenes 10403S (PrfA) and SLCC-5764 (PrfA*) backgrounds. Overnight cultures were diluted 1:10 in BHI medium and grown for 5 h at 37°C in the presence or absence of 1 mM IPTG. Culture supernatants were TCA precipitated, and an equivalent of 8 or 0.32 ml of culture supernatant was separated by SDS-PAGE. PC-PLC activities were detected as described in Materials and Methods. Lane 1, DH-L377 (SLCC-5764 Δ hly ); lane 2, DH-L419 (SLCC-5764 Δ hly Δ plcB ); lane 3, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 4, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; lane 5, DH-L727 (10403S Δ hly , pPL2); lane 6, DP-L726 (10403S Δ hly Δ plcB , pPL2); lane 7, DH-L718 (10403S Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 8, DH-L718 (10403S Δ hly Δ plcB i- plcB ) without IPTG. (C) Intracellular growth in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. Symbols: •, DH-L377 (SLCC-5764 Δ hly ); ○, DH-L419 (SLCC-5764 Δ hly Δ plcB ); ▵, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; ▴, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 10 mM IPTG. The data points in growth curves represent the means ± standard deviations of three coverslips from one of two experiments.

Techniques Used: Expressing, Planar Chromatography, Activity Assay, SDS Page, Infection

Multicopy inducible PC-PLC expression system for L. monocytogenes. (A) Schematic representation of the inducible PC-PLC expression vector pAMi plcB. plcB was cloned into plasmid pAM401 under SPAC/ lac Oid promoter/operator control, together with lacI under constitutive SPO-1 promoter control. (B) PC-PLC activity assays of SLCC-5764 (PrfA*) and 10403S (PrfA) derived strains. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium with or without IPTG at the indicated concentrations and grown for 5 h at 37°C. Culture supernatants were TCA precipitated, an equivalent of 0.2 or 2 ml of culture supernatant was separated by SDS-PAGE, and PC-PLC activities were detected by egg yolk overlay assays. In the upper panel are SLCC-5764 (PrfA*) strain derivatives. Lane 1, DH-L683 (SLCC-5764 Δ hly , pAMspacOid); lane 2, DH-L687 (SLCC-5764 Δ hly Δ plcB , pAMspacOid); lanes 3 to 10, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ; inducible plcB ) grown in the presence of increasing concentrations of IPTG as indicated above the figure. In the lower panel are 10403S (PrfA) strain derivatives. Lane 1, DH-L728 (10403S Δ hly , pAMspacOid); lane 2, DH-L729 (10403S Δ hly Δ plcB , pAMspacOid); lanes 3 to 10, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ; inducible plcB ) grown in the presence of increasing concentrations of IPTG as indicated above the figure.
Figure Legend Snippet: Multicopy inducible PC-PLC expression system for L. monocytogenes. (A) Schematic representation of the inducible PC-PLC expression vector pAMi plcB. plcB was cloned into plasmid pAM401 under SPAC/ lac Oid promoter/operator control, together with lacI under constitutive SPO-1 promoter control. (B) PC-PLC activity assays of SLCC-5764 (PrfA*) and 10403S (PrfA) derived strains. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium with or without IPTG at the indicated concentrations and grown for 5 h at 37°C. Culture supernatants were TCA precipitated, an equivalent of 0.2 or 2 ml of culture supernatant was separated by SDS-PAGE, and PC-PLC activities were detected by egg yolk overlay assays. In the upper panel are SLCC-5764 (PrfA*) strain derivatives. Lane 1, DH-L683 (SLCC-5764 Δ hly , pAMspacOid); lane 2, DH-L687 (SLCC-5764 Δ hly Δ plcB , pAMspacOid); lanes 3 to 10, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ; inducible plcB ) grown in the presence of increasing concentrations of IPTG as indicated above the figure. In the lower panel are 10403S (PrfA) strain derivatives. Lane 1, DH-L728 (10403S Δ hly , pAMspacOid); lane 2, DH-L729 (10403S Δ hly Δ plcB , pAMspacOid); lanes 3 to 10, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ; inducible plcB ) grown in the presence of increasing concentrations of IPTG as indicated above the figure.

Techniques Used: Planar Chromatography, Expressing, Plasmid Preparation, Clone Assay, Activity Assay, Derivative Assay, SDS Page

Intracellular growth of multicopy inducible PC-PLC L. monocytogenes strains in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. (A) SLCC-5764 (PrfA*) strain derivatives. Symbols: •, DH-L683 (SLCC-5764 Δ hly , pAMspacOid); ○, DH-L687 (SLCC-5764 Δ hly Δ plcB , pAMspacOid); ▵, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ) without IPTG; ▴, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ) with 10 mM IPTG. (B) 10403S (PrfA) strain derivatives. Symbols: •, DH-L728 (10403S Δ hly , pAMspacOid); ○, DH-L729 (10403S Δ hly Δ plcB , pAMspacOid); ▵, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ) without IPTG; ▴, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ) with 10 mM IPTG. The data points in the growth curves in panels A and B represent the means ± standard deviations of three coverslips from one of three experiments and from one experiment, respectively.
Figure Legend Snippet: Intracellular growth of multicopy inducible PC-PLC L. monocytogenes strains in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. (A) SLCC-5764 (PrfA*) strain derivatives. Symbols: •, DH-L683 (SLCC-5764 Δ hly , pAMspacOid); ○, DH-L687 (SLCC-5764 Δ hly Δ plcB , pAMspacOid); ▵, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ) without IPTG; ▴, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ) with 10 mM IPTG. (B) 10403S (PrfA) strain derivatives. Symbols: •, DH-L728 (10403S Δ hly , pAMspacOid); ○, DH-L729 (10403S Δ hly Δ plcB , pAMspacOid); ▵, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ) without IPTG; ▴, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ) with 10 mM IPTG. The data points in the growth curves in panels A and B represent the means ± standard deviations of three coverslips from one of three experiments and from one experiment, respectively.

Techniques Used: Planar Chromatography, Infection

2) Product Images from "Pathogen Proliferation Governs the Magnitude but Compromises the Function of CD8 T Cells"

Article Title: Pathogen Proliferation Governs the Magnitude but Compromises the Function of CD8 T Cells

Journal:

doi:

Mutants of ST are attenuated in vivo. C57BL/6J, 129X1SvJ, or B6.129F 1 mice were infected with 10 3 ST-OVA i.v. At various time intervals, spleens were removed and the bacterial burden evaluated by plating serial dilutions on BHI plates ( A ); †,
Figure Legend Snippet: Mutants of ST are attenuated in vivo. C57BL/6J, 129X1SvJ, or B6.129F 1 mice were infected with 10 3 ST-OVA i.v. At various time intervals, spleens were removed and the bacterial burden evaluated by plating serial dilutions on BHI plates ( A ); †,

Techniques Used: In Vivo, Mouse Assay, Infection

3) Product Images from "Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes ▿"

Article Title: Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes ▿

Journal:

doi: 10.1128/JB.00972-06

Western blot analysis of PrfA and PrfA-regulated virulence proteins LLO and ActA. WT EGD-e (A and B), insertion mutants (:: ccpA , :: hprK , and :: ptsH ) and strain P14-A (A), and revertants (R ccpA , R hprK , and R ptsH ) (B) were grown in BHI medium. At an OD
Figure Legend Snippet: Western blot analysis of PrfA and PrfA-regulated virulence proteins LLO and ActA. WT EGD-e (A and B), insertion mutants (:: ccpA , :: hprK , and :: ptsH ) and strain P14-A (A), and revertants (R ccpA , R hprK , and R ptsH ) (B) were grown in BHI medium. At an OD

Techniques Used: Western Blot

Growth of L. monocytogenes EGD-e wild-type, ccpA , hprK , and ptsH insertion mutants. The generation times of WT and insertion mutants are shown to the right. (A) The strains were grown in BHI medium. (B) Strains were shifted from BHI medium to MM with
Figure Legend Snippet: Growth of L. monocytogenes EGD-e wild-type, ccpA , hprK , and ptsH insertion mutants. The generation times of WT and insertion mutants are shown to the right. (A) The strains were grown in BHI medium. (B) Strains were shifted from BHI medium to MM with

Techniques Used:

4) Product Images from "Characterization of the pathogenesis and immune response to Listeria monocytogenes strains isolated from a sustained national outbreak"

Article Title: Characterization of the pathogenesis and immune response to Listeria monocytogenes strains isolated from a sustained national outbreak

Journal: Scientific Reports

doi: 10.1038/s41598-019-56028-3

In vitro characterization of L. monocytogenes strains. ( a ) Growth of L. monocytogenes strains in BHI broth. The indicated strains were grown at 37 °C for 25 hours in BHI broth and the number of bacteria was determined at the indicated times. Data represent the mean ± SD colony forming units (CFU) per milliliter for three experiments performed in duplicate with similar results. ( b–d ) Intracellular infections in HeLa cells. ( b ) HeLa cells were infected with the indicated L. monocytogenes strains for 1 hour prior to intracellular bacteria being quantified by gentamicin protection assay at 2 hours post-infection. Data represent the mean ± SD CFU per well for one of three experiments performed in triplicate with similar results. * P
Figure Legend Snippet: In vitro characterization of L. monocytogenes strains. ( a ) Growth of L. monocytogenes strains in BHI broth. The indicated strains were grown at 37 °C for 25 hours in BHI broth and the number of bacteria was determined at the indicated times. Data represent the mean ± SD colony forming units (CFU) per milliliter for three experiments performed in duplicate with similar results. ( b–d ) Intracellular infections in HeLa cells. ( b ) HeLa cells were infected with the indicated L. monocytogenes strains for 1 hour prior to intracellular bacteria being quantified by gentamicin protection assay at 2 hours post-infection. Data represent the mean ± SD CFU per well for one of three experiments performed in triplicate with similar results. * P

Techniques Used: In Vitro, Infection

5) Product Images from "In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection"

Article Title: In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000449

Macroarray validations. (A) Analysis of the impact of the in vitro culture conditions used as reference. The expression of known and potential virulence factors was analyzed in BHI at 37°C in exponential (BHI log) or stationary (BHI stat) growth phase, or in minimal medium in exponential growth phase (MM log) by real-time RT-PCR, and normalized to expression in mouse spleen. (B) Validation of macroarray data by real-time RT-PCR. Fold changes in in vivo gene expression 48 h p.i. compared to that in BHI were measured by macroarray and real-time RT-PCR, log transformed and compared for correlation analysis. (C) Analysis of the effect of the RNA extraction method on L. monocytogenes gene expression. RNAs from bacteria grown in BHI were prepared using the standard and adapted procedures for RNA extraction. The relative expression of potential virulence genes, cold shock genes and known virulence genes was determined by real-time RT-PCR.
Figure Legend Snippet: Macroarray validations. (A) Analysis of the impact of the in vitro culture conditions used as reference. The expression of known and potential virulence factors was analyzed in BHI at 37°C in exponential (BHI log) or stationary (BHI stat) growth phase, or in minimal medium in exponential growth phase (MM log) by real-time RT-PCR, and normalized to expression in mouse spleen. (B) Validation of macroarray data by real-time RT-PCR. Fold changes in in vivo gene expression 48 h p.i. compared to that in BHI were measured by macroarray and real-time RT-PCR, log transformed and compared for correlation analysis. (C) Analysis of the effect of the RNA extraction method on L. monocytogenes gene expression. RNAs from bacteria grown in BHI were prepared using the standard and adapted procedures for RNA extraction. The relative expression of potential virulence genes, cold shock genes and known virulence genes was determined by real-time RT-PCR.

Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, In Vivo, Transformation Assay, RNA Extraction

In vitro behavior of L. monocytogenes mutants. (A) Growth curves of L. monocytogenes EGDe strains in BHI at 37°C with shaking. (B) Intracellular behavior of L. monocytogenes EGDe strains in J774 cultured cells.
Figure Legend Snippet: In vitro behavior of L. monocytogenes mutants. (A) Growth curves of L. monocytogenes EGDe strains in BHI at 37°C with shaking. (B) Intracellular behavior of L. monocytogenes EGDe strains in J774 cultured cells.

Techniques Used: In Vitro, Cell Culture

6) Product Images from "Indirect Pathogenicity of Haemophilus influenzae and Moraxella catarrhalis in Polymicrobial Otitis Media Occurs via Interspecies Quorum Signaling"

Article Title: Indirect Pathogenicity of Haemophilus influenzae and Moraxella catarrhalis in Polymicrobial Otitis Media Occurs via Interspecies Quorum Signaling

Journal: mBio

doi: 10.1128/mBio.00102-10

Beta-lactam protection in a polymicrobial biofilm. Stationary biofilms of H. influenzae Rd and/or M. catarrhalis were established on chamber slides for 24 h and treated with 100 µg/ml ampicillin or ampicillin with 25 µg/ml clavulanate for an additional 24 h. Biofilms were resuspended in sterile PBS, serially diluted, and plated on sBHI medium plus clarithromycin and BHI medium plates for enumeration of viable H. influenzae Rd and M. catarrhalis bacteria, respectively. Data are represented as means ± SEM. *, P
Figure Legend Snippet: Beta-lactam protection in a polymicrobial biofilm. Stationary biofilms of H. influenzae Rd and/or M. catarrhalis were established on chamber slides for 24 h and treated with 100 µg/ml ampicillin or ampicillin with 25 µg/ml clavulanate for an additional 24 h. Biofilms were resuspended in sterile PBS, serially diluted, and plated on sBHI medium plus clarithromycin and BHI medium plates for enumeration of viable H. influenzae Rd and M. catarrhalis bacteria, respectively. Data are represented as means ± SEM. *, P

Techniques Used:

Polymicrobial infection augments M. catarrhalis persistence in vivo . Chinchillas were infected with 10 3 CFU of H. influenzae or H. influenzae luxS , 10 4 CFU of M. catarrhalis , or a mixture of both species. (A) Middle ear effusion fluids were removed for enumeration of viable H. influenzae and M. catarrhalis bacteria by plating on sBHI medium plus clarithromycin or BHI medium, respectively. (B) Bullae were removed at each time point and homogenized for enumeration of viable H. influenzae and M. catarrhalis bacteria, as described above. Data represent the mean results from four experiments ± SEM. ***, P
Figure Legend Snippet: Polymicrobial infection augments M. catarrhalis persistence in vivo . Chinchillas were infected with 10 3 CFU of H. influenzae or H. influenzae luxS , 10 4 CFU of M. catarrhalis , or a mixture of both species. (A) Middle ear effusion fluids were removed for enumeration of viable H. influenzae and M. catarrhalis bacteria by plating on sBHI medium plus clarithromycin or BHI medium, respectively. (B) Bullae were removed at each time point and homogenized for enumeration of viable H. influenzae and M. catarrhalis bacteria, as described above. Data represent the mean results from four experiments ± SEM. ***, P

Techniques Used: Infection, In Vivo

AI-2 promotes M. catarrhalis biofilm development and antibiotic resistance. (A) M. catarrhalis was cultured in BHI medium or BHI medium supplemented with 0.2 µM synthetic AI-2 (DPD) to determine AI-2 production and depletion, as measured by Vibrio harveyi bioluminescence. H. influenzae luxS was cultured in sBHI medium supplemented with DPD to measure depletion. An uninoculated control of BHI medium with DPD shows the minimal degradation of the AI-2 signal during 6 h of incubation at 37°C. (B) Depletion of DPD by M. catarrhalis biofilms were established for 24 h following incubation with 10 µg/ml tetracycline was measured by bioluminescence over a period of 7 h. (C) M. catarrhalis biofilms were established in the presence or absense of DPD and stained with crystal violet for determination of biofilm biomass at 4, 6, 12, 24, and 48 h. Data represent the mean results from three combined experiments, with three replicate wells per experiment. Error bars represent SEM. (D and E) M. catarrhalis biofilms were established for 24 h in the presence (E) or absence (D) of DPD and stained with a viability kit for CLSM visualization of surface coverage and biofilm thickness. (F and G) Z-series images from panels D and E were compressed to show total viable and nonviable staining of biofilms established in the presence (G) or absence (F) of DPD. (H and I) SEM images of 24-h M. catarrhalis biofilms established with (I) or without (H) DPD. (J) M. catarrhalis biofilms were established for 4 h in the presence or absence of DPD and then treated with 6 µg/ml clarithromycin for 24 h and plated for enumeration of viable bacteria. Data represent the means from three replicates ± SEM. *, P
Figure Legend Snippet: AI-2 promotes M. catarrhalis biofilm development and antibiotic resistance. (A) M. catarrhalis was cultured in BHI medium or BHI medium supplemented with 0.2 µM synthetic AI-2 (DPD) to determine AI-2 production and depletion, as measured by Vibrio harveyi bioluminescence. H. influenzae luxS was cultured in sBHI medium supplemented with DPD to measure depletion. An uninoculated control of BHI medium with DPD shows the minimal degradation of the AI-2 signal during 6 h of incubation at 37°C. (B) Depletion of DPD by M. catarrhalis biofilms were established for 24 h following incubation with 10 µg/ml tetracycline was measured by bioluminescence over a period of 7 h. (C) M. catarrhalis biofilms were established in the presence or absense of DPD and stained with crystal violet for determination of biofilm biomass at 4, 6, 12, 24, and 48 h. Data represent the mean results from three combined experiments, with three replicate wells per experiment. Error bars represent SEM. (D and E) M. catarrhalis biofilms were established for 24 h in the presence (E) or absence (D) of DPD and stained with a viability kit for CLSM visualization of surface coverage and biofilm thickness. (F and G) Z-series images from panels D and E were compressed to show total viable and nonviable staining of biofilms established in the presence (G) or absence (F) of DPD. (H and I) SEM images of 24-h M. catarrhalis biofilms established with (I) or without (H) DPD. (J) M. catarrhalis biofilms were established for 4 h in the presence or absence of DPD and then treated with 6 µg/ml clarithromycin for 24 h and plated for enumeration of viable bacteria. Data represent the means from three replicates ± SEM. *, P

Techniques Used: Cell Culture, Incubation, Staining, Confocal Laser Scanning Microscopy

Polymicrobial biofilm formation protects H. influenzae and M. catarrhalis from antibiotic treatment. Single-species or polymicrobial stationary biofilms were established for 4 h and treated with 60 µg/ml trimethoprim-sulfamethoxazole (TS) (A) or 6 µg/ml clarithromycin (B) for 24 h. Biofilms were resuspended in sterile PBS, serially diluted, and plated on sBHI medium plus clarithromycin or BHI medium for enumeration of viable H. influenzae and M. catarrhalis bacteria, respectively. Data are represented as the mean results from three combined experiments, with two replicates per experiment. Error bars represent SEM. *, P
Figure Legend Snippet: Polymicrobial biofilm formation protects H. influenzae and M. catarrhalis from antibiotic treatment. Single-species or polymicrobial stationary biofilms were established for 4 h and treated with 60 µg/ml trimethoprim-sulfamethoxazole (TS) (A) or 6 µg/ml clarithromycin (B) for 24 h. Biofilms were resuspended in sterile PBS, serially diluted, and plated on sBHI medium plus clarithromycin or BHI medium for enumeration of viable H. influenzae and M. catarrhalis bacteria, respectively. Data are represented as the mean results from three combined experiments, with two replicates per experiment. Error bars represent SEM. *, P

Techniques Used:

7) Product Images from "Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans"

Article Title: Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans

Journal: Microbiology

doi: 10.1099/mic.0.072884-0

TEM analysis. Strep. mutans wild-type, UA159, BrpB-deficient mutant, JB409, BrpB-complemented strain, JB409C, BrpB- and BrpA-deficient double mutant, JB819, and BrpB- and BrpA-complemented strain, JB819C, were grown in BHI, pH 7.4, until mid-exponential
Figure Legend Snippet: TEM analysis. Strep. mutans wild-type, UA159, BrpB-deficient mutant, JB409, BrpB-complemented strain, JB409C, BrpB- and BrpA-deficient double mutant, JB819, and BrpB- and BrpA-complemented strain, JB819C, were grown in BHI, pH 7.4, until mid-exponential

Techniques Used: Transmission Electron Microscopy, Mutagenesis

8) Product Images from "Diaminopimelic Acid Amidation in Corynebacteriales"

Article Title: Diaminopimelic Acid Amidation in Corynebacteriales

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.642843

Growth curves of the wild-type (○) and Δ ltsA (●) C. glutamicum strains in BHI medium at 30 °C.
Figure Legend Snippet: Growth curves of the wild-type (○) and Δ ltsA (●) C. glutamicum strains in BHI medium at 30 °C.

Techniques Used:

9) Product Images from "Functional Analysis of ycfR and ycfQ in Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce ▿ O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce ▿ †"

Article Title: Functional Analysis of ycfR and ycfQ in Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce ▿ O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce ▿ †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02420-10

Chlorine resistance of Sakai, TW14359, and EDL933 wild-type strains and their ycfR mutants. (A) Growth curves derived from the OD 600 recorded on the Bioscreen C automatic microbiology growth curve analysis system for 24 h at 37°C. (a) Wild-type Sakai and polar SK- ycfR :: cat grown in BHI; (b) wild-type Sakai grown in BHI containing 0.59 ppm free chlorine; (c) polar SK- ycfR :: cat grown in BHI containing 0.59 ppm free chlorine; (d) wild-type Sakai grown in BHI containing 0.81 ppm free chlorine; (e) polar SK- ycfR :: cat grown in BHI containing 0.81 ppm free chlorine; (f) wild-type Sakai grown in BHI containing 0.94 ppm free chlorine. Four replicates are shown in different colors for each of the above conditions. (B) Chlorine resistance of Sakai, TW14359, and EDL933 wild-type strains, their polar ycfR :: cat mutants, and mutants complemented with pKD11-1, as assessed by the extension of the lag phase during growth in BHI with chlorine compared with in BHI alone. (C) Chlorine resistance of Sakai, TW14359, and EDL933 wild-type strains and their nonpolar ycfR :: cat mutants. (D) Chlorine resistance of Sakai nonpolar ycfR :: cat mutants containing vector pACYC177 and the mutant with complementing plasmid pKD11-1. The free chlorine concentration in the experiments in panels B to D was 0.81 ppm in BHI. Error bars in this figure represent the standard deviations of the average extensions of lag phase from three independent experiments.
Figure Legend Snippet: Chlorine resistance of Sakai, TW14359, and EDL933 wild-type strains and their ycfR mutants. (A) Growth curves derived from the OD 600 recorded on the Bioscreen C automatic microbiology growth curve analysis system for 24 h at 37°C. (a) Wild-type Sakai and polar SK- ycfR :: cat grown in BHI; (b) wild-type Sakai grown in BHI containing 0.59 ppm free chlorine; (c) polar SK- ycfR :: cat grown in BHI containing 0.59 ppm free chlorine; (d) wild-type Sakai grown in BHI containing 0.81 ppm free chlorine; (e) polar SK- ycfR :: cat grown in BHI containing 0.81 ppm free chlorine; (f) wild-type Sakai grown in BHI containing 0.94 ppm free chlorine. Four replicates are shown in different colors for each of the above conditions. (B) Chlorine resistance of Sakai, TW14359, and EDL933 wild-type strains, their polar ycfR :: cat mutants, and mutants complemented with pKD11-1, as assessed by the extension of the lag phase during growth in BHI with chlorine compared with in BHI alone. (C) Chlorine resistance of Sakai, TW14359, and EDL933 wild-type strains and their nonpolar ycfR :: cat mutants. (D) Chlorine resistance of Sakai nonpolar ycfR :: cat mutants containing vector pACYC177 and the mutant with complementing plasmid pKD11-1. The free chlorine concentration in the experiments in panels B to D was 0.81 ppm in BHI. Error bars in this figure represent the standard deviations of the average extensions of lag phase from three independent experiments.

Techniques Used: Derivative Assay, Plasmid Preparation, Mutagenesis, Concentration Assay

Related Articles

Clone Assay:

Article Title: Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes ▿
Article Snippet: The Escherichia coli strain XL2-Blue was used for cloning and construction of the mutagenesis vectors. .. L. monocytogenes EGD-e strains and L. monocytogenes P14-A ( ) were grown under aerobic conditions in brain heart infusion (BHI) medium (Difco), in Luria-Bertani medium (LB), or in chemically defined minimal medium (MM) ( ) supplemented with different sugars for L. monocytogenes at 37°C or 42°C with antibiotics if required.

Selection:

Article Title: Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells
Article Snippet: L. monocytogenes strains were grown in brain heart infusion (BHI) medium (Difco, Detroit, Mich.). .. Antibiotics were used at the following concentrations: ampicillin at 100 μg/ml; chloramphenicol at 20 μg/ml for selection of pAM401 derivatives and pPL2 derivatives in E. coli , at 10 μg/ml for selection of pAM401 derivatives in L. monocytogenes , and at 7.5 μg/ml for selection of integrated pPL2 derivatives in L. monocytogenes ; kanamycin at 30 μg/ml; streptomycin at 200 μg/ml; and nalidixic acid at 40 μg/ml.

Article Title: Vibrio fischeri DarR Directs Responses to d-Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators
Article Snippet: .. For selection of E. coli with erythromycin (Erm), 150 μg · ml−1 was added to brain heart infusion (BHI) medium (Difco, Sparks, MD). ..

Mutagenesis:

Article Title: Functional Analysis of ycfR and ycfQ in Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce ▿ O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce ▿ †
Article Snippet: E. coli K-12 strains BW25113 wild type and BW25113 ΔycfR mutant were kindly provided by Thomas Wood at Texas A & M University ( ). .. E. coli O157:H7 strains were grown at 37°C in brain heart infusion (BHI) medium (Difco, Detroit, MI).

Article Title: Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes ▿
Article Snippet: The Escherichia coli strain XL2-Blue was used for cloning and construction of the mutagenesis vectors. .. L. monocytogenes EGD-e strains and L. monocytogenes P14-A ( ) were grown under aerobic conditions in brain heart infusion (BHI) medium (Difco), in Luria-Bertani medium (LB), or in chemically defined minimal medium (MM) ( ) supplemented with different sugars for L. monocytogenes at 37°C or 42°C with antibiotics if required.

Article Title: Characterization of the pathogenesis and immune response to Listeria monocytogenes strains isolated from a sustained national outbreak
Article Snippet: L. monocytogenes strains were grown in Brain Heart Infusion (BHI) medium (Difco). .. To generate an isogenic hly deletion mutant of L. monocytogenes LS741, the hly gene was deleted from the genome of L. monocytogenes LS741.

Isolation:

Article Title: Identification through MALDI-TOF mass spectrometry and antimicrobial susceptibility profiling of bacterial pathogens isolated from sow urinary tract infection
Article Snippet: Paragraph title: Bacterial isolation ... Each colony of interest was maintained at −86 °C in brain-heart infusion (BHI) medium (Difco, Sparks, MD, USA) with 30% of glycerol, supplemented with fetal calf serum (5%) when necessary for fastidious pathogens, for further analysis.

Construct:

Article Title: Indirect Pathogenicity of Haemophilus influenzae and Moraxella catarrhalis in Polymicrobial Otitis Media Occurs via Interspecies Quorum Signaling
Article Snippet: M. catarrhalis strains were cultivated in brain heart infusion (BHI) medium (Difco), and H. influenzae strains were cultivated in BHI medium supplemented with hemin (ICN Biochemicals) and NAD (Sigma); this medium is referred to herein as supplemented BHI (sBHI). .. H. influenzae siaB was constructed essentially as described previously for strain 2019 siaB ( ) and confirmed by immunoblotting to have decreased reactivity with Limax flavus (LFA) lectin (EY Laboratories).

Concentration Assay:

Article Title: Mu-driven transposition of recombinant mini-Mu unit DNA in the Corynebacterium glutamicum chromosome
Article Snippet: The Corynebacterium glutamicum strains were grown at 32 °С on Brain Heart Infusion (BHI) medium (Difco, USA). .. When needed, the corresponding antibiotics were added at the following final concentration: 200 μg/mL of ampicillin (Ap), 20 μg/mL of Tc, 50 μg/mL of Sm, 10 μg/mL of Gm, and 40 μg/mL of Km.

Article Title: Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans
Article Snippet: Strep. mutans strains were maintained in brain heart infusion (BHI) medium (Difco Laboratories). .. Solid media were prepared similarly, but Bacto agar (Difco Laboratories) was added at a concentration of 1.5 % (w/v).

Article Title: Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes ▿
Article Snippet: L. monocytogenes EGD-e strains and L. monocytogenes P14-A ( ) were grown under aerobic conditions in brain heart infusion (BHI) medium (Difco), in Luria-Bertani medium (LB), or in chemically defined minimal medium (MM) ( ) supplemented with different sugars for L. monocytogenes at 37°C or 42°C with antibiotics if required. .. Erythromycin was used at a concentration of 5 μg/ml for L. monocytogenes and at 300 μg/ml for E. coli .

Article Title: The Madeira Archipelago As a Significant Source of Marine-Derived Actinomycete Diversity with Anticancer and Antimicrobial Potential
Article Snippet: Crude extracts antimicrobial screening The antimicrobial activity was evaluated for the 400 crude extracts by performing screenings against two of the most important antibiotic-resistant microorganisms that cause nosocomial infections, specifically methicillin-resistant Staphylococcus aureus COL (MRSA; Gill et al., ) and vancomycin-resistant (vanA phenotype) Enterococcus faecium EF82 (VREF; Mato et al., ), using Brain Heart Infusion (BHI) medium (DIFCO Laboratories, Detroit, USA, 1 l DI water). .. The screenings were performed in 96 well plates; each crude extract, previously concentrated at 10 mg ml−1 in DMSO, was added to a log-phase grown culture (OD600 nm = 0.04−0.06) to a 2.5% (v/v) final concentration.

Article Title: Vibrio fischeri DarR Directs Responses to d-Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators
Article Snippet: For selection of E. coli with erythromycin (Erm), 150 μg · ml−1 was added to brain heart infusion (BHI) medium (Difco, Sparks, MD). .. When added for the selection of A. baylyi , Kan was added at a concentration of 25 μg · ml−1 .

Incubation:

Article Title: The Madeira Archipelago As a Significant Source of Marine-Derived Actinomycete Diversity with Anticancer and Antimicrobial Potential
Article Snippet: Crude extracts antimicrobial screening The antimicrobial activity was evaluated for the 400 crude extracts by performing screenings against two of the most important antibiotic-resistant microorganisms that cause nosocomial infections, specifically methicillin-resistant Staphylococcus aureus COL (MRSA; Gill et al., ) and vancomycin-resistant (vanA phenotype) Enterococcus faecium EF82 (VREF; Mato et al., ), using Brain Heart Infusion (BHI) medium (DIFCO Laboratories, Detroit, USA, 1 l DI water). .. After 18 h of incubation at 37°C, minimal inhibitory concentrations were determined by visual inspection and spectrophotometric analysis.

Article Title: Vibrio fischeri DarR Directs Responses to d-Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators
Article Snippet: For selection of E. coli with erythromycin (Erm), 150 μg · ml−1 was added to brain heart infusion (BHI) medium (Difco, Sparks, MD). .. Unless otherwise noted, E. coli and A. baylyi were incubated at 37°C.

Article Title: Identification through MALDI-TOF mass spectrometry and antimicrobial susceptibility profiling of bacterial pathogens isolated from sow urinary tract infection
Article Snippet: The agar plates were incubated under aerobic and microaerophilic conditions for 24–48 h at 37 °C. .. Each colony of interest was maintained at −86 °C in brain-heart infusion (BHI) medium (Difco, Sparks, MD, USA) with 30% of glycerol, supplemented with fetal calf serum (5%) when necessary for fastidious pathogens, for further analysis.

Cell Culture:

Article Title: Indirect Pathogenicity of Haemophilus influenzae and Moraxella catarrhalis in Polymicrobial Otitis Media Occurs via Interspecies Quorum Signaling
Article Snippet: M. catarrhalis strains were cultivated in brain heart infusion (BHI) medium (Difco), and H. influenzae strains were cultivated in BHI medium supplemented with hemin (ICN Biochemicals) and NAD (Sigma); this medium is referred to herein as supplemented BHI (sBHI). .. For experiments using trimethoprim-sulfamethoxazole, H. influenzae and M. catarrhalis were cultured in Morse’s defined medium ( ) supplemented with hemin and NAD.

Article Title: Diaminopimelic Acid Amidation in Corynebacteriales
Article Snippet: .. C. glutamicum strain RES167, a restriction-less derivative of ATCC 13032 , and its Δ ltsA derivative were cultured in brain-heart infusion (BHI) medium (Difco) at 30 °C. .. E. coli cells were grown in 2YT medium ( ) or Luria Bertani (LB) medium (Difco) at 37 °C.

Activity Assay:

Article Title: The Madeira Archipelago As a Significant Source of Marine-Derived Actinomycete Diversity with Anticancer and Antimicrobial Potential
Article Snippet: .. Crude extracts antimicrobial screening The antimicrobial activity was evaluated for the 400 crude extracts by performing screenings against two of the most important antibiotic-resistant microorganisms that cause nosocomial infections, specifically methicillin-resistant Staphylococcus aureus COL (MRSA; Gill et al., ) and vancomycin-resistant (vanA phenotype) Enterococcus faecium EF82 (VREF; Mato et al., ), using Brain Heart Infusion (BHI) medium (DIFCO Laboratories, Detroit, USA, 1 l DI water). .. The screenings were performed in 96 well plates; each crude extract, previously concentrated at 10 mg ml−1 in DMSO, was added to a log-phase grown culture (OD600 nm = 0.04−0.06) to a 2.5% (v/v) final concentration.

Infection:

Article Title: Discovery, Purification, and Characterization of a Temperate Transducing Bacteriophage for Bordetella avium
Article Snippet: B. avium and Bordetella hinzii broth cultures were grown at 37°C with shaking in brain heart infusion (BHI) medium (Difco) as described previously ( ). .. Bacteria to be phage infected were grown overnight to stationary phase.

Expressing:

Article Title: Pathogen Proliferation Governs the Magnitude but Compromises the Function of CD8 T Cells
Article Snippet: Expression of OVA by recombinant wild-type (WT) ST-OVA and the various mutants of ST-OVA was determined by enhanced chemiluminescence detection system as described previously ( ). .. ST-OVA were grown in liquid culture at 37°C under constant shaking in brain heart infusion (BHI) medium (Difco Laboratories).

Modification:

Article Title: In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection
Article Snippet: .. Bacterial strains and growth conditions L. monocytogenes EGDe was grown in Brain Heart Infusion (BHI) medium (BD-Difco) or in a defined minimal medium (modified Welshimer's broth ) at 37°C, under aerobic conditions with shaking. ..

Recombinant:

Article Title: Pathogen Proliferation Governs the Magnitude but Compromises the Function of CD8 T Cells
Article Snippet: Expression of OVA by recombinant wild-type (WT) ST-OVA and the various mutants of ST-OVA was determined by enhanced chemiluminescence detection system as described previously ( ). .. ST-OVA were grown in liquid culture at 37°C under constant shaking in brain heart infusion (BHI) medium (Difco Laboratories).

Chick Chorioallantoic Membrane Assay:

Article Title: Vibrio fischeri DarR Directs Responses to d-Aspartate and Represents a Group of Similar LysR-Type Transcriptional Regulators
Article Snippet: When added to LB medium ( ) for the selection of E. coli , chloramphenicol (Cam) and kanamycin (Kan) were used at concentrations 20 and 40 μg · ml−1 , respectively. .. For selection of E. coli with erythromycin (Erm), 150 μg · ml−1 was added to brain heart infusion (BHI) medium (Difco, Sparks, MD).

Plasmid Preparation:

Article Title: Pathogen Proliferation Governs the Magnitude but Compromises the Function of CD8 T Cells
Article Snippet: The gene for OVA was introduced into virulent (SL1344) and the various mutants ( aroA, phoP, ssaR, and invA ) of ST. Plasmid pKK-OVA ( , ) (10–100 ng of DNA) carrying the full-length OVA was electroporated into ST as described previously ( ). .. ST-OVA were grown in liquid culture at 37°C under constant shaking in brain heart infusion (BHI) medium (Difco Laboratories).

Article Title: Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM
Article Snippet: Escherichia coli was grown in brain heart infusion (BHI) medium (Difco) at 37°C with shaking aeration. .. For upp -based counterselective gene replacement procedures, plasmid-free double recombinants were selected on a glucose semidefined agar medium containing 100 μg/ml 5-fluorouracil (5-FU) (Sigma), as previously described ( ).

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    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: 97/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Inducible expression of LLO and PC-PLC in L. monocytogenes. (A) Hemolytic activity assay. Hemolytic activities were determined from culture supernatants of L. monocytogenes as described in Materials and Methods. LLO was expressed from the inducible SPAC/ lac Oid promoter in the presence of 1 mM IPTG or from the native PrfA- or PrfA*-regulated hly promoter in strains 10403S and SLCC-5764, respectively. (B) PC-PLC activity assay. PC-PLC was expressed under the control of the inducible SPAC/ lac Oid promoter (i- plcB ) or the native PrfA-regulated actA-plcB promoter in the L. monocytogenes 10403S (PrfA) and SLCC-5764 (PrfA*) backgrounds. Overnight cultures were diluted 1:10 in BHI medium and grown for 5 h at 37°C in the presence or absence of 1 mM IPTG. Culture supernatants were TCA precipitated, and an equivalent of 8 or 0.32 ml of culture supernatant was separated by SDS-PAGE. PC-PLC activities were detected as described in Materials and Methods. Lane 1, DH-L377 (SLCC-5764 Δ hly ); lane 2, DH-L419 (SLCC-5764 Δ hly Δ plcB ); lane 3, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 4, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; lane 5, DH-L727 (10403S Δ hly , pPL2); lane 6, DP-L726 (10403S Δ hly Δ plcB , pPL2); lane 7, DH-L718 (10403S Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 8, DH-L718 (10403S Δ hly Δ plcB i- plcB ) without IPTG. (C) Intracellular growth in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. Symbols: •, DH-L377 (SLCC-5764 Δ hly ); ○, DH-L419 (SLCC-5764 Δ hly Δ plcB ); ▵, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; ▴, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 10 mM IPTG. The data points in growth curves represent the means ± standard deviations of three coverslips from one of two experiments.

    Journal: Journal of Bacteriology

    Article Title: Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells

    doi: 10.1128/JB.185.21.6295-6307.2003

    Figure Lengend Snippet: Inducible expression of LLO and PC-PLC in L. monocytogenes. (A) Hemolytic activity assay. Hemolytic activities were determined from culture supernatants of L. monocytogenes as described in Materials and Methods. LLO was expressed from the inducible SPAC/ lac Oid promoter in the presence of 1 mM IPTG or from the native PrfA- or PrfA*-regulated hly promoter in strains 10403S and SLCC-5764, respectively. (B) PC-PLC activity assay. PC-PLC was expressed under the control of the inducible SPAC/ lac Oid promoter (i- plcB ) or the native PrfA-regulated actA-plcB promoter in the L. monocytogenes 10403S (PrfA) and SLCC-5764 (PrfA*) backgrounds. Overnight cultures were diluted 1:10 in BHI medium and grown for 5 h at 37°C in the presence or absence of 1 mM IPTG. Culture supernatants were TCA precipitated, and an equivalent of 8 or 0.32 ml of culture supernatant was separated by SDS-PAGE. PC-PLC activities were detected as described in Materials and Methods. Lane 1, DH-L377 (SLCC-5764 Δ hly ); lane 2, DH-L419 (SLCC-5764 Δ hly Δ plcB ); lane 3, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 4, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; lane 5, DH-L727 (10403S Δ hly , pPL2); lane 6, DP-L726 (10403S Δ hly Δ plcB , pPL2); lane 7, DH-L718 (10403S Δ hly Δ plcB i- plcB ) with 1 mM IPTG; lane 8, DH-L718 (10403S Δ hly Δ plcB i- plcB ) without IPTG. (C) Intracellular growth in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. Symbols: •, DH-L377 (SLCC-5764 Δ hly ); ○, DH-L419 (SLCC-5764 Δ hly Δ plcB ); ▵, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) without IPTG; ▴, DH-L699 (SLCC-5764 Δ hly Δ plcB i- plcB ) with 10 mM IPTG. The data points in growth curves represent the means ± standard deviations of three coverslips from one of two experiments.

    Article Snippet: L. monocytogenes strains were grown in brain heart infusion (BHI) medium (Difco, Detroit, Mich.).

    Techniques: Expressing, Planar Chromatography, Activity Assay, SDS Page, Infection

    Multicopy inducible PC-PLC expression system for L. monocytogenes. (A) Schematic representation of the inducible PC-PLC expression vector pAMi plcB. plcB was cloned into plasmid pAM401 under SPAC/ lac Oid promoter/operator control, together with lacI under constitutive SPO-1 promoter control. (B) PC-PLC activity assays of SLCC-5764 (PrfA*) and 10403S (PrfA) derived strains. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium with or without IPTG at the indicated concentrations and grown for 5 h at 37°C. Culture supernatants were TCA precipitated, an equivalent of 0.2 or 2 ml of culture supernatant was separated by SDS-PAGE, and PC-PLC activities were detected by egg yolk overlay assays. In the upper panel are SLCC-5764 (PrfA*) strain derivatives. Lane 1, DH-L683 (SLCC-5764 Δ hly , pAMspacOid); lane 2, DH-L687 (SLCC-5764 Δ hly Δ plcB , pAMspacOid); lanes 3 to 10, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ; inducible plcB ) grown in the presence of increasing concentrations of IPTG as indicated above the figure. In the lower panel are 10403S (PrfA) strain derivatives. Lane 1, DH-L728 (10403S Δ hly , pAMspacOid); lane 2, DH-L729 (10403S Δ hly Δ plcB , pAMspacOid); lanes 3 to 10, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ; inducible plcB ) grown in the presence of increasing concentrations of IPTG as indicated above the figure.

    Journal: Journal of Bacteriology

    Article Title: Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells

    doi: 10.1128/JB.185.21.6295-6307.2003

    Figure Lengend Snippet: Multicopy inducible PC-PLC expression system for L. monocytogenes. (A) Schematic representation of the inducible PC-PLC expression vector pAMi plcB. plcB was cloned into plasmid pAM401 under SPAC/ lac Oid promoter/operator control, together with lacI under constitutive SPO-1 promoter control. (B) PC-PLC activity assays of SLCC-5764 (PrfA*) and 10403S (PrfA) derived strains. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium with or without IPTG at the indicated concentrations and grown for 5 h at 37°C. Culture supernatants were TCA precipitated, an equivalent of 0.2 or 2 ml of culture supernatant was separated by SDS-PAGE, and PC-PLC activities were detected by egg yolk overlay assays. In the upper panel are SLCC-5764 (PrfA*) strain derivatives. Lane 1, DH-L683 (SLCC-5764 Δ hly , pAMspacOid); lane 2, DH-L687 (SLCC-5764 Δ hly Δ plcB , pAMspacOid); lanes 3 to 10, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ; inducible plcB ) grown in the presence of increasing concentrations of IPTG as indicated above the figure. In the lower panel are 10403S (PrfA) strain derivatives. Lane 1, DH-L728 (10403S Δ hly , pAMspacOid); lane 2, DH-L729 (10403S Δ hly Δ plcB , pAMspacOid); lanes 3 to 10, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ; inducible plcB ) grown in the presence of increasing concentrations of IPTG as indicated above the figure.

    Article Snippet: L. monocytogenes strains were grown in brain heart infusion (BHI) medium (Difco, Detroit, Mich.).

    Techniques: Planar Chromatography, Expressing, Plasmid Preparation, Clone Assay, Activity Assay, Derivative Assay, SDS Page

    Intracellular growth of multicopy inducible PC-PLC L. monocytogenes strains in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. (A) SLCC-5764 (PrfA*) strain derivatives. Symbols: •, DH-L683 (SLCC-5764 Δ hly , pAMspacOid); ○, DH-L687 (SLCC-5764 Δ hly Δ plcB , pAMspacOid); ▵, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ) without IPTG; ▴, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ) with 10 mM IPTG. (B) 10403S (PrfA) strain derivatives. Symbols: •, DH-L728 (10403S Δ hly , pAMspacOid); ○, DH-L729 (10403S Δ hly Δ plcB , pAMspacOid); ▵, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ) without IPTG; ▴, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ) with 10 mM IPTG. The data points in the growth curves in panels A and B represent the means ± standard deviations of three coverslips from one of three experiments and from one experiment, respectively.

    Journal: Journal of Bacteriology

    Article Title: Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells

    doi: 10.1128/JB.185.21.6295-6307.2003

    Figure Lengend Snippet: Intracellular growth of multicopy inducible PC-PLC L. monocytogenes strains in Henle 407 cells. Overnight cultures of L. monocytogenes strains were diluted 1:10 in BHI medium and grown for 2 h at 37°C in the presence or absence of 10 mM IPTG. Monolayers of Henle 407 cells were infected at an MOI of 50:1, and intracellular growth was measured in the presence or absence of 10 mM IPTG as described in Materials and Methods. (A) SLCC-5764 (PrfA*) strain derivatives. Symbols: •, DH-L683 (SLCC-5764 Δ hly , pAMspacOid); ○, DH-L687 (SLCC-5764 Δ hly Δ plcB , pAMspacOid); ▵, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ) without IPTG; ▴, DH-L735 (SLCC-5764 Δ hly Δ plcB , pAMi plcB ) with 10 mM IPTG. (B) 10403S (PrfA) strain derivatives. Symbols: •, DH-L728 (10403S Δ hly , pAMspacOid); ○, DH-L729 (10403S Δ hly Δ plcB , pAMspacOid); ▵, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ) without IPTG; ▴, DH-L824 (10403S Δ hly Δ plcB , pAMi plcB ) with 10 mM IPTG. The data points in the growth curves in panels A and B represent the means ± standard deviations of three coverslips from one of three experiments and from one experiment, respectively.

    Article Snippet: L. monocytogenes strains were grown in brain heart infusion (BHI) medium (Difco, Detroit, Mich.).

    Techniques: Planar Chromatography, Infection

    Mutants of ST are attenuated in vivo. C57BL/6J, 129X1SvJ, or B6.129F 1 mice were infected with 10 3 ST-OVA i.v. At various time intervals, spleens were removed and the bacterial burden evaluated by plating serial dilutions on BHI plates ( A ); †,

    Journal:

    Article Title: Pathogen Proliferation Governs the Magnitude but Compromises the Function of CD8 T Cells

    doi:

    Figure Lengend Snippet: Mutants of ST are attenuated in vivo. C57BL/6J, 129X1SvJ, or B6.129F 1 mice were infected with 10 3 ST-OVA i.v. At various time intervals, spleens were removed and the bacterial burden evaluated by plating serial dilutions on BHI plates ( A ); †,

    Article Snippet: ST-OVA were grown in liquid culture at 37°C under constant shaking in brain heart infusion (BHI) medium (Difco Laboratories).

    Techniques: In Vivo, Mouse Assay, Infection