Structured Review

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
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1) Product Images from "Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes"

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004301

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
Figure Legend 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

Techniques Used: Binding Assay, Incubation

2) 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

3) 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

4) Product Images from "Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei"

Article Title: Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei

Journal: MicrobiologyOpen

doi: 10.1002/mbo3.192

(A) Chromosomally tagged Bacillus anthracis Sterne strains were streaked out on a BHI agar plate. The plate was incubated at 37°C for 24 h and recorded by scanning. The bacteria in each quadrant were Sterne::P ntr - gfp (top left), Sterne::P ntr - rfp (bottom left), Sterne::P0253- gfp (top right), and Sterne::P0253- rfp (bottom right). (B) Fluorescence microscopy of bacteria in each quadrant on the same plate described in (A). Fluorescence images were taken with an exposure time of 1.2 sec for the strains Sterne::P ntr - gfp (top left) and Sterne::P ntr - rfp (bottom left), and with an exposure time of 200 msec for the strains Sterne::P0253- gfp (top right) and Sterne::P0253- rfp (bottom right).
Figure Legend Snippet: (A) Chromosomally tagged Bacillus anthracis Sterne strains were streaked out on a BHI agar plate. The plate was incubated at 37°C for 24 h and recorded by scanning. The bacteria in each quadrant were Sterne::P ntr - gfp (top left), Sterne::P ntr - rfp (bottom left), Sterne::P0253- gfp (top right), and Sterne::P0253- rfp (bottom right). (B) Fluorescence microscopy of bacteria in each quadrant on the same plate described in (A). Fluorescence images were taken with an exposure time of 1.2 sec for the strains Sterne::P ntr - gfp (top left) and Sterne::P ntr - rfp (bottom left), and with an exposure time of 200 msec for the strains Sterne::P0253- gfp (top right) and Sterne::P0253- rfp (bottom right).

Techniques Used: Incubation, Fluorescence, Microscopy, Size-exclusion Chromatography

5) 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:

6) 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

7) Product Images from "Transcriptional Organization and Physiological Contributions of the relQ Operon of Streptococcus mutans"

Article Title: Transcriptional Organization and Physiological Contributions of the relQ Operon of Streptococcus mutans

Journal: Journal of Bacteriology

doi: 10.1128/JB.00037-12

Biofilm formation. S. mutans strains were grown to an OD 600 of 0.5 in BHI broth, diluted 1:50 in BM semidefined medium supplemented with 10 mM sucrose (A) or with 20 mM glucose (B) in a 96-well microtiter plate, and incubated at 37°C with 5% CO
Figure Legend Snippet: Biofilm formation. S. mutans strains were grown to an OD 600 of 0.5 in BHI broth, diluted 1:50 in BM semidefined medium supplemented with 10 mM sucrose (A) or with 20 mM glucose (B) in a 96-well microtiter plate, and incubated at 37°C with 5% CO

Techniques Used: Incubation

8) Product Images from "Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome ▿ Secretome ▿ †"

Article Title: Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome ▿ Secretome ▿ †

Journal:

doi: 10.1128/IAI.02029-06

SERPA of B. anthracis secreted proteins: comparison of Coomassie blue-stained 2-DE (gels 1, 3, and 5) with the corresponding Western blots (gels 2, 4, and 6). (Gels 1 and 2) FAG medium secretome of the Vollum strain. (Gels 3 and 4) BHI medium secretome
Figure Legend Snippet: SERPA of B. anthracis secreted proteins: comparison of Coomassie blue-stained 2-DE (gels 1, 3, and 5) with the corresponding Western blots (gels 2, 4, and 6). (Gels 1 and 2) FAG medium secretome of the Vollum strain. (Gels 3 and 4) BHI medium secretome

Techniques Used: Staining, Western Blot

9) 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

10) Product Images from "LPXTG Protein InlJ, a Newly Identified Internalin Involved in Listeria monocytogenes Virulence "

Article Title: LPXTG Protein InlJ, a Newly Identified Internalin Involved in Listeria monocytogenes Virulence

Journal:

doi: 10.1128/IAI.73.10.6912-6922.2005

Expression of inlI and inlJ . (A) RT-PCR for total RNAs from the end of the exponential phase (OD 600 , 0.8) for cultures in BHI medium at 37°C of L. monocytogenes wild-type strain. Serial dilutions of the RNA templates were used. The gyrA and inlA
Figure Legend Snippet: Expression of inlI and inlJ . (A) RT-PCR for total RNAs from the end of the exponential phase (OD 600 , 0.8) for cultures in BHI medium at 37°C of L. monocytogenes wild-type strain. Serial dilutions of the RNA templates were used. The gyrA and inlA

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

11) Product Images from "Chill Activation of Compatible Solute Transporters in Corynebacterium glutamicum at the Level of Transport Activity"

Article Title: Chill Activation of Compatible Solute Transporters in Corynebacterium glutamicum at the Level of Transport Activity

Journal:

doi: 10.1128/JB.187.14.4752-4759.2005

Temperature-dependent activity profiles of plasmid-encoded BetPΔ25 at low and high osmolality in C. glutamicum DHPF p betP . The cells were grown at 30°C in BHI medium, washed in buffer containing 25 mM potassium phosphate (pH 7.5) and 100
Figure Legend Snippet: Temperature-dependent activity profiles of plasmid-encoded BetPΔ25 at low and high osmolality in C. glutamicum DHPF p betP . The cells were grown at 30°C in BHI medium, washed in buffer containing 25 mM potassium phosphate (pH 7.5) and 100

Techniques Used: Activity Assay, Plasmid Preparation

Temperature-dependent activity profiles of BetP (squares), EctP (triangles), and LcoP (circles) in C. glutamicum DHPF pEKEX2 under hyperosmotic conditions. (A) The cells were grown at 30°C in BHI medium, washed in buffer containing 50 mM potassium
Figure Legend Snippet: Temperature-dependent activity profiles of BetP (squares), EctP (triangles), and LcoP (circles) in C. glutamicum DHPF pEKEX2 under hyperosmotic conditions. (A) The cells were grown at 30°C in BHI medium, washed in buffer containing 50 mM potassium

Techniques Used: Activity Assay

12) 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:

13) Product Images from "Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP"

Article Title: Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

Journal: Journal of Bacteriology

doi:

Stimulation of glycine betaine uptake by C. glutamicum DHPE/pBW4 with NaCl and sorbitol. Cells were grown overnight in BHI medium supplemented with 0.2 mM IPTG. Uptake was started by the addition of 750 μM labeled glycine betaine. The osmolality of the uptake assay medium was increased by the addition of either NaCl (•) or sorbitol in the presence of 50 mM NaCl (○). The uptake rates at an external osmolality of 1.4 or 1.1 osmol/kg were defined as 100% activity for stimulation by NaCl and sorbitol, respectively. The absolute rates were 42.8 nmol/min/mg (dw) at an osmolality of 1.4 osmol/kg (NaCl) and 21.8 nmol/min/mg (dw) at an osmolality of 1.1 osmol/kg (sorbitol).
Figure Legend Snippet: Stimulation of glycine betaine uptake by C. glutamicum DHPE/pBW4 with NaCl and sorbitol. Cells were grown overnight in BHI medium supplemented with 0.2 mM IPTG. Uptake was started by the addition of 750 μM labeled glycine betaine. The osmolality of the uptake assay medium was increased by the addition of either NaCl (•) or sorbitol in the presence of 50 mM NaCl (○). The uptake rates at an external osmolality of 1.4 or 1.1 osmol/kg were defined as 100% activity for stimulation by NaCl and sorbitol, respectively. The absolute rates were 42.8 nmol/min/mg (dw) at an osmolality of 1.4 osmol/kg (NaCl) and 21.8 nmol/min/mg (dw) at an osmolality of 1.1 osmol/kg (sorbitol).

Techniques Used: Labeling, Activity Assay

Stimulation of glycine betaine uptake in C. glutamicum DHPE/pBW4 and DHPE/pGTG by the local anesthetic tetracaine. Cells were grown overnight in BHI medium supplemented with 0.2 mM IPTG. Uptake was started by the addition of 750 μM labeled betaine. Betaine uptake in strain DHPE/pGTG (■) was determined at an external NaCl osmolality of 0.4 osmol/kg. The activity of DHPE/pBW4 (open symbols) was measured at values of external osmolality of 0.4 (○), 0.7 (□), and 1.1 osmol/kg (▵).
Figure Legend Snippet: Stimulation of glycine betaine uptake in C. glutamicum DHPE/pBW4 and DHPE/pGTG by the local anesthetic tetracaine. Cells were grown overnight in BHI medium supplemented with 0.2 mM IPTG. Uptake was started by the addition of 750 μM labeled betaine. Betaine uptake in strain DHPE/pGTG (■) was determined at an external NaCl osmolality of 0.4 osmol/kg. The activity of DHPE/pBW4 (open symbols) was measured at values of external osmolality of 0.4 (○), 0.7 (□), and 1.1 osmol/kg (▵).

Techniques Used: Labeling, Activity Assay

Kinetic analysis of the onset time of activity of BetP and EctP. Cells of C. glutamicum DHPE/pGTG (A) and DHPE/pBW4 (B), grown in BHI medium supplemented with 0.2 mM IPTG and 50 μg of kanamycin per liter were preequilibrated for 1 h at 4°C either in hypo-osmotic (50 mM potassium phosphate; pH 7.5) or in isosmotic medium (50 mM potassium phosphate [pH 7.5], 600 mM NaCl). The transport experiments were started by the addition of high-osmolality buffer (50 mM potassium phosphate [pH 7.5], 600 mM NaCl) together with labeled glycine betaine (100 μM for strain DHPE/pGTG and 1 mM for DHPE/pBW4, because of the different K m values). Blank values were subtracted from the measured values; they were derived by using identical cells under low-osmolality conditions (0.2 osmol/kg) under which both BetP and EctP are not active. The onset time, i.e., the period of time between osmotic shock and the functional response of the transporter, was derived by extrapolation to zero uptake.
Figure Legend Snippet: Kinetic analysis of the onset time of activity of BetP and EctP. Cells of C. glutamicum DHPE/pGTG (A) and DHPE/pBW4 (B), grown in BHI medium supplemented with 0.2 mM IPTG and 50 μg of kanamycin per liter were preequilibrated for 1 h at 4°C either in hypo-osmotic (50 mM potassium phosphate; pH 7.5) or in isosmotic medium (50 mM potassium phosphate [pH 7.5], 600 mM NaCl). The transport experiments were started by the addition of high-osmolality buffer (50 mM potassium phosphate [pH 7.5], 600 mM NaCl) together with labeled glycine betaine (100 μM for strain DHPE/pGTG and 1 mM for DHPE/pBW4, because of the different K m values). Blank values were subtracted from the measured values; they were derived by using identical cells under low-osmolality conditions (0.2 osmol/kg) under which both BetP and EctP are not active. The onset time, i.e., the period of time between osmotic shock and the functional response of the transporter, was derived by extrapolation to zero uptake.

Techniques Used: Activity Assay, Labeling, Derivative Assay, Functional Assay

14) 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

15) Product Images from "Morphogenesis of the Bacillus anthracis Spore ▿"

Article Title: Morphogenesis of the Bacillus anthracis Spore ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00921-06

Effects of coat protein gene mutations on germination and spore survival in macrophages. Spores were suspended in BHI medium, and the percentage of heat-sensitive cotE or cotH mutant or wild-type cells was determined at each time point. Percent germination of the mutant cells was normalized to that of the wild type. Therefore, a ratio of 1 indicates wild-type levels of germination, and a value less than 1 indicates a germination defect (A). Spore survival in macrophages was measured by CFU determinations with or without heat shocking (B and C). All assays were done in triplicate, and standard errors of the means are shown. Am, Am cotE , Am tot, Am hs, Am cotE tot, Am cotE hs, Am cotH , St, and St cotH indicate Ames wild type, Ames cotE mutant (JAB4), Ames wild-type total, Ames wild type after heat shock, Ames cotE mutant (JAB4) total, Ames cotE mutant (JAB4) after heat shock, Ames cotH mutant (JAB3), Sterne wild type (RG1), and Sterne cotH mutant (RG73), respectively.
Figure Legend Snippet: Effects of coat protein gene mutations on germination and spore survival in macrophages. Spores were suspended in BHI medium, and the percentage of heat-sensitive cotE or cotH mutant or wild-type cells was determined at each time point. Percent germination of the mutant cells was normalized to that of the wild type. Therefore, a ratio of 1 indicates wild-type levels of germination, and a value less than 1 indicates a germination defect (A). Spore survival in macrophages was measured by CFU determinations with or without heat shocking (B and C). All assays were done in triplicate, and standard errors of the means are shown. Am, Am cotE , Am tot, Am hs, Am cotE tot, Am cotE hs, Am cotH , St, and St cotH indicate Ames wild type, Ames cotE mutant (JAB4), Ames wild-type total, Ames wild type after heat shock, Ames cotE mutant (JAB4) total, Ames cotE mutant (JAB4) after heat shock, Ames cotH mutant (JAB3), Sterne wild type (RG1), and Sterne cotH mutant (RG73), respectively.

Techniques Used: Mutagenesis

16) Product Images from "Core-Gene-Encoded Peptide Regulating Virulence-Associated Traits in Streptococcus mutans"

Article Title: Core-Gene-Encoded Peptide Regulating Virulence-Associated Traits in Streptococcus mutans

Journal: Journal of Bacteriology

doi: 10.1128/JB.00189-13

Biofilm formation. S. mutans wild-type and mutant strains were grown to an OD 600 of 0.5 in BHI broth, diluted 1:50 in BM semidefined medium supplemented with 20 mM glucose (A) or with 10 mM sucrose (B) in a 96-well microtiter plate, and incubated at 37°C
Figure Legend Snippet: Biofilm formation. S. mutans wild-type and mutant strains were grown to an OD 600 of 0.5 in BHI broth, diluted 1:50 in BM semidefined medium supplemented with 20 mM glucose (A) or with 10 mM sucrose (B) in a 96-well microtiter plate, and incubated at 37°C

Techniques Used: Mutagenesis, Incubation

Genetic competence and complementation of SMU.1147. (A and B) S. mutans wild-type and mutant strains were grown to an OD 600 of 0.15 in BHI broth at 37°C with 5% CO 2 . Cells were incubated for 30 min in the absence (A) or presence (B) of 200 nM
Figure Legend Snippet: Genetic competence and complementation of SMU.1147. (A and B) S. mutans wild-type and mutant strains were grown to an OD 600 of 0.15 in BHI broth at 37°C with 5% CO 2 . Cells were incubated for 30 min in the absence (A) or presence (B) of 200 nM

Techniques Used: Mutagenesis, Incubation

17) Product Images from "N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeriamonocytogenes"

Article Title: N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeriamonocytogenes

Journal: Nature microbiology

doi: 10.1038/nmicrobiol.2017.5

A proteogenomic approach to map translation initiation sites (TISs). a , Schematic representation of bacterial protein translation. b , Dose-dependent Listeria growth inhibition by actinonin. Values are expressed as mean ± s.e.m. of three replicates from one representative experiment. c , Schematic representation of our proteogenomic approach. L. monocytogenes EGD-e was grown in BHI medium under different conditions and actinonin (50 μg ml −1 ) was added for 90 min before collecting the bacteria. Extracted proteins were digested with trypsin or endoGluC and (formylated) N-terminal peptides were isolated by COFRADIC before LC-MS/MS analysis. Over 1,000,000 MS/MS spectra were recorded and searched against a database generated by six-frame translation of the EGD-e genome sequence from every possible start codon. Two different search algorithms—Mascot and MaxQuant—were used to identify more than 12,000 peptides that were mapped on the Listeria genome, leading to the identification of TISs at a single-codon resolution.
Figure Legend Snippet: A proteogenomic approach to map translation initiation sites (TISs). a , Schematic representation of bacterial protein translation. b , Dose-dependent Listeria growth inhibition by actinonin. Values are expressed as mean ± s.e.m. of three replicates from one representative experiment. c , Schematic representation of our proteogenomic approach. L. monocytogenes EGD-e was grown in BHI medium under different conditions and actinonin (50 μg ml −1 ) was added for 90 min before collecting the bacteria. Extracted proteins were digested with trypsin or endoGluC and (formylated) N-terminal peptides were isolated by COFRADIC before LC-MS/MS analysis. Over 1,000,000 MS/MS spectra were recorded and searched against a database generated by six-frame translation of the EGD-e genome sequence from every possible start codon. Two different search algorithms—Mascot and MaxQuant—were used to identify more than 12,000 peptides that were mapped on the Listeria genome, leading to the identification of TISs at a single-codon resolution.

Techniques Used: Inhibition, Isolation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Generated, Sequencing

18) Product Images from "A Deficiency in Arabinogalactan Biosynthesis Affects Corynebacterium glutamicum Mycolate Outer Membrane Stability ▿"

Article Title: A Deficiency in Arabinogalactan Biosynthesis Affects Corynebacterium glutamicum Mycolate Outer Membrane Stability ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00009-10

Identification of membrane fragments in the culture medium of the Δ aftB mutant. (A and B) Cryo-electron microscopy images of C. glutamicum 13032 and Δ aftB cells grown on BHI medium, respectively. Culture supernatant from the wild type, the Δ aftB mutant, or the complemented strain was ultracentrifuged as described in Materials and Methods. An important pellet is clearly visible for the mutant strain but not for the wild type or the complemented strain (C). Membrane fragments released by the Δ aftB strain were then purified on a sucrose gradient (D) and were observed directly by cryo-electron microscopy (E). Fragments of different sizes and forms are indicated by arrows. The percentages of sucrose (wt/wt) at specific positions are indicated next to the gradient. Bars, 200 nm.
Figure Legend Snippet: Identification of membrane fragments in the culture medium of the Δ aftB mutant. (A and B) Cryo-electron microscopy images of C. glutamicum 13032 and Δ aftB cells grown on BHI medium, respectively. Culture supernatant from the wild type, the Δ aftB mutant, or the complemented strain was ultracentrifuged as described in Materials and Methods. An important pellet is clearly visible for the mutant strain but not for the wild type or the complemented strain (C). Membrane fragments released by the Δ aftB strain were then purified on a sucrose gradient (D) and were observed directly by cryo-electron microscopy (E). Fragments of different sizes and forms are indicated by arrows. The percentages of sucrose (wt/wt) at specific positions are indicated next to the gradient. Bars, 200 nm.

Techniques Used: Mutagenesis, Electron Microscopy, Purification

19) 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:

20) 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.

Centrifugation:

Article Title: Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome ▿ Secretome ▿ †
Article Snippet: Cells were grown under aerobic conditions in FAG medium , in brain heart infusion (BHI) medium (Difco/Becton Dickinson, Maryland), or in NBY medium (0.8% [wt/vol] nutrient broth [Difco], 0.3% yeast extract [Difco], 0.5% glucose) for up to 24 h at 37°C with vigorous agitation or under semiaerobic conditions in NBY medium supplemented with 0.9% NaHCO3 in hermetically sealed filled flasks with slow agitation. .. One hundred milliliters of conditioned FAG or BHI medium or 200 ml of NBY medium was incubated at 4°C overnight in the presence of 10% trichloroacetic acid, and then the proteins were precipitated by centrifugation for 30 min in a Sorvall S34 (12,000 rpm).

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.

Sample Prep:

Article Title: Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome ▿ Secretome ▿ †
Article Snippet: Paragraph title: Bacterial cultures and sample preparation for 2-DE. ... Cells were grown under aerobic conditions in FAG medium , in brain heart infusion (BHI) medium (Difco/Becton Dickinson, Maryland), or in NBY medium (0.8% [wt/vol] nutrient broth [Difco], 0.3% yeast extract [Difco], 0.5% glucose) for up to 24 h at 37°C with vigorous agitation or under semiaerobic conditions in NBY medium supplemented with 0.9% NaHCO3 in hermetically sealed filled flasks with slow agitation.

Mutagenesis:

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.

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: Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei
Article Snippet: Bacillus anthracis sp. and their derivatives were grown aerobically at 37°C in brain heart infusion (BHI) medium (Difco, Franklin Lakes, NJ, USA.). .. Yersina pseudotuberculosis, Y . pestis , and their derivatives were cultured aerobically at 37°C in tryptic soy broth (TSB, Difco).

Article Title: LPXTG Protein InlJ, a Newly Identified Internalin Involved in Listeria monocytogenes Virulence
Article Snippet: Bacteria were grown at 37°C in brain heart infusion (BHI) medium (Difco) supplemented with erythromycin (5 μg ml−1 ) when they carried pMAD derivatives. .. Cell lines were cultured in modified Eagle medium (Gibco), Dulbecco modified Eagle medium (DMEM), or Ham's F12K medium supplemented with fetal bovine serum (10% or 20%), 1 mM pyruvate, 2 mM glutamine, and 1% nonessential amino acids (Gibco) according to American Type Culture Collection recommendations.

Infection:

Article Title: Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome ▿ Secretome ▿ †
Article Snippet: Cells were grown under aerobic conditions in FAG medium , in brain heart infusion (BHI) medium (Difco/Becton Dickinson, Maryland), or in NBY medium (0.8% [wt/vol] nutrient broth [Difco], 0.3% yeast extract [Difco], 0.5% glucose) for up to 24 h at 37°C with vigorous agitation or under semiaerobic conditions in NBY medium supplemented with 0.9% NaHCO3 in hermetically sealed filled flasks with slow agitation. .. The low-nutrient (compared to FAG or BHI medium) NBY-CO2 medium promotes efficient toxin production (as detected by Western analysis) and capsule synthesis (which was visualized by negative staining using India ink [Becton Dickinson, Maryland]) and thus is considered to mimic infection conditions.

Concentration Assay:

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: Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei
Article Snippet: When necessary, diaminopimelate (DAP) was supplemented at a final concentration of 200 μ g/mL. .. Bacillus anthracis sp. and their derivatives were grown aerobically at 37°C in brain heart infusion (BHI) medium (Difco, Franklin Lakes, NJ, USA.).

Incubation:

Article Title: Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome ▿ Secretome ▿ †
Article Snippet: Cells were grown under aerobic conditions in FAG medium , in brain heart infusion (BHI) medium (Difco/Becton Dickinson, Maryland), or in NBY medium (0.8% [wt/vol] nutrient broth [Difco], 0.3% yeast extract [Difco], 0.5% glucose) for up to 24 h at 37°C with vigorous agitation or under semiaerobic conditions in NBY medium supplemented with 0.9% NaHCO3 in hermetically sealed filled flasks with slow agitation. .. One hundred milliliters of conditioned FAG or BHI medium or 200 ml of NBY medium was incubated at 4°C overnight in the presence of 10% trichloroacetic acid, and then the proteins were precipitated by centrifugation for 30 min in a Sorvall S34 (12,000 rpm).

other:

Article Title: Transcriptional Organization and Physiological Contributions of the relQ Operon of Streptococcus mutans
Article Snippet: S. mutans UA159 and its derivatives were maintained in brain heart infusion (BHI) medium (Difco Laboratories, Detroit, MI) supplemented with spectinomycin (Sp; 1 mg ml−1 ), erythromycin (Erm; 10 μg ml−1 ), and/or kanamycin (Km; 1 mg ml−1 ) (Sigma-Aldrich, St. Louis, MO), when necessary.

Article Title: Chill Activation of Compatible Solute Transporters in Corynebacterium glutamicum at the Level of Transport Activity
Article Snippet: C. glutamicum cells were grown in either brain heart infusion (BHI) medium (Difco, Detroit, Mich.) or minimal medium ( ) at 30°C, and E. coli cells were cultivated in Luria-Bertani (LB) medium at 37°C.

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

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).

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. ..

Article Title: LPXTG Protein InlJ, a Newly Identified Internalin Involved in Listeria monocytogenes Virulence
Article Snippet: Bacteria were grown at 37°C in brain heart infusion (BHI) medium (Difco) supplemented with erythromycin (5 μg ml−1 ) when they carried pMAD derivatives. .. Cell lines were cultured in modified Eagle medium (Gibco), Dulbecco modified Eagle medium (DMEM), or Ham's F12K medium supplemented with fetal bovine serum (10% or 20%), 1 mM pyruvate, 2 mM glutamine, and 1% nonessential amino acids (Gibco) according to American Type Culture Collection recommendations.

Western Blot:

Article Title: Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome ▿ Secretome ▿ †
Article Snippet: Cells were grown under aerobic conditions in FAG medium , in brain heart infusion (BHI) medium (Difco/Becton Dickinson, Maryland), or in NBY medium (0.8% [wt/vol] nutrient broth [Difco], 0.3% yeast extract [Difco], 0.5% glucose) for up to 24 h at 37°C with vigorous agitation or under semiaerobic conditions in NBY medium supplemented with 0.9% NaHCO3 in hermetically sealed filled flasks with slow agitation. .. The low-nutrient (compared to FAG or BHI medium) NBY-CO2 medium promotes efficient toxin production (as detected by Western analysis) and capsule synthesis (which was visualized by negative staining using India ink [Becton Dickinson, Maryland]) and thus is considered to mimic infection conditions.

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).

Negative Staining:

Article Title: Identification of In Vivo-Expressed Immunogenic Proteins by Serological Proteome Analysis of the Bacillus anthracis Secretome ▿ Secretome ▿ †
Article Snippet: Cells were grown under aerobic conditions in FAG medium , in brain heart infusion (BHI) medium (Difco/Becton Dickinson, Maryland), or in NBY medium (0.8% [wt/vol] nutrient broth [Difco], 0.3% yeast extract [Difco], 0.5% glucose) for up to 24 h at 37°C with vigorous agitation or under semiaerobic conditions in NBY medium supplemented with 0.9% NaHCO3 in hermetically sealed filled flasks with slow agitation. .. The low-nutrient (compared to FAG or BHI medium) NBY-CO2 medium promotes efficient toxin production (as detected by Western analysis) and capsule synthesis (which was visualized by negative staining using India ink [Becton Dickinson, Maryland]) and thus is considered to mimic infection conditions.

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).

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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: 98/100, 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