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  • 99
    Thermo Fisher middlebrook 7h10 agar
    garA is required for growth of M . tuberculosis in vitro , survival in macrophages, and virulence in mice. ( A ) M . tuberculosis lacking garA was unable to grow on standard <t>7H10</t> medium unless supplemented with asparagine. Plasmid-borne garA restored the defect, but variants of garA with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in <t>Middlebrook</t> 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. ( B ) M . tuberculosis lacking garA (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental M . tuberculosis H37Rv (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. ( C ) M . tuberculosis lacking garA was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 10 5 bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with Δ garA Mt (red squares) was significantly lower than those infected with M . tuberculosis H37Rv (black circles), or complemented Δ garA Mt (black triangles) at all time points from day 7 (p
    Middlebrook 7h10 Agar, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 7h10 agar base
    garA is required for growth of M . tuberculosis in vitro , survival in macrophages, and virulence in mice. ( A ) M . tuberculosis lacking garA was unable to grow on standard <t>7H10</t> medium unless supplemented with asparagine. Plasmid-borne garA restored the defect, but variants of garA with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in <t>Middlebrook</t> 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. ( B ) M . tuberculosis lacking garA (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental M . tuberculosis H37Rv (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. ( C ) M . tuberculosis lacking garA was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 10 5 bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with Δ garA Mt (red squares) was significantly lower than those infected with M . tuberculosis H37Rv (black circles), or complemented Δ garA Mt (black triangles) at all time points from day 7 (p
    7h10 Agar Base, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Difco 7h 10 agar
    Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on <t>7H10</t> agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P
    7h 10 Agar, supplied by Difco, used in various techniques. Bioz Stars score: 79/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    MiddleBrook Pharmaceuticals 7h 10 medium
    Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on <t>7H10</t> agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P
    7h 10 Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 83/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    MiddleBrook Pharmaceuticals 7h 10 agar plates
    Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on <t>7H10</t> agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P
    7h 10 Agar Plates, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    MiddleBrook Pharmaceuticals middlebrook 7h 10 agar
    Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on <t>7H10</t> agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P
    Middlebrook 7h 10 Agar, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Difco 7h 10 agar medium
    Δ tatA and Δ tatC mutants have growth defects. (A) Single colonies of wild-type (mc 2 155, pMB198), Δ tatA mutant (MB692, pMB198), and complemented Δ tatA attB :: tatA (MB692, pJM124) strains are shown on <t>7H10</t> agar medium containing 0.1% Tween 80, grown at 37°C. Not shown are the Δ tatC mutant and the complemented Δ tatC strain, which display the same growth phenotypes. (B) Representative growth curves for wild-type (WT) (mc 2 155, pMV261), Δ tatC mutant (JM567, pMV261), and complemented Δ tatC (JM567, pJM120) strains in 7H9 liquid medium containing 0.1% Tween 80, grown at 37°C, are shown as OD 600 . (C) Viable-count measurements from the same cultures shown in panel B, shown here as the log 10 of viable CFU/ml.
    7h 10 Agar Medium, supplied by Difco, used in various techniques. Bioz Stars score: 80/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    MiddleBrook Pharmaceuticals 7h 10 solid medium
    Δ tatA and Δ tatC mutants have growth defects. (A) Single colonies of wild-type (mc 2 155, pMB198), Δ tatA mutant (MB692, pMB198), and complemented Δ tatA attB :: tatA (MB692, pJM124) strains are shown on <t>7H10</t> agar medium containing 0.1% Tween 80, grown at 37°C. Not shown are the Δ tatC mutant and the complemented Δ tatC strain, which display the same growth phenotypes. (B) Representative growth curves for wild-type (WT) (mc 2 155, pMV261), Δ tatC mutant (JM567, pMV261), and complemented Δ tatC (JM567, pJM120) strains in 7H9 liquid medium containing 0.1% Tween 80, grown at 37°C, are shown as OD 600 . (C) Viable-count measurements from the same cultures shown in panel B, shown here as the log 10 of viable CFU/ml.
    7h 10 Solid Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson 7h10
    NADH:NAD + sensor (Peredox) was overexpressed in Msmeg mc 2 155 using IMT100 with no visible effects on bacterial growth. (A) Msmeg expressing the reporter (MSP) colony on <t>7H10</t> agar plate under normal and UV-light demonstrating expression of the sensor. WT- Msmeg having pMV762; MSP-Msmeg containing IMT-100 and overexpressing Peredox. (B) Growth curve of Msmeg reporter strain (MSP) and Msmeg transformed with vector pMV762 only. (C,D) Representative excitation (C) and emission spectra (D) of Msmeg overexpressing Peredox-mCherry sensor showing excitation max at 400 and 587 nm, respectively, and emission max at 510 and 615 nm, respectively. T-sapphire indicates the fluorescence due to NADH:NAD + responsive Peredox and mCherry indicates the fluorescence due to NADH:NAD + unresponsive mCherry.
    7h10, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Difco 7h10 oadc
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
    7h10 Oadc, supplied by Difco, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Difco middelbrooks 7h10
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
    Middelbrooks 7h10, supplied by Difco, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson 7h10 broth
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
    7h10 Broth, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Becton Dickinson 7h10 plates
    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto <t>7H10-OADC</t> or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p
    7h10 Plates, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 98/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco 7h10 plates
    Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis . Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on <t>7H10</t> agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.
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    Becton Dickinson middelbrook 7h10
    Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis . Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on <t>7H10</t> agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.
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    Becton Dickinson middlesbrook 7h10
    Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis . Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on <t>7H10</t> agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.
    Middlesbrook 7h10, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals 7h10 broth
    Effect of deletion of MMAR2333 on colony morphology. Colonies of M. marinum wild-type (1218R), mutant (Δ MMAR2333 ), and complemented (Δ MMAR2333 -C) strains on <t>7H10</t> agar plates. Bar, 1 mm.
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    Difco 7h10 broth
    Effect of deletion of MMAR2333 on colony morphology. Colonies of M. marinum wild-type (1218R), mutant (Δ MMAR2333 ), and complemented (Δ MMAR2333 -C) strains on <t>7H10</t> agar plates. Bar, 1 mm.
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    Millipore 7h10 media
    Antibiotic resistance patterns of CDC1551 deletion strains. M. tuberculosis strains were streaked on <t>7H10</t> agar containing either 5 μg/ml KAN (A) or 2 μg/ml STR (B) and incubated for 28 days at 37°C.
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    Thermo Fisher 7h10 agar plates
    Kinyoun stain of patient isolate cultured on <t>7H10</t> agar at 35°C. Magnification, ×1,000.
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    Becton Dickinson 7h10 medium
    ATP binding by Rv2624c is required for its ability to increase survival in infected THP-1 cells. ( A ) The ATP-binding deficiencies of Rv2624c mutants did not influence growth on <t>7H10</t> medium. The mycobacterial strains indicated were serially diluted and were spotted onto solid 7H10 medium with 10% OADC. Pictures were taken after 3 d of incubation at 37 °C. ( B ) ATP-binding ability affects the survival of mycobacterial strains in THP-1 cells. Rv2624c mutant D17E with abrogated ATP-binding ability attenuated survival in THP-1 cells. The viability of Rv2624c mutant G109A, which had reduced ATP-binding ability, was comparable with that of wild-type Rv2624c. All results presented are representative of three independent experiments. p
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    Becton Dickinson middlebrook 7h10
    Survival of M. tuberculosis ( Mtb ) inside human macrophages. Human MDMs infected with Mtb H37Rv (MOI 5). The percentages of intracellular and not phagocytosed bacilli immediately after infection were determined by CFU assay and compared to inoculum (a). Lysed infected macrophages and released bacteria in the culture supernatants at 1, 3, 7 and 11 days after infection were plated on <t>Middlebrook</t> <t>7H10</t> with OADC and the CFU were counted after 14 days (b). Cytotoxicity induced by Mtb in human MDMs was measured by LDH activity (c). Values are the means ± SD from four independent experiments. The percentage of infected cells was measured, after 3 hr of infection (time 0) and 1, 3, 7 days after infection, by acridine-orange staining (d). and the representative pictures of infected MDMs, are shown (e). A significant difference was detected with respect to 1 and 3 days post-infection (* P
    Middlebrook 7h10, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebrook 7h10
    mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in <t>Middlebrook</t> 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook <t>7H10-OADC-glycerol-PLAM</t> plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).
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    HiMedia Laboratories middlebrook 7h10
    mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in <t>Middlebrook</t> 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook <t>7H10-OADC-glycerol-PLAM</t> plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).
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    Teknova 7h10 agar plates
    mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in <t>Middlebrook</t> 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook <t>7H10-OADC-glycerol-PLAM</t> plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).
    7h10 Agar Plates, supplied by Teknova, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebook 7h10 agar
    mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in <t>Middlebrook</t> 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook <t>7H10-OADC-glycerol-PLAM</t> plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).
    Middlebook 7h10 Agar, supplied by Difco, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco solid 7h10 agar
    Isolation and visualization of viable mycobacteria in the RASC. (A) M . smegmatis :: gfp growth on solid <t>7H10</t> agar plates from the Six-Stage Viable Andersen Cascade Impactor after wet release of 200 μl diluted culture into the RASC (30 000, 3000 and 300 colony forming units—CFU). The columns indicate the particle sizes captured on each plate across the 6 stages of the impactor, and the rows indicate the estimated total number of CFU passing through the impactor. Each release was repeated three times and the mean and SD for each plate are presented below the typical growth pattern distribution seen in the particle release. In all the releases the sampling was run for 5 minutes at 28 l/min resulting in the potential total capture of 3000, 300 and 30 CFU respectively. (B) SEM (left) and fluorescent microscopy (right) of M . smegmatis :: gfp isolated on a PM10 impactor following experimental release.
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    Becton Dickinson 7h10 solid agar
    Isolation and visualization of viable mycobacteria in the RASC. (A) M . smegmatis :: gfp growth on solid <t>7H10</t> agar plates from the Six-Stage Viable Andersen Cascade Impactor after wet release of 200 μl diluted culture into the RASC (30 000, 3000 and 300 colony forming units—CFU). The columns indicate the particle sizes captured on each plate across the 6 stages of the impactor, and the rows indicate the estimated total number of CFU passing through the impactor. Each release was repeated three times and the mean and SD for each plate are presented below the typical growth pattern distribution seen in the particle release. In all the releases the sampling was run for 5 minutes at 28 l/min resulting in the potential total capture of 3000, 300 and 30 CFU respectively. (B) SEM (left) and fluorescent microscopy (right) of M . smegmatis :: gfp isolated on a PM10 impactor following experimental release.
    7h10 Solid Agar, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HiMedia Laboratories 7h10
    M. smegmatis expressing PPE2 with intact NLS inhibited NO production and survived significantly longer inside macrophages. About 0.5 million peritoneal macrophages from C57BL/6 mice ( A and C ) or RAW 264.7 macrophages ( B and D ) were infected with either M. smegmatis harbouring pVV16 vector alone (Msmeg-pVV16), or pVV16-PPE2 (Msmeg-PPE2) or pVV16-ΔNLS-PPE2 (Msmeg-ΔNLS-PPE2) at 1:10 multiplicities of infection (MOI) and supernatants were removed for quantification of nitrite accumulation at 24 and 48 hours ( A and B ). The infected cells were lysed at 4, 24 and 48 hour post-infection. The cell lysates were plated on <t>7H10</t> plates supplemented with 10% ADC and 0.05% of Tween 80 and incubated for 4 days for counting the number of colony forming units (CFUs) ( C and D ). Data were expressed as mean ± SD of 3 independent experiments.
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    BVA Scientific 7h10 agar plates
    M. smegmatis expressing PPE2 with intact NLS inhibited NO production and survived significantly longer inside macrophages. About 0.5 million peritoneal macrophages from C57BL/6 mice ( A and C ) or RAW 264.7 macrophages ( B and D ) were infected with either M. smegmatis harbouring pVV16 vector alone (Msmeg-pVV16), or pVV16-PPE2 (Msmeg-PPE2) or pVV16-ΔNLS-PPE2 (Msmeg-ΔNLS-PPE2) at 1:10 multiplicities of infection (MOI) and supernatants were removed for quantification of nitrite accumulation at 24 and 48 hours ( A and B ). The infected cells were lysed at 4, 24 and 48 hour post-infection. The cell lysates were plated on <t>7H10</t> plates supplemented with 10% ADC and 0.05% of Tween 80 and incubated for 4 days for counting the number of colony forming units (CFUs) ( C and D ). Data were expressed as mean ± SD of 3 independent experiments.
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    Image Search Results


    garA is required for growth of M . tuberculosis in vitro , survival in macrophages, and virulence in mice. ( A ) M . tuberculosis lacking garA was unable to grow on standard 7H10 medium unless supplemented with asparagine. Plasmid-borne garA restored the defect, but variants of garA with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in Middlebrook 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. ( B ) M . tuberculosis lacking garA (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental M . tuberculosis H37Rv (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. ( C ) M . tuberculosis lacking garA was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 10 5 bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with Δ garA Mt (red squares) was significantly lower than those infected with M . tuberculosis H37Rv (black circles), or complemented Δ garA Mt (black triangles) at all time points from day 7 (p

    Journal: PLoS Pathogens

    Article Title: PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis

    doi: 10.1371/journal.ppat.1006399

    Figure Lengend Snippet: garA is required for growth of M . tuberculosis in vitro , survival in macrophages, and virulence in mice. ( A ) M . tuberculosis lacking garA was unable to grow on standard 7H10 medium unless supplemented with asparagine. Plasmid-borne garA restored the defect, but variants of garA with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in Middlebrook 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. ( B ) M . tuberculosis lacking garA (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental M . tuberculosis H37Rv (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. ( C ) M . tuberculosis lacking garA was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 10 5 bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with Δ garA Mt (red squares) was significantly lower than those infected with M . tuberculosis H37Rv (black circles), or complemented Δ garA Mt (black triangles) at all time points from day 7 (p

    Article Snippet: Bacterial strains, media, and culture M . tuberculosis H37Rv and M . smegmatis mc2 155 were routinely cultured on Middlebrook 7H10 agar (Oxoid) with 10% ADN (0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl) and Middlebrook 7H9 medium (Oxoid) with 10% ADN and 0.05% Tween 80.

    Techniques: In Vitro, Mouse Assay, Plasmid Preparation, Variant Assay, Standard Deviation, Infection

    Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P

    Journal: mBio

    Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

    doi: 10.1128/mBio.00333-17

    Figure Lengend Snippet: Strategy for identification of in vivo induced exported proteins. (a) Identification of in vivo induced exported proteins. Spleens from β-lactam-treated mice infected with the EXIT library were harvested after 2 weeks of infection. Spleen homogenates were plated in parallel on 7H10 agar without β-lactam to recover all clones (red Venn diagram) and on 7H10 agar containing β-lactam to recover clones exporting ‘BlaTEM fusion proteins during in vivo growth and in vitro growth (purple Venn diagram). The population of clones identified only or in significantly greater abundance on media lacking β-lactams represents proteins whose export was induced during infection (blue). (b) Sequenced read count values recovered from agar with or without β-lactam for the 593 EXIT proteins were plotted to compare abundances after β-lactam treatment in vivo , with the abundance after dual β-lactam treatment in vivo and in vitro indicated. The majority of proteins identified as exported in vivo remained highly abundant after additional β-lactam treatment in vitro (black). A total of 38 genes (highlighted in red) were identified as statistically less abundant after in vitro β-lactam selection, representing proteins exported significantly more in vivo than in vitro (see Materials and Methods for details on statistical analysis). (c) In vivo induced exported proteins with roles promoting growth in macrophages ( rv1508 :: tn , rv3707c:tn , rv0559c :: tn , and rv2536 :: tn ). Murine bone marrow-derived macrophages were infected with M. tuberculosis CDC1551 transposon mutants lacking individual in vivo induced exported proteins. At specific times postinfection, macrophage lysates were plated to measure intracellular CFU. The fold change in CFU over the course of the infection is plotted relative to the bacterial burden at day 0 postinfection. Statistical significance was determined by one-way analysis of variance (ANOVA) with multiple comparisons performed by the use of the Holm-Sidak (normal by Shapiro-Wilk) or Student-Newman-Keuls (nonnormal) test (*, P

    Article Snippet: At time points postinfection, the macrophages were lysed using 1% Triton X-100 (Sigma), and the lysates were diluted and plated for CFU determinations on 7H10 (Difco) or 7H11 (Sigma) plates supplemented with 0.05% Tween 80, 0.5% glycerol, 1× albumin dextrose saline (ADS), and 20 µg/ml kanamycin (Acros).

    Techniques: In Vivo, Mouse Assay, Infection, Clone Assay, In Vitro, Selection, Derivative Assay

    (a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.

    Journal: mBio

    Article Title: The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

    doi: 10.1128/mBio.00333-17

    Figure Lengend Snippet: (a) The ‘BlaTEM reporter. The ‘BlaTEM reporter is compatible with proteins localized to the bacterial cytoplasmic membrane or cell wall or secreted from the bacterial cell. The right panel indicates in-frame fusions to categories of exported proteins that confer β-lactam resistance (red). In-frame fusions to cytoplasmic proteins or the cytoplasmic domain of integral membrane proteins (purple) do not confer β-lactam resistance. (b) EXIT strategy. In step 1, a comprehensive library of 5 × 10 6 plasmids containing fragments of M. tuberculosis (Mtb) genomic DNA fused to the ‘ bla TEM reporter was constructed. The plasmid library was transformed into the ΔblaC β-lactamase-sensitive mutant of M. tuberculosis , and 5 × 10 6 transformants were pooled to generate the EXIT library. In step 2, mice were infected by intravenous injection with the EXIT library and treated with β-lactam antibiotics (oral gavage twice daily) to select for EXIT clones exporting ‘BlaTEM fusion proteins. β-lactam treatment began 1 day after infection and continued to 2 weeks after infection. Mice were sacrificed, and spleens and lungs were harvested and homogenized. In step 3, organ homogenates were plated on 7H10 agar and grown to recover M. tuberculosis clones that survived β-lactam treatment during infection. Plates were scraped, and colonies were pooled separately for lungs and spleens. In step 4, plasmids from the recovered bacteria and the input samples were isolated and the fusion junction was sequenced using next-generation sequencing. Sequencing primers were designed to read out of the ‘ bla TEM reporter and sequence the immediately adjacent M. tuberculosis DNA. Sequences were aligned to the M. tuberculosis genome. Unique sequences were counted to identify the abundance of each fusion junction site within the population. The genes that were most highly abundant after in vivo β-lactam treatment were identified, and the results corresponded to plasmids producing in-frame exported ‘BlaTEM fusion proteins.

    Article Snippet: At time points postinfection, the macrophages were lysed using 1% Triton X-100 (Sigma), and the lysates were diluted and plated for CFU determinations on 7H10 (Difco) or 7H11 (Sigma) plates supplemented with 0.05% Tween 80, 0.5% glycerol, 1× albumin dextrose saline (ADS), and 20 µg/ml kanamycin (Acros).

    Techniques: Transmission Electron Microscopy, Construct, Plasmid Preparation, Transformation Assay, Mutagenesis, Mouse Assay, Infection, Injection, Clone Assay, Isolation, Next-Generation Sequencing, Sequencing, In Vivo

    Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

    Journal: Journal of Bacteriology

    Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    doi: 10.1128/JB.00759-13

    Figure Lengend Snippet: Rel Mtb -mediated (p)ppGpp hydrolysis is required for growth in vitro and maintenance of ATP and GTP levels when (p)ppGpp is being produced. (A and B) An M. tuberculosis Δ rel Mtb strain containing an episomal vector expressing the WT TetR that induces expression of rel Mtb H80A from a cassette integrated at the attB site in the genome in the presence of ATc (Tet-Rel Mtb H80A strain, designated H80A here) and a WT M. tuberculosis strain containing an empty vector in the attB site and transformed with the same TetR-expressing plasmid (control strain). (A) Transcript levels in exponential-growth-phase cultures in liquid 7H9 media. rel Mtb H80A transcripts from the cassette at the attB site were detected in the Tet-Rel Mtb H80A strain, and transcripts from the endogenous rel Mtb gene were detected in the control strain. The first comparison shows the ratio of transcript levels in the presence compared to the absence of ATc in each strain (+ATc/−ATc), and it illustrates the induction of rel Mtb H80A in the Tet-Rel Mtb H80A strain when exposed to ATc and the unresponsiveness of the rel Mtb gene to ATc in the control strain. The second comparison shows the ratio of transcript levels in the Tet-Rel Mtb H80A strain compared to the control strain (H80A/control) under each condition to illustrate the low level of transcription of rel Mtb H80A in the absence of ATc compared to that of induced cultures. (B) M. tuberculosis Tet-Rel Mtb H80A and control strains were diluted and plated on 7H10 plates in the absence or presence of ATc. (C to G) M. smegmatis Δ rel Msm strains containing an episomal vector expressing the WT TetR that turns on expression of rel Mtb WT (Tet-Rel Mtb WT strain; designated the control) or rel Mtb H80A (Tet-Rel Mtb H80A strain; designated H80A here) from cassettes integrated at the attB site in the genome in the presence of ATc. (C and D) M. smegmatis strains were diluted and plated on 7H10 (C) and LB (D) plates in the absence or presence of ATc. (E) Graphic representation of the number of M. smegmatis CFU that grew in the presence compared to the absence of ATc when grown on LB, where all Tet-Rel Mtb H80A strain bacteria that grew in the presence of ATc on LB were suppressors and were no longer responsive to ATc treatment. Data are means ± SEM from 13 replicates. (F) ATP and GTP levels in M. smegmatis strains grown in the absence or presence of ATc for 6 h to an ODλ 600 of ∼0.6 in LB liquid media were measured by LC-MS/MS. The ratio of levels in the Tet-Rel Mtb H80A strain (H80A) to those in the Tet-Rel Mtb WT -expressing strain (control) under the same conditions is graphed. Data are the means ± SEM from 4 to 6 replicates. (E and F) The significance of differences was determined by calculating P values by Student's t tests; one asterisk indicates significance with a P value of

    Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

    Techniques: In Vitro, Produced, Plasmid Preparation, Expressing, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

    Journal: Journal of Bacteriology

    Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    doi: 10.1128/JB.00759-13

    Figure Lengend Snippet: Rv1366 is not necessary for M. tuberculosis growth or biofilm formation in vitro . (A) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rv1366 strain (lanes 2 to 4) using EcoRI-digested genomic DNA and a probe that spans nucleotides 1537706 to 1538406 of the M. tuberculosis genome. EcoRI digestion of WT M. tuberculosis yields a band at 5,083 bp. EcoRI digestion of Δ rv1366 results in a 2,226-bp band due to the replacement of the rv1366 gene with a hygromycin resistance cassette. (B) Southern blot analysis of WT M. tuberculosis Erdman (lane 1) and the Δ rel Mtb Δ rv1366 strain (lanes 2 to 6) using SmaI-digested genomic DNA and a probe that spans nucleotides 2910191 to 2910844 of the M. tuberculosis genome. SmaI digestion of WT M. tuberculosis yields a band at 2,257 bp. SmaI digestion of the Δ rel Mtb Δ rv1366 strain results in a 4,769-bp band due to the replacement of the rel Mtb gene with a hygromycin resistance cassette. (C and D) M. tuberculosis Δ rel Mtb , Δ rv1366 , and Δ rel Mtb Δ rv1366 strains containing a vector that constitutively expresses Rel Mtb WT or Rv1366 or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (C) or grown in Sauton's media in static, biofilm-forming conditions in a 24-well dish (D). (C) 7H10 plates were incubated for 3 weeks at 37°C. (D) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

    Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

    Techniques: In Vitro, Southern Blot, Plasmid Preparation, Incubation, Labeling

    Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

    Journal: Journal of Bacteriology

    Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    doi: 10.1128/JB.00759-13

    Figure Lengend Snippet: Rel Mtb -mediated (p)ppGpp synthesis is necessary for optimal M. tuberculosis growth and biofilm formation in vitro. M. tuberculosis Δ rel Mtb strains containing a vector that constitutively expresses Rel Mtb WT (WT) or Rel Mtb H344Y (H344Y) or containing an empty vector (−) integrated at the attB site in the genome were normalized to the same ODλ 600 and either diluted and plated on 7H10 plates (A) or grown in Sauton's medium in static, biofilm-forming conditions in a 24-well dish (B). (A) 7H10 plates were incubated for 3 weeks at 37°C. (B) Biofilms were incubated for 4 weeks at 37°C. The parent strain used is labeled above each photo, and the protein expressed from the attB site is labeled below the photo.

    Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

    Techniques: In Vitro, Plasmid Preparation, Incubation, Labeling

    Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

    Journal: Journal of Bacteriology

    Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

    doi: 10.1128/JB.00759-13

    Figure Lengend Snippet: Rel Mtb (p)ppGpp hydrolase activity is essential for acute and chronic M. tuberculosis infection of mice. Bacterial titers in the lungs (A, E, and G) and spleens (B and H) of C57BL/6 mice infected with either control or Tet-Rel Mtb H80A (designated H80A in the figure) M. tuberculosis strains. The parent strain for the control is WT M. tuberculosis Erdman, and the parent strain for Tet-Rel Mtb H80A is M. tuberculosis Δ rel Mtb . Both strains express TetR from a plasmid that confers streptomycin resistance and contain a kanamycin resistance cassette integrated into the attB site that, in the case of Tet-Rel Mtb H80A , carries the rel Mtb H80A allele. The strain symbols in panel A are the same for all panels. (A to C) Mice were given normal mouse chow throughout infection. (E and F) Mice were administered doxycycline-containing mouse chow starting at day 1 postinfection (designated by the arrow). (G to I) Mice were administered doxycycline-containing mouse chow starting at day 63 postinfection (designated by the arrow). (C, F, and I) The ratio of CFU from the lungs or spleens grown on 7H10 plates containing streptomycin and kanamycin compared to 7H10 containing no antibiotics (No AB). ND denotes when no colonies were recovered after plating 5% of the lung homogenate (limit of detection, 20 CFU). Data are means ± SEM of 6 Tet-Rel Mtb H80A strain-infected mice and 3 control strain-infected mice per time point from two replicate experiments. (E, G, and H) The significance of differences were determined by calculating P values by Student's t tests; two asterisks indicate significance with a P value of

    Article Snippet: Bacterial burden was determined by plating serial dilutions of lung and spleen homogenates onto 7H10 agar plates in the absence or presence of kanamycin and streptomycin.

    Techniques: Activity Assay, Infection, Mouse Assay, Plasmid Preparation

    Macroscopic morphology of M. smegmatis mc 2 155 strain spreading on the surface of a motility agar plate. mc 2 155 was grown in 7H10, and a single colony was transferred with a toothpick to the center of a 0.3% agar plate containing 7H9 basal medium without any added carbon source. The plate was sealed with parafilm and incubated at 37°C for 2 weeks.

    Journal: Journal of Bacteriology

    Article Title: Sliding Motility in Mycobacteria

    doi:

    Figure Lengend Snippet: Macroscopic morphology of M. smegmatis mc 2 155 strain spreading on the surface of a motility agar plate. mc 2 155 was grown in 7H10, and a single colony was transferred with a toothpick to the center of a 0.3% agar plate containing 7H9 basal medium without any added carbon source. The plate was sealed with parafilm and incubated at 37°C for 2 weeks.

    Article Snippet: Middlebrook 7H9 and 7H10 media (Difco) supplemented with ADC ( ) were used to grow M. avium.

    Techniques: Incubation

    Spreading phenotype of M. smegmatis colony morphology variants. (A) Colony morphology of mc 2 155, Sm-1, and Rg-1 on 7H10 agar plates. (B and C) Spreading phenotype of the morphology variants in 0.3% agarose plates containing M63 salts (B) or M63 with 2% glucose and 0.5% Casamino Acids (C). For both plates: mc 2 155, top left; Sm-1, top right; Rg-1, bottom.

    Journal: Journal of Bacteriology

    Article Title: Sliding Motility in Mycobacteria

    doi:

    Figure Lengend Snippet: Spreading phenotype of M. smegmatis colony morphology variants. (A) Colony morphology of mc 2 155, Sm-1, and Rg-1 on 7H10 agar plates. (B and C) Spreading phenotype of the morphology variants in 0.3% agarose plates containing M63 salts (B) or M63 with 2% glucose and 0.5% Casamino Acids (C). For both plates: mc 2 155, top left; Sm-1, top right; Rg-1, bottom.

    Article Snippet: Middlebrook 7H9 and 7H10 media (Difco) supplemented with ADC ( ) were used to grow M. avium.

    Techniques:

    Course of M. tuberculosis aerosol challenge in the lungs of memory and naive mice. Memory immune (●) and naive (□) mice were infected with ∼60 M. tuberculosis bacilli via aerosol. The numbers of viable bacilli were determined by plating serial dilutions of lung homogenates onto 7H10 plates and counting the colonies after 3 weeks at 37°C. Each time point represents four mice, and the experiment was repeated once. The standard error bars are too small to see on this graph. ∗, P ≤ 0.001, comparing naive and memory mice at each time point.

    Journal: Infection and Immunity

    Article Title: CD8+ T Cells Participate in the Memory Immune Response to Mycobacterium tuberculosis

    doi: 10.1128/IAI.69.7.4320-4328.2001

    Figure Lengend Snippet: Course of M. tuberculosis aerosol challenge in the lungs of memory and naive mice. Memory immune (●) and naive (□) mice were infected with ∼60 M. tuberculosis bacilli via aerosol. The numbers of viable bacilli were determined by plating serial dilutions of lung homogenates onto 7H10 plates and counting the colonies after 3 weeks at 37°C. Each time point represents four mice, and the experiment was repeated once. The standard error bars are too small to see on this graph. ∗, P ≤ 0.001, comparing naive and memory mice at each time point.

    Article Snippet: Mice were infected intravenously (i.v.) via tail vein with 2 × 105 live bacilli in 100 μl or by aerosol with approximately 100 live bacilli as determined by viable counts on 7H10 agar plates (Difco Laboratories, Detroit, Mich.).

    Techniques: Mouse Assay, Infection

    a) M. smegmatis , M. abscessus , M. fortuitum subsp. fortuitum , M. chelonae , and M. goodii were patched onto 7H10 agar media (Difco) at pH 7.0, 6.0, 5.5 or 5.0 to determine pigment production . b) M. avium intracellulare and M. avium subsps. avium were patched onto 7H10 agar media at pH 7.0, 6.0, and 5.5 to determine pigment production. c) From left to right: acetone only, M. smegmatis pH 7.0 extracted w/acetone, M. smegmatis pH 6.0 extracted w/acetone, M. smegmatis pH 5.5 extracted with acetone. d) Fluorescence specific activity units of M. smegmatis bearing empty reporter plasmid pFPV27 or the M. smegmatis probable carotenoid promoter upstream of gfp ( pCar ) at pH 7.0, 6.0 or 5.5.

    Journal: BMC Research Notes

    Article Title: Acidochromogenicity is a common characteristic in nontuberculous mycobacteria

    doi: 10.1186/1756-0500-4-466

    Figure Lengend Snippet: a) M. smegmatis , M. abscessus , M. fortuitum subsp. fortuitum , M. chelonae , and M. goodii were patched onto 7H10 agar media (Difco) at pH 7.0, 6.0, 5.5 or 5.0 to determine pigment production . b) M. avium intracellulare and M. avium subsps. avium were patched onto 7H10 agar media at pH 7.0, 6.0, and 5.5 to determine pigment production. c) From left to right: acetone only, M. smegmatis pH 7.0 extracted w/acetone, M. smegmatis pH 6.0 extracted w/acetone, M. smegmatis pH 5.5 extracted with acetone. d) Fluorescence specific activity units of M. smegmatis bearing empty reporter plasmid pFPV27 or the M. smegmatis probable carotenoid promoter upstream of gfp ( pCar ) at pH 7.0, 6.0 or 5.5.

    Article Snippet: Isolated colonies of mycobacteria were patched onto 7H10 agar media (Difco) adjusted to pH 5.5 or 6.0 with HCl, or pH 7.0 with NaOH and containing 10% ADC (bovine serum albumin, dextrose and NaCl) and 0.2% glycerol and were incubated for 72 hours at 37°C in the dark.

    Techniques: Fluorescence, Activity Assay, Plasmid Preparation

    Δ tatA and Δ tatC mutants have growth defects. (A) Single colonies of wild-type (mc 2 155, pMB198), Δ tatA mutant (MB692, pMB198), and complemented Δ tatA attB :: tatA (MB692, pJM124) strains are shown on 7H10 agar medium containing 0.1% Tween 80, grown at 37°C. Not shown are the Δ tatC mutant and the complemented Δ tatC strain, which display the same growth phenotypes. (B) Representative growth curves for wild-type (WT) (mc 2 155, pMV261), Δ tatC mutant (JM567, pMV261), and complemented Δ tatC (JM567, pJM120) strains in 7H9 liquid medium containing 0.1% Tween 80, grown at 37°C, are shown as OD 600 . (C) Viable-count measurements from the same cultures shown in panel B, shown here as the log 10 of viable CFU/ml.

    Journal: Journal of Bacteriology

    Article Title: The Twin-Arginine Translocation Pathway of Mycobacterium smegmatis Is Functional and Required for the Export of Mycobacterial ?-Lactamases

    doi: 10.1128/JB.187.22.7667-7679.2005

    Figure Lengend Snippet: Δ tatA and Δ tatC mutants have growth defects. (A) Single colonies of wild-type (mc 2 155, pMB198), Δ tatA mutant (MB692, pMB198), and complemented Δ tatA attB :: tatA (MB692, pJM124) strains are shown on 7H10 agar medium containing 0.1% Tween 80, grown at 37°C. Not shown are the Δ tatC mutant and the complemented Δ tatC strain, which display the same growth phenotypes. (B) Representative growth curves for wild-type (WT) (mc 2 155, pMV261), Δ tatC mutant (JM567, pMV261), and complemented Δ tatC (JM567, pJM120) strains in 7H9 liquid medium containing 0.1% Tween 80, grown at 37°C, are shown as OD 600 . (C) Viable-count measurements from the same cultures shown in panel B, shown here as the log 10 of viable CFU/ml.

    Article Snippet: Middlebrook 7H9 or 7H10 medium (Difco; BD Biosciences) was used for the culturing of M. smegmatis and M. tuberculosis .

    Techniques: Mutagenesis

    Increased release of membrane vesicles from the Δ pstA1 mutant requires RegX3. Wild-type M. tuberculosis Erdman (WT) and the Δ pstA1 , Δ pstA1 Δ regX3 , and Δ pstA1 Δ regX3 pND regX3 strains were grown for 5 days in Sauton’s complete medium without Tween 80. (A) Cellular proteins (5 µg), secreted proteins depleted of MV (5 µg), and MV suspension (20 µl) were separated and analyzed by Western blotting to detect the indicated proteins. (B) Nanoparticle tracking analyses of culture supernatants. The results were normalized to numbers of CFU per milliliter determined from a control culture grown in Sauton’s medium with Tween 80 and plated on 7H10 medium. Data are means ± standard deviations for three independent cultures. **, P

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity

    doi: 10.1128/mBio.00778-18

    Figure Lengend Snippet: Increased release of membrane vesicles from the Δ pstA1 mutant requires RegX3. Wild-type M. tuberculosis Erdman (WT) and the Δ pstA1 , Δ pstA1 Δ regX3 , and Δ pstA1 Δ regX3 pND regX3 strains were grown for 5 days in Sauton’s complete medium without Tween 80. (A) Cellular proteins (5 µg), secreted proteins depleted of MV (5 µg), and MV suspension (20 µl) were separated and analyzed by Western blotting to detect the indicated proteins. (B) Nanoparticle tracking analyses of culture supernatants. The results were normalized to numbers of CFU per milliliter determined from a control culture grown in Sauton’s medium with Tween 80 and plated on 7H10 medium. Data are means ± standard deviations for three independent cultures. **, P

    Article Snippet: Bacteria were routinely cultured in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 or on Middlebrook 7H10 agar medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) and 0.5% glycerol.

    Techniques: Mutagenesis, Western Blot

    Depletion of EccD 5 prevents ESX-5 secretion but does not affect membrane vesicle production. The eccD 5 Tet-OFF and Δ pstA1 eccD 5 Tet-OFF mutants were grown for 5 days in Sauton’s complete medium without Tween 80. Anhydrotetracycline hydrochloride (ATc; 100 ng/ml) was added as indicated to repress eccD 5 . (A) Cellular proteins (5 µg), secreted proteins depleted of MV (5 µg), and MV suspension (20 µl) were analyzed by Western blotting to detect the indicated proteins. (B) Nanoparticle tracking analysis of culture supernatants. Numbers of particles per milliliter were normalized to numbers of CFU per milliliter determined from a control culture of each strain grown with Tween 80 and plated on 7H10 complete medium. The data are means ± standard deviations for three independent cultures.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity

    doi: 10.1128/mBio.00778-18

    Figure Lengend Snippet: Depletion of EccD 5 prevents ESX-5 secretion but does not affect membrane vesicle production. The eccD 5 Tet-OFF and Δ pstA1 eccD 5 Tet-OFF mutants were grown for 5 days in Sauton’s complete medium without Tween 80. Anhydrotetracycline hydrochloride (ATc; 100 ng/ml) was added as indicated to repress eccD 5 . (A) Cellular proteins (5 µg), secreted proteins depleted of MV (5 µg), and MV suspension (20 µl) were analyzed by Western blotting to detect the indicated proteins. (B) Nanoparticle tracking analysis of culture supernatants. Numbers of particles per milliliter were normalized to numbers of CFU per milliliter determined from a control culture of each strain grown with Tween 80 and plated on 7H10 complete medium. The data are means ± standard deviations for three independent cultures.

    Article Snippet: Bacteria were routinely cultured in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 or on Middlebrook 7H10 agar medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) and 0.5% glycerol.

    Techniques: Western Blot

    Increased vesicle release from the Δ pstA1 mutant is independent of VirR. Wild-type M. tuberculosis Erdman (WT) and the Δ pstA1 , Δ virR , Δ pstA1 Δ virR , WT p virR , and Δ pstA1 p virR strains were grown for 5 days in Sauton’s complete medium without Tween 80. (A) Cellular proteins (5 µg) and MV suspension (20 µl) were analyzed by Western blotting to detect the indicated proteins. (B) Nanoparticle tracking analysis of culture supernatants. Numbers of particles per milliliter were normalized to numbers of CFU per milliliter determined from a control culture grown in Sauton’s medium with Tween 80 and plated on 7H10 medium. Data are means ± standard deviations for three independent cultures. The results of all statistical analyses are compared to WT levels unless otherwise indicated. *, P

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity

    doi: 10.1128/mBio.00778-18

    Figure Lengend Snippet: Increased vesicle release from the Δ pstA1 mutant is independent of VirR. Wild-type M. tuberculosis Erdman (WT) and the Δ pstA1 , Δ virR , Δ pstA1 Δ virR , WT p virR , and Δ pstA1 p virR strains were grown for 5 days in Sauton’s complete medium without Tween 80. (A) Cellular proteins (5 µg) and MV suspension (20 µl) were analyzed by Western blotting to detect the indicated proteins. (B) Nanoparticle tracking analysis of culture supernatants. Numbers of particles per milliliter were normalized to numbers of CFU per milliliter determined from a control culture grown in Sauton’s medium with Tween 80 and plated on 7H10 medium. Data are means ± standard deviations for three independent cultures. The results of all statistical analyses are compared to WT levels unless otherwise indicated. *, P

    Article Snippet: Bacteria were routinely cultured in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 or on Middlebrook 7H10 agar medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) and 0.5% glycerol.

    Techniques: Mutagenesis, Western Blot

    Incomplete repression of eccD 5 transcription has moderate effects on growth. (A to D) Wild-type M. tuberculosis Erdman (WT) and the Δ pstA1 , eccD 5 Tet-OFF, and Δ pstA1 eccD 5 Tet-OFF strains were inoculated in 7H9 complete medium at an OD 600 of 0.05 and grown at 37°C with aeration. Anhydrotetracycline hydrochloride (ATc; 100 ng/ml) was added at day 0 and day 7 as indicated. Growth was monitored by daily OD 600 measurements (A and C) and by plating serially diluted cultures on 7H10 medium to determine numbers of viable CFU per milliliter on days 0, 3, 6, 9, 12, and 15 (B and D). The key in panel B applies to panels A and B; the key in panel D applies to panels C and D. **, P

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity

    doi: 10.1128/mBio.00778-18

    Figure Lengend Snippet: Incomplete repression of eccD 5 transcription has moderate effects on growth. (A to D) Wild-type M. tuberculosis Erdman (WT) and the Δ pstA1 , eccD 5 Tet-OFF, and Δ pstA1 eccD 5 Tet-OFF strains were inoculated in 7H9 complete medium at an OD 600 of 0.05 and grown at 37°C with aeration. Anhydrotetracycline hydrochloride (ATc; 100 ng/ml) was added at day 0 and day 7 as indicated. Growth was monitored by daily OD 600 measurements (A and C) and by plating serially diluted cultures on 7H10 medium to determine numbers of viable CFU per milliliter on days 0, 3, 6, 9, 12, and 15 (B and D). The key in panel B applies to panels A and B; the key in panel D applies to panels C and D. **, P

    Article Snippet: Bacteria were routinely cultured in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 or on Middlebrook 7H10 agar medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) and 0.5% glycerol.

    Techniques:

    NADH:NAD + sensor (Peredox) was overexpressed in Msmeg mc 2 155 using IMT100 with no visible effects on bacterial growth. (A) Msmeg expressing the reporter (MSP) colony on 7H10 agar plate under normal and UV-light demonstrating expression of the sensor. WT- Msmeg having pMV762; MSP-Msmeg containing IMT-100 and overexpressing Peredox. (B) Growth curve of Msmeg reporter strain (MSP) and Msmeg transformed with vector pMV762 only. (C,D) Representative excitation (C) and emission spectra (D) of Msmeg overexpressing Peredox-mCherry sensor showing excitation max at 400 and 587 nm, respectively, and emission max at 510 and 615 nm, respectively. T-sapphire indicates the fluorescence due to NADH:NAD + responsive Peredox and mCherry indicates the fluorescence due to NADH:NAD + unresponsive mCherry.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Imaging the NADH:NAD+ Homeostasis for Understanding the Metabolic Response of Mycobacterium to Physiologically Relevant Stresses

    doi: 10.3389/fcimb.2016.00145

    Figure Lengend Snippet: NADH:NAD + sensor (Peredox) was overexpressed in Msmeg mc 2 155 using IMT100 with no visible effects on bacterial growth. (A) Msmeg expressing the reporter (MSP) colony on 7H10 agar plate under normal and UV-light demonstrating expression of the sensor. WT- Msmeg having pMV762; MSP-Msmeg containing IMT-100 and overexpressing Peredox. (B) Growth curve of Msmeg reporter strain (MSP) and Msmeg transformed with vector pMV762 only. (C,D) Representative excitation (C) and emission spectra (D) of Msmeg overexpressing Peredox-mCherry sensor showing excitation max at 400 and 587 nm, respectively, and emission max at 510 and 615 nm, respectively. T-sapphire indicates the fluorescence due to NADH:NAD + responsive Peredox and mCherry indicates the fluorescence due to NADH:NAD + unresponsive mCherry.

    Article Snippet: Middlebrook 7H9, 7H10, and 7H11 media and O-ADC were from BD biosciences.

    Techniques: Expressing, Transformation Assay, Plasmid Preparation, Fluorescence

    CD40 engagement of DCs enhances control of Mtb infection. B6 DCs were left uninfected or infected with heat-killed Mtb in the presence or absence of CD40LT treatment (1 μg/ml), or infected with heat-killed hip1 mutant Mtb, each for 24 hours. Cells were washed twice and reconstituted in PBS to deliver 1x10 6 DC per mouse intratracheally. (A) Diagram of experimental design. Lung responses were assessed 6 and 12 days post-instillation. Mice were infected through the aerosol route with ~100 CFU Mtb 15 days post-instillation and bacterial burden was assessed 35 days (5 weeks) post-challenge. (B) Frequencies of CD44 + CD4 T cells in the lungs 6 and 12 days after intratracheal instillation of DCs. (C) Frequencies of IL-17 + (top) and IFN-γ + (bottom) CD4 + cells in the lungs 6 and 12 days after intratracheal instillation of DCs. Cells were stimulated with PMA (80 ng/ml) and ionomycin (500 ng/ml). (D) Lung bacterial burden 35 days post-challenge (overall day 50 post-DC intratracheal instillation). Bacterial burden was assessed by plating homogenized lungs on 7H10 agar plates and counting CFU. (E) Lung CD4 + IL-17 + and IFN-γ + frequencies at day 50 following Mtb whole cell lysate (10 μg/ml) restimulation. (F) Correlation plots showing association between lung bacterial burden and IL-17 (left) or IFN-γ (right) responses to WCL restimulation. A linear regression was utilized to generate a best-fit line and Spearman’s correlation coefficient calculated. 4–5 mice were used for each group. Statistical significance (B-E) was determined using one-way analysis of variance correcting for multiple comparisons. * p

    Journal: PLoS Pathogens

    Article Title: Engaging the CD40-CD40L pathway augments T-helper cell responses and improves control of Mycobacterium tuberculosis infection

    doi: 10.1371/journal.ppat.1006530

    Figure Lengend Snippet: CD40 engagement of DCs enhances control of Mtb infection. B6 DCs were left uninfected or infected with heat-killed Mtb in the presence or absence of CD40LT treatment (1 μg/ml), or infected with heat-killed hip1 mutant Mtb, each for 24 hours. Cells were washed twice and reconstituted in PBS to deliver 1x10 6 DC per mouse intratracheally. (A) Diagram of experimental design. Lung responses were assessed 6 and 12 days post-instillation. Mice were infected through the aerosol route with ~100 CFU Mtb 15 days post-instillation and bacterial burden was assessed 35 days (5 weeks) post-challenge. (B) Frequencies of CD44 + CD4 T cells in the lungs 6 and 12 days after intratracheal instillation of DCs. (C) Frequencies of IL-17 + (top) and IFN-γ + (bottom) CD4 + cells in the lungs 6 and 12 days after intratracheal instillation of DCs. Cells were stimulated with PMA (80 ng/ml) and ionomycin (500 ng/ml). (D) Lung bacterial burden 35 days post-challenge (overall day 50 post-DC intratracheal instillation). Bacterial burden was assessed by plating homogenized lungs on 7H10 agar plates and counting CFU. (E) Lung CD4 + IL-17 + and IFN-γ + frequencies at day 50 following Mtb whole cell lysate (10 μg/ml) restimulation. (F) Correlation plots showing association between lung bacterial burden and IL-17 (left) or IFN-γ (right) responses to WCL restimulation. A linear regression was utilized to generate a best-fit line and Spearman’s correlation coefficient calculated. 4–5 mice were used for each group. Statistical significance (B-E) was determined using one-way analysis of variance correcting for multiple comparisons. * p

    Article Snippet: Briefly, bacteria were grown at 37°C in Middlebrook 7H9 broth or 7H10 agar supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (Becton Dickinson, Franklin Lakes, NJ), 0.5% glycerol, and 0.05% Tween 80 (for broth), with the addition of 25 μg/ml kanamycin (Sigma-Aldrich, St. Louis, MO) for hip1 mutant Mtb.

    Techniques: Infection, Mutagenesis, Mouse Assay

    The anti-bacillus effect of vitamin B1 (VB1) in macrophages. (A,B) Bone marrow-derived macrophages were pretreated with phosphate buffer saline, VB1, Rosi, or mixture of VB1 and Rosi for 24 h and then challenged with Mycobacterium tuberculosis H37Rv (MOI 5) for 1 h. (A) Intracellular viable bacteria were detected with colony-forming unit (CFU) assays at 0, 1, 2, 3 day post-infection. (B) Intracellular viable bacteria were detected with CFU assays at 72 h post-infection. (C,D) PPAR-γ fl/fl or PPAR-γ fl/fl -Lys2-cre mice were infected with H37Rv (~200 bacteria/mouse). Oral administration with water (Ctrl), VB1, INH, or Rosi ( n = 5 mice/group) was started from the day after infection (day 1) and continued for 2 weeks. CFUs were obtained from the lung cell lysates by serial dilution and plating on 7H10 agars in triplicate. The colonies were counted after 4 weeks. (C) C57BL/6J mice. (D) PPAR-γ fl/fl or PPAR-γ fl/fl -Lys2-cre mice. Data shown are the mean ± SD. * P

    Journal: Frontiers in Immunology

    Article Title: Vitamin B1 Helps to Limit Mycobacterium tuberculosis Growth via Regulating Innate Immunity in a Peroxisome Proliferator-Activated Receptor-γ-Dependent Manner

    doi: 10.3389/fimmu.2018.01778

    Figure Lengend Snippet: The anti-bacillus effect of vitamin B1 (VB1) in macrophages. (A,B) Bone marrow-derived macrophages were pretreated with phosphate buffer saline, VB1, Rosi, or mixture of VB1 and Rosi for 24 h and then challenged with Mycobacterium tuberculosis H37Rv (MOI 5) for 1 h. (A) Intracellular viable bacteria were detected with colony-forming unit (CFU) assays at 0, 1, 2, 3 day post-infection. (B) Intracellular viable bacteria were detected with CFU assays at 72 h post-infection. (C,D) PPAR-γ fl/fl or PPAR-γ fl/fl -Lys2-cre mice were infected with H37Rv (~200 bacteria/mouse). Oral administration with water (Ctrl), VB1, INH, or Rosi ( n = 5 mice/group) was started from the day after infection (day 1) and continued for 2 weeks. CFUs were obtained from the lung cell lysates by serial dilution and plating on 7H10 agars in triplicate. The colonies were counted after 4 weeks. (C) C57BL/6J mice. (D) PPAR-γ fl/fl or PPAR-γ fl/fl -Lys2-cre mice. Data shown are the mean ± SD. * P

    Article Snippet: Bacterial burden was determined by plating serial dilutions of spleen and lung homogenates onto 7H10 agar plates (BD Biosciences, USA) with 10% OADC.

    Techniques: Derivative Assay, Infection, Mouse Assay, Serial Dilution

    The anti-bacillus effect of vitamin B1 (VB1) in mice with Mycobacterium tuberculosis infection. C57BL/6J mice were infected with H37Rv (~200 bacteria/mouse). Oral administration with water (Ctrl), VB1, and INH ( n = 15 mice/group) was started from the day after infection (day 1) and continued for 1, 2, and 4 weeks alternatively. The lungs and spleens were analyzed at indicated time. Colony-forming units (CFUs) were obtained from the lung and spleen cell lysates by serial dilution and plating on 7H10 agars in triplicate. The colonies were counted after 4 weeks. Data shown are the mean ± SD. * P

    Journal: Frontiers in Immunology

    Article Title: Vitamin B1 Helps to Limit Mycobacterium tuberculosis Growth via Regulating Innate Immunity in a Peroxisome Proliferator-Activated Receptor-γ-Dependent Manner

    doi: 10.3389/fimmu.2018.01778

    Figure Lengend Snippet: The anti-bacillus effect of vitamin B1 (VB1) in mice with Mycobacterium tuberculosis infection. C57BL/6J mice were infected with H37Rv (~200 bacteria/mouse). Oral administration with water (Ctrl), VB1, and INH ( n = 15 mice/group) was started from the day after infection (day 1) and continued for 1, 2, and 4 weeks alternatively. The lungs and spleens were analyzed at indicated time. Colony-forming units (CFUs) were obtained from the lung and spleen cell lysates by serial dilution and plating on 7H10 agars in triplicate. The colonies were counted after 4 weeks. Data shown are the mean ± SD. * P

    Article Snippet: Bacterial burden was determined by plating serial dilutions of spleen and lung homogenates onto 7H10 agar plates (BD Biosciences, USA) with 10% OADC.

    Techniques: Mouse Assay, Infection, Serial Dilution

    Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto 7H10-OADC or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p

    Journal: PLoS Pathogens

    Article Title: Methylfolate Trap Promotes Bacterial Thymineless Death by Sulfa Drugs

    doi: 10.1371/journal.ppat.1005949

    Figure Lengend Snippet: Methylfolate trap in Mycobacterium tuberculosis . ( A ) SULFA sensitivity of H37Rv-derived strains in 7H9-S medium, in the absence or presence of exogenous B 12 and/or methionine (Met), was analyzed using the MTT method. Cultures grown to an OD 600 of 2 were washed and diluted in 7H9-S. Wells were inoculated with 10 4 cells in the presence of 1.56 μg/ml SMZ supplemented with 0.3 mM B 12 alone and in combination with 1 mM methionine. Plates were incubated for 7 days at 37°C. MTT solution prepared in 1X PBS, pH 6.8, was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( B ) H37Rv-derived strains were grown to OD 600 of 1 and 5 μl cultures were spotted onto 7H10-OADC or the same medium supplemented with 5.7 μg/ml SCP, 0.5 mM B 12 , and 1 mM methionine. Plates were incubated at 37°C for 4 weeks. The spotted cell suspension for each strain under both conditions was collected and suspended in 7H9-OADC. Suspensions underwent 10-fold serial dilutions from which 100 μl aliquots were plated onto 7H10-OADC in triplicate. After 4 weeks of incubation at 37°C, viability was determined by counting colony forming unit (c.f.u.) and normalized to the c.f.u. values of the input inoculum. The y-axis represents c.f.u. fold-change on a log10 scale. Bars represent standard deviations from experimental triplicates. P values are shown above the bars and were calculated using unpaired Student’s t-test; ns, no significant difference compared to corresponding H37Rv sample in same condition. Representative 10 −6 dilution plates provide a visual comparison between strains in viability (top). ( C ) Domain alignment of MetH proteins from H37Rv and CDC1551 using PROSITE ( http://prosite.expasy.org ). Domains are labeled as the cofactors to which they bind. ( D ) SULFA sensitivity of CDC1551-derived strains in Dubos medium in the absence or presence of B 12 and methionine was analyzed using the MTT method. Cultures growing at an OD 600 of 2 were washed and diluted in Dubos medium. Wells containing two-fold increasing SMZ concentrations (0–8 μg/ml) were inoculated with 10 4 cells of each strain, as indicated in the box on the left. Test plates, supplemented with varying concentrations of B 12 (0.25–1 μM), without or with 1 mM methionine, were incubated for 7 days at 37°C. MTT solution was added to each well and incubated for 24 hours. The reaction was stopped by adding SDS-DMF solution followed by incubation at 37°C for an additional 24 hours. Purple formazan indicates living cells. ( E ) Survival of H37Rv (Red), its derived metH mutant (RvΔ metH , Blue) and the complemented strain (RvΔ metH / metH , Green) in macrophages, non-treated or treated with 40 μg/ml SMZ. Presented data are the c.f.u. values of internalized bacteria at 0 h (0) and after 72 h chase without (-) or with (+) 40 μg/ml SMZ. Shown are means of biological triplicates with standard deviations. ** p

    Article Snippet: M . tuberculosis strains were grown in 7H10-OADC or Dubos-ADC media (Difco).

    Techniques: Derivative Assay, MTT Assay, Incubation, Labeling, Mutagenesis

    Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis . Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on 7H10 agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.

    Journal: Journal of Clinical Microbiology

    Article Title: Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis

    doi: 10.1128/JCM.41.7.3252-3259.2003

    Figure Lengend Snippet: Diagnostic nitrate reductase activity of M. tuberculosis and the narG mutant of M. tuberculosis . Three-week-old cultures from M. tuberculosis wild-type and the narG mutant of M. tuberculosis on 7H10 agar plates were used to inoculate three loops (tube a), one loop (tube b), and one-third loop (tube c) of bacilli into phosphate buffer containing 10 mM nitrate and tested for the accumulation of nitrite after 2 h at 37°C.

    Article Snippet: All strains were cultured in 7H9 broth or on 7H10 plates (Difco Laboratories, Inc., Detroit, Mich.) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% ADS (0.5% bovine albumin fraction V, 0.2% glucose, 140 mM NaCl) unless indicated otherwise.

    Techniques: Diagnostic Assay, Activity Assay, Mutagenesis

    Effect of deletion of MMAR2333 on colony morphology. Colonies of M. marinum wild-type (1218R), mutant (Δ MMAR2333 ), and complemented (Δ MMAR2333 -C) strains on 7H10 agar plates. Bar, 1 mm.

    Journal: Journal of Bacteriology

    Article Title: Identification of a Glycosyltransferase from Mycobacterium marinum Involved in Addition of a Caryophyllose Moiety in Lipooligosaccharides ▿ Involved in Addition of a Caryophyllose Moiety in Lipooligosaccharides ▿ †

    doi: 10.1128/JB.00065-11

    Figure Lengend Snippet: Effect of deletion of MMAR2333 on colony morphology. Colonies of M. marinum wild-type (1218R), mutant (Δ MMAR2333 ), and complemented (Δ MMAR2333 -C) strains on 7H10 agar plates. Bar, 1 mm.

    Article Snippet: To assess the effects of the loss of MMAR2333 function on cell wall lipid composition, cultures of wild-type, mutant, and complemented strains grown at 30°C in 7H10 broth (Middlebrook 7H10 minus agar) were first pulsed with 1 μCi/ml [14 C]acetate (57 mCi/mmol) to label lipids.

    Techniques: Mutagenesis

    Antibiotic resistance patterns of CDC1551 deletion strains. M. tuberculosis strains were streaked on 7H10 agar containing either 5 μg/ml KAN (A) or 2 μg/ml STR (B) and incubated for 28 days at 37°C.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Aminoglycoside Cross-Resistance in Mycobacterium tuberculosis Due to Mutations in the 5? Untranslated Region of whiB7

    doi: 10.1128/AAC.02191-12

    Figure Lengend Snippet: Antibiotic resistance patterns of CDC1551 deletion strains. M. tuberculosis strains were streaked on 7H10 agar containing either 5 μg/ml KAN (A) or 2 μg/ml STR (B) and incubated for 28 days at 37°C.

    Article Snippet: The susceptibilities to KAN and STR were determined according to guidelines and definitions stated by the Clinical and Laboratory Standards Institute , using 7H10 agar containing KAN (Sigma) at concentrations of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, and 80 μg/ml and STR, isoniazid (INH), capreomycin (CAP), rifampin (RIF), and ofloxacin (OFX) at 0.25, 0.5, 1, 2, 4, 8, 10, and 16 μg/ml.

    Techniques: Incubation

    Kinyoun stain of patient isolate cultured on 7H10 agar at 35°C. Magnification, ×1,000.

    Journal: Journal of Clinical Microbiology

    Article Title: Brown-Pigmented Mycobacterium mageritense as a Cause of Prosthetic Valve Endocarditis and Bloodstream Infection

    doi: 10.1128/JCM.01041-15

    Figure Lengend Snippet: Kinyoun stain of patient isolate cultured on 7H10 agar at 35°C. Magnification, ×1,000.

    Article Snippet: Positive blood cultures were subcultured to Middlebrook 7H10 agar plates (Remel, Lenexa, KS) and Lowenstein-Jensen (LJ) agar slants (Becton Dickinson, Sparks, MD) and incubated at 35°C.

    Techniques: Staining, Cell Culture

    ATP binding by Rv2624c is required for its ability to increase survival in infected THP-1 cells. ( A ) The ATP-binding deficiencies of Rv2624c mutants did not influence growth on 7H10 medium. The mycobacterial strains indicated were serially diluted and were spotted onto solid 7H10 medium with 10% OADC. Pictures were taken after 3 d of incubation at 37 °C. ( B ) ATP-binding ability affects the survival of mycobacterial strains in THP-1 cells. Rv2624c mutant D17E with abrogated ATP-binding ability attenuated survival in THP-1 cells. The viability of Rv2624c mutant G109A, which had reduced ATP-binding ability, was comparable with that of wild-type Rv2624c. All results presented are representative of three independent experiments. p

    Journal: Scientific Reports

    Article Title: Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria

    doi: 10.1038/srep35462

    Figure Lengend Snippet: ATP binding by Rv2624c is required for its ability to increase survival in infected THP-1 cells. ( A ) The ATP-binding deficiencies of Rv2624c mutants did not influence growth on 7H10 medium. The mycobacterial strains indicated were serially diluted and were spotted onto solid 7H10 medium with 10% OADC. Pictures were taken after 3 d of incubation at 37 °C. ( B ) ATP-binding ability affects the survival of mycobacterial strains in THP-1 cells. Rv2624c mutant D17E with abrogated ATP-binding ability attenuated survival in THP-1 cells. The viability of Rv2624c mutant G109A, which had reduced ATP-binding ability, was comparable with that of wild-type Rv2624c. All results presented are representative of three independent experiments. p

    Article Snippet: Reagents and growth media Mycobacterial strains were grown in liquid Middlebrook 7H9 medium (Becton Dickinson, Sparks, MD, USA) containing 10% OADC supplement (oleic acid, albumin, dextrose, catalase; Becton Dickinson, Sparks, MD, USA) and 0.05% Tween 80 (Sigma, St. Louis, MO, USA), or on solid 7H10 medium (Becton Dickinson, Sparks, MD, USA) containing 10% OADC supplement.

    Techniques: Binding Assay, Infection, Incubation, Mutagenesis

    In vitro characterization of Δ rv2623 . (A) Growth curves of cultures inoculated (10 6 CFU/ml) into 7H9+10% OADC+0.05% Tween 80 (top) and in minimal Sauton's media (bottom); Erdman (closed boxes, solid line) and Δ rv2623 (open boxes, dashed line) cultures. Error bars represent the standard error of the means; each curve is a combination of at least three independent experiments. (B) Overexpression of Rv2623 in M. smegmatis . The top panel represents serial dilutions (1∶10) of the empty vector pMV261-containing negative control strain and Rv2623-overexpressing strain harboring pMV261:: rv2623 . Diluted stationary phase M. smegmatis culture was spotted (5 µl) onto solid 7H10 media supplemented with 10% OADC and kanamycin (40 µg/mL). Photographs were taken after three days incubation at 37°C. Growth of corresponding strains in liquid medium was assessed based on the time to detection determined using a BD BACTEC 9000MB system (bottom). The various strains were inoculated at 10 4 CFU/ml in triplicates. Data shown are representative of several independent experiments. ***p

    Journal: PLoS Pathogens

    Article Title: Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP-Binding: Requirement for Establishing Chronic Persistent Infection

    doi: 10.1371/journal.ppat.1000460

    Figure Lengend Snippet: In vitro characterization of Δ rv2623 . (A) Growth curves of cultures inoculated (10 6 CFU/ml) into 7H9+10% OADC+0.05% Tween 80 (top) and in minimal Sauton's media (bottom); Erdman (closed boxes, solid line) and Δ rv2623 (open boxes, dashed line) cultures. Error bars represent the standard error of the means; each curve is a combination of at least three independent experiments. (B) Overexpression of Rv2623 in M. smegmatis . The top panel represents serial dilutions (1∶10) of the empty vector pMV261-containing negative control strain and Rv2623-overexpressing strain harboring pMV261:: rv2623 . Diluted stationary phase M. smegmatis culture was spotted (5 µl) onto solid 7H10 media supplemented with 10% OADC and kanamycin (40 µg/mL). Photographs were taken after three days incubation at 37°C. Growth of corresponding strains in liquid medium was assessed based on the time to detection determined using a BD BACTEC 9000MB system (bottom). The various strains were inoculated at 10 4 CFU/ml in triplicates. Data shown are representative of several independent experiments. ***p

    Article Snippet: For the determination of the number of colony forming units (CFU) and examination of growth on solid media, Middlebrook 7H10 agar medium (Becton Dickinson) supplemented with 0.5% glycerol and 10% OADC was used.

    Techniques: In Vitro, Over Expression, Plasmid Preparation, Negative Control, Incubation

    Survival of M. tuberculosis ( Mtb ) inside human macrophages. Human MDMs infected with Mtb H37Rv (MOI 5). The percentages of intracellular and not phagocytosed bacilli immediately after infection were determined by CFU assay and compared to inoculum (a). Lysed infected macrophages and released bacteria in the culture supernatants at 1, 3, 7 and 11 days after infection were plated on Middlebrook 7H10 with OADC and the CFU were counted after 14 days (b). Cytotoxicity induced by Mtb in human MDMs was measured by LDH activity (c). Values are the means ± SD from four independent experiments. The percentage of infected cells was measured, after 3 hr of infection (time 0) and 1, 3, 7 days after infection, by acridine-orange staining (d). and the representative pictures of infected MDMs, are shown (e). A significant difference was detected with respect to 1 and 3 days post-infection (* P

    Journal: Immunology

    Article Title: Gene expression profiling of human macrophages at late time of infection with Mycobacterium tuberculosis

    doi: 10.1111/j.1365-2567.2006.02378.x

    Figure Lengend Snippet: Survival of M. tuberculosis ( Mtb ) inside human macrophages. Human MDMs infected with Mtb H37Rv (MOI 5). The percentages of intracellular and not phagocytosed bacilli immediately after infection were determined by CFU assay and compared to inoculum (a). Lysed infected macrophages and released bacteria in the culture supernatants at 1, 3, 7 and 11 days after infection were plated on Middlebrook 7H10 with OADC and the CFU were counted after 14 days (b). Cytotoxicity induced by Mtb in human MDMs was measured by LDH activity (c). Values are the means ± SD from four independent experiments. The percentage of infected cells was measured, after 3 hr of infection (time 0) and 1, 3, 7 days after infection, by acridine-orange staining (d). and the representative pictures of infected MDMs, are shown (e). A significant difference was detected with respect to 1 and 3 days post-infection (* P

    Article Snippet: Representative samples were thawed and colony-forming units (CFUs) per ml were enumerated by plating on Middlebrook 7H10 (Becton Dickinson, Cockeysville, MD) supplemented with Middlebrook oleic acid-albumin-dextrose-catalase (OADC).

    Techniques: Infection, Colony-forming Unit Assay, Activity Assay, Staining

    mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).

    Journal: mBio

    Article Title: Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy

    doi: 10.1128/mBio.00938-18

    Figure Lengend Snippet: mc 2 7901 and mc 2 7902 grow similarly to virulent M. tuberculosis in vitro . (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100, and growth was followed by recording optical density at 600 nm (OD 600 ) over time. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1 with mc 2 7901, mc 2 7902, or H37Rv. At the indicated time points, macrophages were lysed, and bacterial titers were determined by plating for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) Growth of mc 2 7901 and mc 2 7902 in RAW 264.7 macrophages relative to the inocula (same experiment as in panel B). Mean with standard deviation is plotted ( n = 2).

    Article Snippet: Middlebrook 7H10 (Difco) supplemented with 10% (vol/vol) OADC and 0.2% (vol/vol) glycerol was used as solid medium.

    Techniques: In Vitro, Standard Deviation, Infection

    mc 2 7901- and mc 2 7902-derived MDR strains grow slower than parental strains in vitro and are killed by second-line TB drugs or nutrient starvation. (A) Log-phase cultures were diluted 1/100, and growth was followed by recording optical density at 600 nm. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1. At the indicated time points, macrophages were lysed to determine bacterial titers. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) PLAM-supplemented log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were treated with EOK (ethionamide [25 mg/liter], ofloxacin [5 mg/liter], kanamycin [20 mg/liter]). (D) Log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were washed five times in PBS and resuspended in Middlebrook 7H9-OADC-glycerol-tyloxapol containing PLAM (dilution factor 1/100) or not. In experiments in panels B, C, and D, the strains were initially grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM. Samples were taken at the indicated time points, diluted, and plated for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. Means with standard deviations are plotted ( n = 3).

    Journal: mBio

    Article Title: Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy

    doi: 10.1128/mBio.00938-18

    Figure Lengend Snippet: mc 2 7901- and mc 2 7902-derived MDR strains grow slower than parental strains in vitro and are killed by second-line TB drugs or nutrient starvation. (A) Log-phase cultures were diluted 1/100, and growth was followed by recording optical density at 600 nm. Mean with standard deviation is plotted ( n = 2). (B) RAW 264.7 macrophages were infected at an MOI of 1. At the indicated time points, macrophages were lysed to determine bacterial titers. PLAM was added to the macrophage growth medium, and the medium was changed at each time point. (C) PLAM-supplemented log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were treated with EOK (ethionamide [25 mg/liter], ofloxacin [5 mg/liter], kanamycin [20 mg/liter]). (D) Log-phase cultures of mc 2 7901, mc 2 7902, mc 2 8248, and mc 2 8251 were washed five times in PBS and resuspended in Middlebrook 7H9-OADC-glycerol-tyloxapol containing PLAM (dilution factor 1/100) or not. In experiments in panels B, C, and D, the strains were initially grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM. Samples were taken at the indicated time points, diluted, and plated for CFU on Middlebrook 7H10-OADC-glycerol-PLAM plates. Means with standard deviations are plotted ( n = 3).

    Article Snippet: Middlebrook 7H10 (Difco) supplemented with 10% (vol/vol) OADC and 0.2% (vol/vol) glycerol was used as solid medium.

    Techniques: Derivative Assay, In Vitro, Standard Deviation, Infection

    Isolation and visualization of viable mycobacteria in the RASC. (A) M . smegmatis :: gfp growth on solid 7H10 agar plates from the Six-Stage Viable Andersen Cascade Impactor after wet release of 200 μl diluted culture into the RASC (30 000, 3000 and 300 colony forming units—CFU). The columns indicate the particle sizes captured on each plate across the 6 stages of the impactor, and the rows indicate the estimated total number of CFU passing through the impactor. Each release was repeated three times and the mean and SD for each plate are presented below the typical growth pattern distribution seen in the particle release. In all the releases the sampling was run for 5 minutes at 28 l/min resulting in the potential total capture of 3000, 300 and 30 CFU respectively. (B) SEM (left) and fluorescent microscopy (right) of M . smegmatis :: gfp isolated on a PM10 impactor following experimental release.

    Journal: PLoS ONE

    Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)

    doi: 10.1371/journal.pone.0146658

    Figure Lengend Snippet: Isolation and visualization of viable mycobacteria in the RASC. (A) M . smegmatis :: gfp growth on solid 7H10 agar plates from the Six-Stage Viable Andersen Cascade Impactor after wet release of 200 μl diluted culture into the RASC (30 000, 3000 and 300 colony forming units—CFU). The columns indicate the particle sizes captured on each plate across the 6 stages of the impactor, and the rows indicate the estimated total number of CFU passing through the impactor. Each release was repeated three times and the mean and SD for each plate are presented below the typical growth pattern distribution seen in the particle release. In all the releases the sampling was run for 5 minutes at 28 l/min resulting in the potential total capture of 3000, 300 and 30 CFU respectively. (B) SEM (left) and fluorescent microscopy (right) of M . smegmatis :: gfp isolated on a PM10 impactor following experimental release.

    Article Snippet: For the pilot TB studies, the Andersen impactor contained solid 7H10 agar (Difco) plates supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex.

    Techniques: Isolation, Sampling, Microscopy

    M. smegmatis expressing PPE2 with intact NLS inhibited NO production and survived significantly longer inside macrophages. About 0.5 million peritoneal macrophages from C57BL/6 mice ( A and C ) or RAW 264.7 macrophages ( B and D ) were infected with either M. smegmatis harbouring pVV16 vector alone (Msmeg-pVV16), or pVV16-PPE2 (Msmeg-PPE2) or pVV16-ΔNLS-PPE2 (Msmeg-ΔNLS-PPE2) at 1:10 multiplicities of infection (MOI) and supernatants were removed for quantification of nitrite accumulation at 24 and 48 hours ( A and B ). The infected cells were lysed at 4, 24 and 48 hour post-infection. The cell lysates were plated on 7H10 plates supplemented with 10% ADC and 0.05% of Tween 80 and incubated for 4 days for counting the number of colony forming units (CFUs) ( C and D ). Data were expressed as mean ± SD of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: The PPE2 protein of Mycobacterium tuberculosis translocates to host nucleus and inhibits nitric oxide production

    doi: 10.1038/srep39706

    Figure Lengend Snippet: M. smegmatis expressing PPE2 with intact NLS inhibited NO production and survived significantly longer inside macrophages. About 0.5 million peritoneal macrophages from C57BL/6 mice ( A and C ) or RAW 264.7 macrophages ( B and D ) were infected with either M. smegmatis harbouring pVV16 vector alone (Msmeg-pVV16), or pVV16-PPE2 (Msmeg-PPE2) or pVV16-ΔNLS-PPE2 (Msmeg-ΔNLS-PPE2) at 1:10 multiplicities of infection (MOI) and supernatants were removed for quantification of nitrite accumulation at 24 and 48 hours ( A and B ). The infected cells were lysed at 4, 24 and 48 hour post-infection. The cell lysates were plated on 7H10 plates supplemented with 10% ADC and 0.05% of Tween 80 and incubated for 4 days for counting the number of colony forming units (CFUs) ( C and D ). Data were expressed as mean ± SD of 3 independent experiments.

    Article Snippet: Serial dilutions of cell extracts were plated on 7H10 plates supplemented with 10% ADC (HIMEDIA, India) and 25 μg/ml Kanamycin, 50 μg/ml Hygromycin.

    Techniques: Expressing, Mouse Assay, Infection, Plasmid Preparation, Incubation

    Effect of NO inhibitor, aminoguanidine (AG) and NO donor sodium nitroprusside (SNP) on survival of M. smegmatis harbouring either backbone vector (Msmeg-pVV16), or expressing PPE2 (Msmeg-PPE2) or ΔNLS-PPE2 (Msmeg-ΔNLS-PPE2). Murine peritoneal macrophages (0.5 × 10 6 ) were either cultured in medium alone or pre-treated with either AG (300 μg/ml) or SNP (100 μM) followed by infection with either Msmeg-pVV16, or Msmeg-PPE2 or Msmeg-ΔNLS-PPE2 at 1:10 multiplicities of infection (MOI). Twenty four hours after infection, supernatants were removed for nitrite determination and the cells were lysed for CFU assay. The cell lysates were plated on 7H10 plates supplemented with 10% ADC, Kanamycin (25 μg/ml), Hygromycin B (50 μg/ml) and 0.05% of Tween 80 and incubated for 4 days for counting the number of colony forming units (CFUs). Data were expressed as mean ± SD of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: The PPE2 protein of Mycobacterium tuberculosis translocates to host nucleus and inhibits nitric oxide production

    doi: 10.1038/srep39706

    Figure Lengend Snippet: Effect of NO inhibitor, aminoguanidine (AG) and NO donor sodium nitroprusside (SNP) on survival of M. smegmatis harbouring either backbone vector (Msmeg-pVV16), or expressing PPE2 (Msmeg-PPE2) or ΔNLS-PPE2 (Msmeg-ΔNLS-PPE2). Murine peritoneal macrophages (0.5 × 10 6 ) were either cultured in medium alone or pre-treated with either AG (300 μg/ml) or SNP (100 μM) followed by infection with either Msmeg-pVV16, or Msmeg-PPE2 or Msmeg-ΔNLS-PPE2 at 1:10 multiplicities of infection (MOI). Twenty four hours after infection, supernatants were removed for nitrite determination and the cells were lysed for CFU assay. The cell lysates were plated on 7H10 plates supplemented with 10% ADC, Kanamycin (25 μg/ml), Hygromycin B (50 μg/ml) and 0.05% of Tween 80 and incubated for 4 days for counting the number of colony forming units (CFUs). Data were expressed as mean ± SD of 3 independent experiments.

    Article Snippet: Serial dilutions of cell extracts were plated on 7H10 plates supplemented with 10% ADC (HIMEDIA, India) and 25 μg/ml Kanamycin, 50 μg/ml Hygromycin.

    Techniques: Plasmid Preparation, Expressing, Cell Culture, Infection, Colony-forming Unit Assay, Incubation