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Difco brain heart infusion medium bhi
pPD1 enhances EF competition for an intestinal niche (a) Bacteriocin assay by the soft agar method with EF r +pPD1::Δ bacAB, bacA-E +, EF r +pPD1 and EF r +pPD1::Δ bacAB spotted on a lawn of susceptible EF. Mice (N=5/group) were given EF r or EF r +pPD1 or EF r +pPD1::Δ bacAB or sterile drinking water for 14 days, at which time all mice were given sterile water. (b) One week and (c) four weeks after withdrawal of EF from drinking water, fecal samples were collected and abundance of total enterococci was determined by enumeration on m-Enterococcus selective agar <t>(Ent</t> agar, ○). Lab strain of EF was enumerated using Ent agar with rifampicin (Rf) (○), or <t>BHI</t> agar with rifampicin (○). (d and e and Fig. 1e ) At the end of week four, animals were euthanized and abundance of EF was determined in distal small intestine (DSI) and large intestine (LI). The results shown are representative of two biologically independent experiments. Horizontal lines indicate geometric mean. Each symbol represents an individual animal.
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1) Product Images from "Bacteriocin production augments niche competition by enterococci in the mammalian GI tract"

Article Title: Bacteriocin production augments niche competition by enterococci in the mammalian GI tract

Journal: Nature

doi: 10.1038/nature15524

pPD1 enhances EF competition for an intestinal niche (a) Bacteriocin assay by the soft agar method with EF r +pPD1::Δ bacAB, bacA-E +, EF r +pPD1 and EF r +pPD1::Δ bacAB spotted on a lawn of susceptible EF. Mice (N=5/group) were given EF r or EF r +pPD1 or EF r +pPD1::Δ bacAB or sterile drinking water for 14 days, at which time all mice were given sterile water. (b) One week and (c) four weeks after withdrawal of EF from drinking water, fecal samples were collected and abundance of total enterococci was determined by enumeration on m-Enterococcus selective agar (Ent agar, ○). Lab strain of EF was enumerated using Ent agar with rifampicin (Rf) (○), or BHI agar with rifampicin (○). (d and e and Fig. 1e ) At the end of week four, animals were euthanized and abundance of EF was determined in distal small intestine (DSI) and large intestine (LI). The results shown are representative of two biologically independent experiments. Horizontal lines indicate geometric mean. Each symbol represents an individual animal.
Figure Legend Snippet: pPD1 enhances EF competition for an intestinal niche (a) Bacteriocin assay by the soft agar method with EF r +pPD1::Δ bacAB, bacA-E +, EF r +pPD1 and EF r +pPD1::Δ bacAB spotted on a lawn of susceptible EF. Mice (N=5/group) were given EF r or EF r +pPD1 or EF r +pPD1::Δ bacAB or sterile drinking water for 14 days, at which time all mice were given sterile water. (b) One week and (c) four weeks after withdrawal of EF from drinking water, fecal samples were collected and abundance of total enterococci was determined by enumeration on m-Enterococcus selective agar (Ent agar, ○). Lab strain of EF was enumerated using Ent agar with rifampicin (Rf) (○), or BHI agar with rifampicin (○). (d and e and Fig. 1e ) At the end of week four, animals were euthanized and abundance of EF was determined in distal small intestine (DSI) and large intestine (LI). The results shown are representative of two biologically independent experiments. Horizontal lines indicate geometric mean. Each symbol represents an individual animal.

Techniques Used: Mouse Assay

EF+pPD1 but not EF r +pCF10 dominates the intestinal enterococcal population pCF10 is a well-studied pheromone-inducible conjugative plasmid of EF that encodes resistance to tetracycline but does not encode a known bacteriocin determinant. (a) EF r : (o, N=3 mice, no plasmid), EF r +pPD1: (☐, N=3 mice, pPD1) or EF r +pCF10, (∇, N=5 mice, pCF10) was added to drinking water for 14 days, and then replaced by sterile drinking water. (a) Fecal samples were taken from each animal at the transition to sterile drinking water (week 0) and then weekly. EF abundance was determined by enumeration on BHI agar with rifampicin. (b) Four weeks after withdrawal of EF from drinking water, animals were euthanized and abundance of EF r +pCF10 was determined in each segment of the GI tract (distal small intestine, DSI; cecum; and large intestine, LI). Mice colonized with EF r +pCF10 maintained long-term fecal shedding of EF r +pCF10 similar to EF r , and persistent colonization throughout the GI tract. Abundance of enterococci in the feces was determined by enumeration on m-Enterococcus agar (Ent agar, o), Ent agar with rifampicin (Rf) (○), or BHI agar with rifampicin (○) at week 1 (c) and week 4 (d). Unlike EF r +pPD1 that dominated the enterococcal niche in the GI tract ( Fig. 1e ), EF r +pCF10 did not outcompete the indigenous enterococci, colonizing at levels comparable to EF r (c and d). (a) The lines are fitted using an exponential decay model, and there are significant differences in rate of decay for the “pCF10” group vs. “pPD1” group ( P =0.012) and for the “pPD1” vs. “no plasmid” group ( P =0.007). Horizontal lines in (b, c, and d) indicate geometric mean. Each symbol represents an individual animal; data are obtained from one experiment.
Figure Legend Snippet: EF+pPD1 but not EF r +pCF10 dominates the intestinal enterococcal population pCF10 is a well-studied pheromone-inducible conjugative plasmid of EF that encodes resistance to tetracycline but does not encode a known bacteriocin determinant. (a) EF r : (o, N=3 mice, no plasmid), EF r +pPD1: (☐, N=3 mice, pPD1) or EF r +pCF10, (∇, N=5 mice, pCF10) was added to drinking water for 14 days, and then replaced by sterile drinking water. (a) Fecal samples were taken from each animal at the transition to sterile drinking water (week 0) and then weekly. EF abundance was determined by enumeration on BHI agar with rifampicin. (b) Four weeks after withdrawal of EF from drinking water, animals were euthanized and abundance of EF r +pCF10 was determined in each segment of the GI tract (distal small intestine, DSI; cecum; and large intestine, LI). Mice colonized with EF r +pCF10 maintained long-term fecal shedding of EF r +pCF10 similar to EF r , and persistent colonization throughout the GI tract. Abundance of enterococci in the feces was determined by enumeration on m-Enterococcus agar (Ent agar, o), Ent agar with rifampicin (Rf) (○), or BHI agar with rifampicin (○) at week 1 (c) and week 4 (d). Unlike EF r +pPD1 that dominated the enterococcal niche in the GI tract ( Fig. 1e ), EF r +pCF10 did not outcompete the indigenous enterococci, colonizing at levels comparable to EF r (c and d). (a) The lines are fitted using an exponential decay model, and there are significant differences in rate of decay for the “pCF10” group vs. “pPD1” group ( P =0.012) and for the “pPD1” vs. “no plasmid” group ( P =0.007). Horizontal lines in (b, c, and d) indicate geometric mean. Each symbol represents an individual animal; data are obtained from one experiment.

Techniques Used: Plasmid Preparation, Mouse Assay

Complementation of Bac-21 production restores colonization phenotype by providing competitive advantage Bacteriocin activity was restored upon ectopic expression of bacABCDE (from pAM401) in EF+pPD1::Δ bacAB but not in EF lacking pPD1, indicating that the distal part of the bac operon ( bacFGHI) is necessary for bacteriocin expression and that the bacAB in-frame deletion is not polar on downstream genes. Mice (N=5) were given EF r +pPD1::Δ bacAB, bacABCDE + as described in the methods and abundance was determined by enumeration on m-Enterococcus agar (Ent agar), Ent agar plus rifampicin (Rf), or BHI agar with rifampicin. The presence of pAM401A:: bacABCDE+ (complementing plasmid) was determined by enumerating CFU on BHI agar with rifampicin and chloramphenicol (Cm). Fecal samples were obtained at week 1 (a) and week 4 (b) after transition to sterile drinking water. Horizontal lines indicate geometric mean. Each symbol represents an individual animal and data is a representative of two biologically independent experiments. EF s +pPD1:: ΔbacAB bacABCDE + stably colonized the GI tract (a), although in the absence of chloramphenicol selection, pAM401:: bacABCDE was gradually lost from the population (b). Over time, loss of pAM401:: bacABCDE resulted in the complemented strain reverting back to the bacteriocin-defective Δ bacAB strain, with the loss of bacteriocin activity. Nevertheless, this strain persisted in the gut, suggesting that Bac-21 was essential for clearing a niche for EF, but once cleared, EF uses other mechanisms to maintain colonization (a and b).
Figure Legend Snippet: Complementation of Bac-21 production restores colonization phenotype by providing competitive advantage Bacteriocin activity was restored upon ectopic expression of bacABCDE (from pAM401) in EF+pPD1::Δ bacAB but not in EF lacking pPD1, indicating that the distal part of the bac operon ( bacFGHI) is necessary for bacteriocin expression and that the bacAB in-frame deletion is not polar on downstream genes. Mice (N=5) were given EF r +pPD1::Δ bacAB, bacABCDE + as described in the methods and abundance was determined by enumeration on m-Enterococcus agar (Ent agar), Ent agar plus rifampicin (Rf), or BHI agar with rifampicin. The presence of pAM401A:: bacABCDE+ (complementing plasmid) was determined by enumerating CFU on BHI agar with rifampicin and chloramphenicol (Cm). Fecal samples were obtained at week 1 (a) and week 4 (b) after transition to sterile drinking water. Horizontal lines indicate geometric mean. Each symbol represents an individual animal and data is a representative of two biologically independent experiments. EF s +pPD1:: ΔbacAB bacABCDE + stably colonized the GI tract (a), although in the absence of chloramphenicol selection, pAM401:: bacABCDE was gradually lost from the population (b). Over time, loss of pAM401:: bacABCDE resulted in the complemented strain reverting back to the bacteriocin-defective Δ bacAB strain, with the loss of bacteriocin activity. Nevertheless, this strain persisted in the gut, suggesting that Bac-21 was essential for clearing a niche for EF, but once cleared, EF uses other mechanisms to maintain colonization (a and b).

Techniques Used: BAC Assay, Activity Assay, Expressing, Mouse Assay, Plasmid Preparation, Stable Transfection, Selection

Related Articles

Mutagenesis:

Article Title: Identification of Genes Induced in Listeria monocytogenes during Growth and Attachment to Cut Cabbage, Using Differential Display
Article Snippet: L. monocytogenes strain 10403 ( ) and derivatives of this strain were grown in brain heart infusion medium (BHI) (Difco, Becton Dickinson, Franklin Lakes, NJ) or Listeria minimal medium (LMM) ( ). .. RM2980 is a strain 10403 derivative that has an insertion of transposon Tn 917 -LTV3 into an unknown gene within a flagellar biosynthetic operon, which results in a mutant that has no flagella and is nonmotile ( ).

Isolation:

Article Title: Identification of Genes Induced in Listeria monocytogenes during Growth and Attachment to Cut Cabbage, Using Differential Display
Article Snippet: L. monocytogenes strain 10403 ( ) and derivatives of this strain were grown in brain heart infusion medium (BHI) (Difco, Becton Dickinson, Franklin Lakes, NJ) or Listeria minimal medium (LMM) ( ). .. L. monocytogenes colonies were isolated and enumerated on modified Oxford agar (MOX) (Difco), which inhibits gram-negative bacteria and many gram-positive bacteria.

Cell Culture:

Article Title: Bacteriocin production augments niche competition by enterococci in the mammalian GI tract
Article Snippet: Brain heart infusion medium (BHI) and m-Enterococcus agar (Difco™) (Ent-agar) were prepared as described by the manufacturer (Becton Dickinson). .. EF was cultured in BHI media at 37°C.

Article Title: The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model
Article Snippet: .. The pathogen was cultured in Brain-Heart Infusion medium (BHI) (Difco) containing 5 mg/ml yeast extract and 0.1% L-cysteine at 37°C for 72 h in an anaerobic chamber (Coy Laboratory Products). ..

Plasmid Preparation:

Article Title: The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model
Article Snippet: Paragraph title: Plasmid, C. difficile culture, cell lines ... The pathogen was cultured in Brain-Heart Infusion medium (BHI) (Difco) containing 5 mg/ml yeast extract and 0.1% L-cysteine at 37°C for 72 h in an anaerobic chamber (Coy Laboratory Products).

Modification:

Article Title: Identification of Genes Induced in Listeria monocytogenes during Growth and Attachment to Cut Cabbage, Using Differential Display
Article Snippet: L. monocytogenes strain 10403 ( ) and derivatives of this strain were grown in brain heart infusion medium (BHI) (Difco, Becton Dickinson, Franklin Lakes, NJ) or Listeria minimal medium (LMM) ( ). .. L. monocytogenes colonies were isolated and enumerated on modified Oxford agar (MOX) (Difco), which inhibits gram-negative bacteria and many gram-positive bacteria.

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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