middlebrook 7h9 Search Results


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  • 99
    Thermo Fisher middlebrook 7h9
    Disruption of pknG or removal of the phosphorylation motif of garA caused a nutrient-dependent growth defect in M . smegmatis . ( A ) All strains grew at the same rate on standard <t>Middlebrook</t> <t>7H9</t> medium. ( B ) Δ garA Ms grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH 4 Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” garA ). ( C + D ) Δ pknG Ms grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon ( C ) or nitrogen source ( D ) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. ( E ) Δ pknG Ms formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve ( D ) imaged at 60 hours. Growth of Δ garA Ms complemented with phosphoablative GarA (EAAS) was equivalent to that of Δ pknG Ms complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.
    Middlebrook 7h9, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    middlebrook 7h9 - by Bioz Stars, 2020-03
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    Millipore middlebrook 7h9 broth
    Disruption of pknG or removal of the phosphorylation motif of garA caused a nutrient-dependent growth defect in M . smegmatis . ( A ) All strains grew at the same rate on standard <t>Middlebrook</t> <t>7H9</t> medium. ( B ) Δ garA Ms grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH 4 Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” garA ). ( C + D ) Δ pknG Ms grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon ( C ) or nitrogen source ( D ) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. ( E ) Δ pknG Ms formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve ( D ) imaged at 60 hours. Growth of Δ garA Ms complemented with phosphoablative GarA (EAAS) was equivalent to that of Δ pknG Ms complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.
    Middlebrook 7h9 Broth, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher middlebrook 7h9 broth
    Disruption of pknG or removal of the phosphorylation motif of garA caused a nutrient-dependent growth defect in M . smegmatis . ( A ) All strains grew at the same rate on standard <t>Middlebrook</t> <t>7H9</t> medium. ( B ) Δ garA Ms grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH 4 Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” garA ). ( C + D ) Δ pknG Ms grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon ( C ) or nitrogen source ( D ) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. ( E ) Δ pknG Ms formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve ( D ) imaged at 60 hours. Growth of Δ garA Ms complemented with phosphoablative GarA (EAAS) was equivalent to that of Δ pknG Ms complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.
    Middlebrook 7h9 Broth, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebrook 7h9
    Toxic effect of the five toxins on the growth of M. smegmatis . (A) Transformation of pMV306AC-toxin plasmid into M. smegmatis mc 2 155. One microgram of DNA was electrophorated and cells were plated on <t>Middlebrook</t> 7H10 solid media. The plates were incubated for 3 days at 37°C. (B) Effect of toxin on the induction on colony formation of M. smegmatis mc 2 155. Each transformant from (A) was grown for 3 days at 37°C. The culture was diluted 100-fold into Middlebrook <t>7H9</t> liquid media and streaked on Middlebrook 7H10 solid media in the absence and presence of 0.2% acetamide. The numbers in the circle correspond to those in (A) .
    Middlebrook 7h9, supplied by Difco, used in various techniques. Bioz Stars score: 99/100, based on 334 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson middlebrook 7h9
    Activities of anti-TB TB drugs in the DAC system are not affected by inoculum size. (A) The MIC values for the first-line drugs according to various inoculum sizes. MTB H37Rv ATCC 27294 cells from ~10 4 to ~10 7 CFU/mL were inoculated in the DAC system and the MIC values were determined. The spots (circle, triangle and square) of each drug indicate the MICs values from three independently repeated experiments. The tested concentrations for each drug were a two-fold dilution scale. The breakpoints of the BACTEC 460 TB and MGIT 960 systems based on the <t>Middlebrook</t> <t>7H9</t> broth were adopted for the DAC system; 0.1 μg/mL for INH, 1.0 μg/mL for RIF, 1.0 μg/mL for STR, and 5.0 μg/mL for EMB. The red horizontal line indicates the breakpoints for each drug. All MIC values were determined under the breakpoints. (B) The comparison of an inoculum effect for the first-line drugs against H37Rv between the DAC system and two routine methods, the L-J method (solid) and MGIT 960 method (liquid). The various inoculum sizes from ~10 4 to ~10 7 CFU/mL were tested. The DST results were represented as resistant (R) or susceptible (S). The DST results were consistently “S” regardless of the various inoculum sizes in the DAC system, whereas they were changed from “S” to “R” or “Error” over 5 × 10 6 CFU/mL in the two routine methods. (C) The MICs values from clinical isolates in the various inoculum sizes. The MIC values from two pan-susceptible and two resistant strains were estimated for the first-line drugs. The inoculum sizes were from ~10 3 CFU/mL to ~10 8 CFU/mL for two drug susceptible strains and two drug resistant strains. There was no inoculum effect with the clinical isolates in the DAC system.
    Middlebrook 7h9, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore middlebrook 7h9
    Activities of anti-TB TB drugs in the DAC system are not affected by inoculum size. (A) The MIC values for the first-line drugs according to various inoculum sizes. MTB H37Rv ATCC 27294 cells from ~10 4 to ~10 7 CFU/mL were inoculated in the DAC system and the MIC values were determined. The spots (circle, triangle and square) of each drug indicate the MICs values from three independently repeated experiments. The tested concentrations for each drug were a two-fold dilution scale. The breakpoints of the BACTEC 460 TB and MGIT 960 systems based on the <t>Middlebrook</t> <t>7H9</t> broth were adopted for the DAC system; 0.1 μg/mL for INH, 1.0 μg/mL for RIF, 1.0 μg/mL for STR, and 5.0 μg/mL for EMB. The red horizontal line indicates the breakpoints for each drug. All MIC values were determined under the breakpoints. (B) The comparison of an inoculum effect for the first-line drugs against H37Rv between the DAC system and two routine methods, the L-J method (solid) and MGIT 960 method (liquid). The various inoculum sizes from ~10 4 to ~10 7 CFU/mL were tested. The DST results were represented as resistant (R) or susceptible (S). The DST results were consistently “S” regardless of the various inoculum sizes in the DAC system, whereas they were changed from “S” to “R” or “Error” over 5 × 10 6 CFU/mL in the two routine methods. (C) The MICs values from clinical isolates in the various inoculum sizes. The MIC values from two pan-susceptible and two resistant strains were estimated for the first-line drugs. The inoculum sizes were from ~10 3 CFU/mL to ~10 8 CFU/mL for two drug susceptible strains and two drug resistant strains. There was no inoculum effect with the clinical isolates in the DAC system.
    Middlebrook 7h9, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals 7h9 middlebrook
    Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in <t>Middlebrook</t> <t>7H9</t> medium. Error bars show standard errors of the
    7h9 Middlebrook, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 98/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fluka Chemie middlebrook 7h9
    Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in <t>Middlebrook</t> <t>7H9</t> medium. Error bars show standard errors of the
    Middlebrook 7h9, supplied by Fluka Chemie, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc middlebrook 7h9
    Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in <t>Middlebrook</t> <t>7H9</t> medium. Error bars show standard errors of the
    Middlebrook 7h9, supplied by Illumina Inc, 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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    MiddleBrook Pharmaceuticals middlebrook 7h9
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 1645 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad middlebrook 7h9
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific middlebrook 7h9
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson middlebrook 7h9 powder
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9 Powder, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson middlebrook 7h9 liquid
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9 Liquid, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hardy Diagnostics middlebrook 7h9 broth
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9 Broth, supplied by Hardy Diagnostics, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Avantor middlebrook 7h9 media
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9 Media, supplied by Avantor, used in various techniques. Bioz Stars score: 86/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HiMedia Laboratories middlebrook 7h9 broth
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9 Broth, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 96/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebrooke 7h9 broth
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrooke 7h9 Broth, supplied by Difco, used in various techniques. Bioz Stars score: 83/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM middlebrook 7h9 broth
    (A) Growth curves of TB101 in <t>Middlebrook</t> <t>7H9</t> containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.
    Middlebrook 7h9 Broth, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals 7h9 broth middlebrook
    The Mycobacterium tuberculosis (Mtb) RpoB H526D mutant strain exhibits slow growth rate and altered colony morphology. (A) Growth kinetics of Mtb strains in supplemented <t>Middlebrook</t> <t>7H9</t> broth. ∗∗ p
    7h9 Broth Middlebrook, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 89/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals middlebrook 7h9 adn
    The Mycobacterium tuberculosis (Mtb) RpoB H526D mutant strain exhibits slow growth rate and altered colony morphology. (A) Growth kinetics of Mtb strains in supplemented <t>Middlebrook</t> <t>7H9</t> broth. ∗∗ p
    Middlebrook 7h9 Adn, supplied by MiddleBrook Pharmaceuticals, 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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    MiddleBrook Pharmaceuticals middlebrook 7h9 oadc
    V-13–012725 and V-13–011503 inhibit cholesterol breakdown. (A) Growth of wild type Mtb is not inhibited in <t>7H9</t> <t>OADC</t> containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC 50 values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.
    Middlebrook 7h9 Oadc, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebrook 7h9 medium
    Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in <t>Middlebrook</t> <t>7H9</t> medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation
    Middlebrook 7h9 Medium, supplied by Difco, used in various techniques. Bioz Stars score: 99/100, based on 839 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson middlebrook 7h9 medium
    Cell surface of Msmeg-PE11 is highly hydrophobic. ( a ) Pellicle formation of Msmeg-pVV or Msmeg-PE11 was monitored by growing standing cultures of the strains without shaking in <t>Middlebrook</t> <t>7H9</t> medium in absence of Tween 80 at 37 °C for various time points. ( b ) Biofilm formation was quantified by crystal violet staining for which Msmeg-pVV or Msmeg-PE11 cells were washed, stained with 1% crystal violet and ethanol extract was spectrophotometrically measured at 570 nm. ( c ) Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 medium with (upper panel) or without (lower panel) 0.05% Tween-80 at 37 °C with shaking for 24 h and 48 h. Cultures were then allowed to settle at room temperature for 30 min. ( d ) In another experiment, Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 broth with congo red (100 μg/ml) and 0.05% Tween 80 for 24 h, 48 h and 72 h at 37 °C. Cells were next washed and re-suspended in acetone. Congo red in the supernatant was spectrophotometrically measured at 490 nm. Data are representative of mean ± SD of three different experiments.
    Middlebrook 7h9 Medium, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore middlebrook 7h9 medium
    Cell surface of Msmeg-PE11 is highly hydrophobic. ( a ) Pellicle formation of Msmeg-pVV or Msmeg-PE11 was monitored by growing standing cultures of the strains without shaking in <t>Middlebrook</t> <t>7H9</t> medium in absence of Tween 80 at 37 °C for various time points. ( b ) Biofilm formation was quantified by crystal violet staining for which Msmeg-pVV or Msmeg-PE11 cells were washed, stained with 1% crystal violet and ethanol extract was spectrophotometrically measured at 570 nm. ( c ) Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 medium with (upper panel) or without (lower panel) 0.05% Tween-80 at 37 °C with shaking for 24 h and 48 h. Cultures were then allowed to settle at room temperature for 30 min. ( d ) In another experiment, Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 broth with congo red (100 μg/ml) and 0.05% Tween 80 for 24 h, 48 h and 72 h at 37 °C. Cells were next washed and re-suspended in acetone. Congo red in the supernatant was spectrophotometrically measured at 490 nm. Data are representative of mean ± SD of three different experiments.
    Middlebrook 7h9 Medium, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals middlebrook 7h9 medium
    FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in <t>Middlebrook</t> <t>7H9</t> medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
    Middlebrook 7h9 Medium, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 99/100, based on 2844 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLife Solutions middlebrook 7h9 medium
    FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in <t>Middlebrook</t> <t>7H9</t> medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
    Middlebrook 7h9 Medium, supplied by BioLife Solutions, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HiMedia Laboratories middlebrook 7h9
    FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in <t>Middlebrook</t> <t>7H9</t> medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
    Middlebrook 7h9, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co middlebrook 7h9 medium
    FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in <t>Middlebrook</t> <t>7H9</t> medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.
    Middlebrook 7h9 Medium, supplied by Merck & Co, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson middlebrook 7h9 oadc broth
    Growth rates of wild-type M. chelonae ATCC 35752, its isogenic porin knock-out mutants and complemented MCH_4691 mutant strains in <t>7H9-OADC-Tween</t> 80 broth at 30°C. Shown are representative results of two to three independent experiments using different culture batches.
    Middlebrook 7h9 Oadc Broth, supplied by Becton Dickinson, 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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    Image Search Results


    Disruption of pknG or removal of the phosphorylation motif of garA caused a nutrient-dependent growth defect in M . smegmatis . ( A ) All strains grew at the same rate on standard Middlebrook 7H9 medium. ( B ) Δ garA Ms grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH 4 Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” garA ). ( C + D ) Δ pknG Ms grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon ( C ) or nitrogen source ( D ) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. ( E ) Δ pknG Ms formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve ( D ) imaged at 60 hours. Growth of Δ garA Ms complemented with phosphoablative GarA (EAAS) was equivalent to that of Δ pknG Ms complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.

    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: Disruption of pknG or removal of the phosphorylation motif of garA caused a nutrient-dependent growth defect in M . smegmatis . ( A ) All strains grew at the same rate on standard Middlebrook 7H9 medium. ( B ) Δ garA Ms grew slower than wild type on minimal Sauton’s medium containing 20 mM propionate, 20 mM NH 4 Cl plus 0.05% tyloxapol, and this growth defect could be fully complemented by GarA lacking phosphorylation sites (truncated “trunc.” garA ). ( C + D ) Δ pknG Ms grew slower than wild type and formed clumps (inset photo) on medium containing glutamate as sole carbon ( C ) or nitrogen source ( D ) (minimal Sauton’s with either 30 mM glutamate plus tyloxapol, or 1% glycerol, 10 mM glutamate plus tyloxapol). Data plotted are the mean and standard deviation of at least 3 independent experiments. ( E ) Δ pknG Ms formed clumps when glutamate was the sole carbon or nitrogen source. The photograph shows a microplate from growth curve ( D ) imaged at 60 hours. Growth of Δ garA Ms complemented with phosphoablative GarA (EAAS) was equivalent to that of Δ pknG Ms complemented with truncated GarA in all tested conditions so only the dataset for truncated GarA is shown for clarity.

    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: Mass Spectrometry, Standard Deviation

    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

    Toxic effect of the five toxins on the growth of M. smegmatis . (A) Transformation of pMV306AC-toxin plasmid into M. smegmatis mc 2 155. One microgram of DNA was electrophorated and cells were plated on Middlebrook 7H10 solid media. The plates were incubated for 3 days at 37°C. (B) Effect of toxin on the induction on colony formation of M. smegmatis mc 2 155. Each transformant from (A) was grown for 3 days at 37°C. The culture was diluted 100-fold into Middlebrook 7H9 liquid media and streaked on Middlebrook 7H10 solid media in the absence and presence of 0.2% acetamide. The numbers in the circle correspond to those in (A) .

    Journal: Frontiers in Microbiology

    Article Title: Functional Studies of Five Toxin-Antitoxin Modules in Mycobacterium tuberculosis H37Rv

    doi: 10.3389/fmicb.2016.02071

    Figure Lengend Snippet: Toxic effect of the five toxins on the growth of M. smegmatis . (A) Transformation of pMV306AC-toxin plasmid into M. smegmatis mc 2 155. One microgram of DNA was electrophorated and cells were plated on Middlebrook 7H10 solid media. The plates were incubated for 3 days at 37°C. (B) Effect of toxin on the induction on colony formation of M. smegmatis mc 2 155. Each transformant from (A) was grown for 3 days at 37°C. The culture was diluted 100-fold into Middlebrook 7H9 liquid media and streaked on Middlebrook 7H10 solid media in the absence and presence of 0.2% acetamide. The numbers in the circle correspond to those in (A) .

    Article Snippet: M. smegmatis mc2 155 was grown in Middlebrook 7H9 (a liquid medium, Difco) containing 0.2% glycerol and 0.02% Tween 80 or Middlebrook 7H10 (a solid agar medium, Difco) containing 0.2% glycerol.

    Techniques: Transformation Assay, Plasmid Preparation, Incubation

    Sources of RNA samples. ( A ) MTB H37Rv was grown in Middlebrook 7H9 medium. Samples were withdrawn for OD 600 measurements and RNA extraction; sigA mRNA and 16S rRNA were quantified by QRT-PCR. •, OD 600 ; □, sigA /16S ratio × 10,000 ± SD ( n ≥ 3). ( B ) C57BL/6 mice were aerosol-infected with ≈500 cfu of MTB H37Rv. Lungs were collected for cfu enumeration and RNA extraction; sigA mRNA and 16S rRNA were quantified by QRT-PCR. •, cfu ± SD ( n ≥ 4); □, sigA /16S ratio × 10,000 ± SD ( n ≥ 3). ( C ) Multilayered organization of granulomatous tissue bordering a 4-cm-diameter cavity in the left upper lung lobe of patient 2 (hematoxylin/eosin-stained). At the periphery were residual airspace and lymphocytic aggregates ( i ). A capsule of fibroblasts and fibrin with scattered epithelioid macrophages ( ii ) surrounded a cellular zone containing clusters of multinucleated giant cells ( iii ). Giant cells were also prominent at the border between the cellular zone and the underlying zone of caseation necrosis ( iv ). The surface of the cavity wall bordering on the necrotic zone showed extensive leukocytic infiltration and numerous acid-fast bacilli, not revealed by hematoxylin/eosin staining ( v ). Magnification: ×4( Left ), ×40 ( Right ). Similar pathology was observed in lung specimens from all four patients analyzed in this study.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients

    doi: 10.1073/pnas.2436197100

    Figure Lengend Snippet: Sources of RNA samples. ( A ) MTB H37Rv was grown in Middlebrook 7H9 medium. Samples were withdrawn for OD 600 measurements and RNA extraction; sigA mRNA and 16S rRNA were quantified by QRT-PCR. •, OD 600 ; □, sigA /16S ratio × 10,000 ± SD ( n ≥ 3). ( B ) C57BL/6 mice were aerosol-infected with ≈500 cfu of MTB H37Rv. Lungs were collected for cfu enumeration and RNA extraction; sigA mRNA and 16S rRNA were quantified by QRT-PCR. •, cfu ± SD ( n ≥ 4); □, sigA /16S ratio × 10,000 ± SD ( n ≥ 3). ( C ) Multilayered organization of granulomatous tissue bordering a 4-cm-diameter cavity in the left upper lung lobe of patient 2 (hematoxylin/eosin-stained). At the periphery were residual airspace and lymphocytic aggregates ( i ). A capsule of fibroblasts and fibrin with scattered epithelioid macrophages ( ii ) surrounded a cellular zone containing clusters of multinucleated giant cells ( iii ). Giant cells were also prominent at the border between the cellular zone and the underlying zone of caseation necrosis ( iv ). The surface of the cavity wall bordering on the necrotic zone showed extensive leukocytic infiltration and numerous acid-fast bacilli, not revealed by hematoxylin/eosin staining ( v ). Magnification: ×4( Left ), ×40 ( Right ). Similar pathology was observed in lung specimens from all four patients analyzed in this study.

    Article Snippet: MTB H37Rv and clinical strains were grown in plastic roller bottles at 37°C in Middlebrook 7H9 broth (Difco) containing 10% oleic acid-albumin-dextrose-catalase (OADC) (Difco), 0.5% glycerol, and 0.05% Tween 80.

    Techniques: RNA Extraction, Quantitative RT-PCR, Mouse Assay, Infection, Staining

    Activities of anti-TB TB drugs in the DAC system are not affected by inoculum size. (A) The MIC values for the first-line drugs according to various inoculum sizes. MTB H37Rv ATCC 27294 cells from ~10 4 to ~10 7 CFU/mL were inoculated in the DAC system and the MIC values were determined. The spots (circle, triangle and square) of each drug indicate the MICs values from three independently repeated experiments. The tested concentrations for each drug were a two-fold dilution scale. The breakpoints of the BACTEC 460 TB and MGIT 960 systems based on the Middlebrook 7H9 broth were adopted for the DAC system; 0.1 μg/mL for INH, 1.0 μg/mL for RIF, 1.0 μg/mL for STR, and 5.0 μg/mL for EMB. The red horizontal line indicates the breakpoints for each drug. All MIC values were determined under the breakpoints. (B) The comparison of an inoculum effect for the first-line drugs against H37Rv between the DAC system and two routine methods, the L-J method (solid) and MGIT 960 method (liquid). The various inoculum sizes from ~10 4 to ~10 7 CFU/mL were tested. The DST results were represented as resistant (R) or susceptible (S). The DST results were consistently “S” regardless of the various inoculum sizes in the DAC system, whereas they were changed from “S” to “R” or “Error” over 5 × 10 6 CFU/mL in the two routine methods. (C) The MICs values from clinical isolates in the various inoculum sizes. The MIC values from two pan-susceptible and two resistant strains were estimated for the first-line drugs. The inoculum sizes were from ~10 3 CFU/mL to ~10 8 CFU/mL for two drug susceptible strains and two drug resistant strains. There was no inoculum effect with the clinical isolates in the DAC system.

    Journal: Scientific Reports

    Article Title: A rapid culture system uninfluenced by an inoculum effect increases reliability and convenience for drug susceptibility testing of Mycobacterium tuberculosis

    doi: 10.1038/s41598-018-26419-z

    Figure Lengend Snippet: Activities of anti-TB TB drugs in the DAC system are not affected by inoculum size. (A) The MIC values for the first-line drugs according to various inoculum sizes. MTB H37Rv ATCC 27294 cells from ~10 4 to ~10 7 CFU/mL were inoculated in the DAC system and the MIC values were determined. The spots (circle, triangle and square) of each drug indicate the MICs values from three independently repeated experiments. The tested concentrations for each drug were a two-fold dilution scale. The breakpoints of the BACTEC 460 TB and MGIT 960 systems based on the Middlebrook 7H9 broth were adopted for the DAC system; 0.1 μg/mL for INH, 1.0 μg/mL for RIF, 1.0 μg/mL for STR, and 5.0 μg/mL for EMB. The red horizontal line indicates the breakpoints for each drug. All MIC values were determined under the breakpoints. (B) The comparison of an inoculum effect for the first-line drugs against H37Rv between the DAC system and two routine methods, the L-J method (solid) and MGIT 960 method (liquid). The various inoculum sizes from ~10 4 to ~10 7 CFU/mL were tested. The DST results were represented as resistant (R) or susceptible (S). The DST results were consistently “S” regardless of the various inoculum sizes in the DAC system, whereas they were changed from “S” to “R” or “Error” over 5 × 10 6 CFU/mL in the two routine methods. (C) The MICs values from clinical isolates in the various inoculum sizes. The MIC values from two pan-susceptible and two resistant strains were estimated for the first-line drugs. The inoculum sizes were from ~10 3 CFU/mL to ~10 8 CFU/mL for two drug susceptible strains and two drug resistant strains. There was no inoculum effect with the clinical isolates in the DAC system.

    Article Snippet: The critical concentrations (CCs) of the DAC system were adopted as the breakpoints of the BACTEC 460 TB and MGIT 960 systems, based on the Middlebrook 7H9 (BD BBL, MD, USA) broth, because it was reported that the CCs were dependent on the medium , and the DAC system was also based on the Middlebrook 7H9 broth.

    Techniques:

    L,D-transpeptidases in mycobacteria and expression of Ldt Mav2 . (A) Cladogram of L,D-transpeptidases from various mycobacteria. Multiple alignment and phylogeny analysis of L,D-transpeptidases from Mycobacterium tuberculosis (O), Mycobacterium smegmatis (□), Mycobacterium absessus (Δ), Mycobacterium leprae (∇), Mycobacterium marinum (◊) and Mycobacterium avium (□). Ldt Mav2 is highlighted as a red-closed square. (B) Western blot analysis of cell wall and cytosolic fractions of M. avium harvested at logarithmic (log) and stationary (stat) phases of growth and probed with antibody against Ldt Mav2 . His6-tagged Ldt Mav2 is included as a control (far right). (C) Levels of Ldt Mav2 in the presence of 100 µM iron (lane 1), 40 µM iron (lane 2), 0.05% SDS (lane 3), 2 µg/ml crystal violet (lane 4), 1 µg/ml tebipenem (lane 5), H 2 O (lane 6), Middlebrook 7H9-enriched medium (lane 7) and His6-tagged Ldt Mav2 as a control (lane 8).

    Journal: Future Microbiology

    Article Title: LdtMav2, a nonclassical transpeptidase and susceptibility of Mycobacterium avium to carbapenems

    doi: 10.2217/fmb-2016-0208

    Figure Lengend Snippet: L,D-transpeptidases in mycobacteria and expression of Ldt Mav2 . (A) Cladogram of L,D-transpeptidases from various mycobacteria. Multiple alignment and phylogeny analysis of L,D-transpeptidases from Mycobacterium tuberculosis (O), Mycobacterium smegmatis (□), Mycobacterium absessus (Δ), Mycobacterium leprae (∇), Mycobacterium marinum (◊) and Mycobacterium avium (□). Ldt Mav2 is highlighted as a red-closed square. (B) Western blot analysis of cell wall and cytosolic fractions of M. avium harvested at logarithmic (log) and stationary (stat) phases of growth and probed with antibody against Ldt Mav2 . His6-tagged Ldt Mav2 is included as a control (far right). (C) Levels of Ldt Mav2 in the presence of 100 µM iron (lane 1), 40 µM iron (lane 2), 0.05% SDS (lane 3), 2 µg/ml crystal violet (lane 4), 1 µg/ml tebipenem (lane 5), H 2 O (lane 6), Middlebrook 7H9-enriched medium (lane 7) and His6-tagged Ldt Mav2 as a control (lane 8).

    Article Snippet: • Bacterial strains & growth conditions M. avium 104, a clinical isolate [ ], was used in this study and was grown in Middlebrook 7H9 broth (Becton and Dickinson, Sparks, MD, USA) supplemented with 10% (v/v) oleic acid/albumin/dextrose/catalase (OADC), 0.2% glycerol, 0.05% Tween-80 and 50 µg/ml cycloheximide (7H9 complete medium) at 37°C with constant shaking.

    Techniques: Expressing, Western Blot

    Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the

    Journal:

    Article Title: A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

    doi: 10.1128/JB.00393-06

    Figure Lengend Snippet: Growth of wild-type (▪ and □) and Δ mshD (• and ○) M. tuberculosis cells at pH 7.0 (▪ and •) and pH 5.5 (□ and ○) in Middlebrook 7H9 medium. Error bars show standard errors of the

    Article Snippet: Bacteria grown in Sauton medium were exposed to 5 mM hydrogen peroxide for 4 h or 6 h and then plated onto 7H9 Middlebrook plates plus OADC to quantitate number of surviving bacteria.

    Techniques:

    Growth (OD 600 ) of wild-type (▴) and Δ mshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.

    Journal:

    Article Title: A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

    doi: 10.1128/JB.00393-06

    Figure Lengend Snippet: Growth (OD 600 ) of wild-type (▴) and Δ mshD (○) M. tuberculosis cells in Middlebrook 7H9 medium plus ADS versus time.

    Article Snippet: Bacteria grown in Sauton medium were exposed to 5 mM hydrogen peroxide for 4 h or 6 h and then plated onto 7H9 Middlebrook plates plus OADC to quantitate number of surviving bacteria.

    Techniques:

    Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and Δ mshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.

    Journal:

    Article Title: A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

    doi: 10.1128/JB.00393-06

    Figure Lengend Snippet: Total thiol (▴ and ▵) and disulfide (▪ and □) content of wild-type (▴ and ▪) and Δ mshD M. tuberculosis cells grown in Middlebrook 7H9 medium as a function of time. CoA data were not included.

    Article Snippet: Bacteria grown in Sauton medium were exposed to 5 mM hydrogen peroxide for 4 h or 6 h and then plated onto 7H9 Middlebrook plates plus OADC to quantitate number of surviving bacteria.

    Techniques:

    Colony appearance of wild-type (A and B) and Δ mshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10 −1 (upper right quadrant), 10 −2

    Journal:

    Article Title: A Mycothiol Synthase Mutant of Mycobacterium tuberculosis Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress

    doi: 10.1128/JB.00393-06

    Figure Lengend Snippet: Colony appearance of wild-type (A and B) and Δ mshD M. tuberculosis (C and D) cells grown on Middlebrook 7H9 plates supplemented with ADS (A and C) or OADC (B and D). Cells were plated at dilutions of 10 −1 (upper right quadrant), 10 −2

    Article Snippet: Bacteria grown in Sauton medium were exposed to 5 mM hydrogen peroxide for 4 h or 6 h and then plated onto 7H9 Middlebrook plates plus OADC to quantitate number of surviving bacteria.

    Techniques:

    (A) Growth curves of TB101 in Middlebrook 7H9 containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.

    Journal: Journal of Bacteriology

    Article Title: The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

    doi: 10.1128/JB.01346-13

    Figure Lengend Snippet: (A) Growth curves of TB101 in Middlebrook 7H9 containing different concentrations of ATc. The optical density at 540 nm was recorded at different time points and used to compile the growth curves. (B) Killing curves of the pimA conditional mutant TB101 in the presence of ATc. The conditional mutant was grown in Middlebrook 7H9 containing 200 ng/ml ATc. Starting from day 1, samples were collected at different time points and plated on Middlebrook 7H10 to determine the viable counts. Each experiment was repeated at least twice, giving similar results. Filled squares, optical density; open squares, viable counts.

    Article Snippet: M. tuberculosis H37Rv and its derivative, TB38, were grown at 37°C in Middlebrook 7H9 (liquid medium) or 7H10 (solid medium) (Difco) supplemented with 0.05% (vol/vol) Tween 80 (Sigma-Aldrich), 0.2% (vol/vol) glycerol (Sigma-Aldrich), and 10% ADN (2% glucose, 5% bovine serum albumin, 0.85% NaCl).

    Techniques: Mutagenesis

    MSMEG_4247 deletion or overexpression results in loss of acid-fast staining. (A and B) Growth and viability of MSMEG_4247 deletion and overexpression mutants. Stationary cells were diluted 100-fold in Middlebrook 7H9 broth, and growth was monitored by OD 600 measurement. Viability was determined by counting CFU on Middlebrook 7H10 agar plates. Experiments were performed in triplicate, and standard deviations are shown. (C) Transmission electron micrographs of Δ4247 and Δ4247+ Phsp60 4247 strains compared to the Δ pimE mutant. Bars, 500 nm. The graph indicates the frequency of cells with membrane invaginations. (D) Sensitivity of M. smegmatis mutants to membrane-permeable compounds malachite green, SDS, and crystal violet. (E) Acid-fast staining of MSMEG_4247 deletion and overexpression mutants.

    Journal: mBio

    Article Title: Critical Roles for Lipomannan and Lipoarabinomannan in Cell Wall Integrity of Mycobacteria and Pathogenesis of Tuberculosis

    doi: 10.1128/mBio.00472-12

    Figure Lengend Snippet: MSMEG_4247 deletion or overexpression results in loss of acid-fast staining. (A and B) Growth and viability of MSMEG_4247 deletion and overexpression mutants. Stationary cells were diluted 100-fold in Middlebrook 7H9 broth, and growth was monitored by OD 600 measurement. Viability was determined by counting CFU on Middlebrook 7H10 agar plates. Experiments were performed in triplicate, and standard deviations are shown. (C) Transmission electron micrographs of Δ4247 and Δ4247+ Phsp60 4247 strains compared to the Δ pimE mutant. Bars, 500 nm. The graph indicates the frequency of cells with membrane invaginations. (D) Sensitivity of M. smegmatis mutants to membrane-permeable compounds malachite green, SDS, and crystal violet. (E) Acid-fast staining of MSMEG_4247 deletion and overexpression mutants.

    Article Snippet: For the alamarBlue growth assay (Invitrogen, Carlsbad, CA), antimicrobials were serially diluted with Middlebrook 7H9 broth in 96-well microtiter plates, and either M. smegmatis or M. tuberculosis cells were inoculated at 5 × 104 CFU or 2 × 105 CFU per well, respectively.

    Techniques: Over Expression, Staining, Transmission Assay, Mutagenesis

    Establishment and analysis of MSMEG_4241 Tet-off cells. (A) Strategy to establish MSMEG_4241 Tet-off cells and Southern blot assay confirming homologous recombination. See Materials and Methods for details. B, BamHI site. The gray bar indicates the region used as a Southern blot probe. (B) Western blotting using anti-MSMEG_4241 and anti-MSMEG_4247 antibodies, showing that the expression of MSMEG_4241 was effectively turned off by the addition of atc. (C) LM/LAM profiles of Tet-off cells. LM and LAM were extracted from cells at the 23-h time point, analyzed by SDS-PAGE, and visualized by ProQ Emerald staining. (D) MALDI-TOF-MS analysis of LM accumulating at 23 h after addition of atc. The number of mannose residues is indicated for each LM peak. (E) Growth of bacteria monitored by OD 600 in Middlebrook 7H9 broth. (F) Transmission electron micrographs of cells with or without atc induction. (G) Sensitivity of cells to malachite green, SDS, and crystal violet. For tetracycline induction, agar plates were supplemented with 40 ng/ml atc. (H) Cells at the 27-h time point were subjected to acid-fast staining.

    Journal: mBio

    Article Title: Critical Roles for Lipomannan and Lipoarabinomannan in Cell Wall Integrity of Mycobacteria and Pathogenesis of Tuberculosis

    doi: 10.1128/mBio.00472-12

    Figure Lengend Snippet: Establishment and analysis of MSMEG_4241 Tet-off cells. (A) Strategy to establish MSMEG_4241 Tet-off cells and Southern blot assay confirming homologous recombination. See Materials and Methods for details. B, BamHI site. The gray bar indicates the region used as a Southern blot probe. (B) Western blotting using anti-MSMEG_4241 and anti-MSMEG_4247 antibodies, showing that the expression of MSMEG_4241 was effectively turned off by the addition of atc. (C) LM/LAM profiles of Tet-off cells. LM and LAM were extracted from cells at the 23-h time point, analyzed by SDS-PAGE, and visualized by ProQ Emerald staining. (D) MALDI-TOF-MS analysis of LM accumulating at 23 h after addition of atc. The number of mannose residues is indicated for each LM peak. (E) Growth of bacteria monitored by OD 600 in Middlebrook 7H9 broth. (F) Transmission electron micrographs of cells with or without atc induction. (G) Sensitivity of cells to malachite green, SDS, and crystal violet. For tetracycline induction, agar plates were supplemented with 40 ng/ml atc. (H) Cells at the 27-h time point were subjected to acid-fast staining.

    Article Snippet: For the alamarBlue growth assay (Invitrogen, Carlsbad, CA), antimicrobials were serially diluted with Middlebrook 7H9 broth in 96-well microtiter plates, and either M. smegmatis or M. tuberculosis cells were inoculated at 5 × 104 CFU or 2 × 105 CFU per well, respectively.

    Techniques: Southern Blot, Homologous Recombination, Western Blot, Expressing, Laser Capture Microdissection, SDS Page, Staining, Mass Spectrometry, Transmission Assay

    Loss of mce operons increases clumping. Aggregation of M. smegmatis mc 2 155 and its Δ6 mce ] Panel A: MH; Panel B: Middlebrook 7H9-ADS-glycerol. Panel C: determination of the

    Journal: Microbes and infection / Institut Pasteur

    Article Title: Impact of the deletion of the six mce operons in Mycobacterium smegmatis

    doi: 10.1016/j.micinf.2012.01.007

    Figure Lengend Snippet: Loss of mce operons increases clumping. Aggregation of M. smegmatis mc 2 155 and its Δ6 mce ] Panel A: MH; Panel B: Middlebrook 7H9-ADS-glycerol. Panel C: determination of the

    Article Snippet: Colony morphology was analyzed by plating 2 µl spots or aliquots (≈ 100 colony forming units (CFU)) of the parental and the Δ6 mce mutant strains on solid Mueller Hinton, Hartmans-de Bont or Middlebrook 7H9 media with the addition of glycerol, NaCl, albumin and dextrose in different combinations.

    Techniques:

    The Mycobacterium tuberculosis (Mtb) RpoB H526D mutant strain exhibits slow growth rate and altered colony morphology. (A) Growth kinetics of Mtb strains in supplemented Middlebrook 7H9 broth. ∗∗ p

    Journal: Frontiers in Microbiology

    Article Title: Altered Mycobacterium tuberculosis Cell Wall Metabolism and Physiology Associated With RpoB Mutation H526D

    doi: 10.3389/fmicb.2018.00494

    Figure Lengend Snippet: The Mycobacterium tuberculosis (Mtb) RpoB H526D mutant strain exhibits slow growth rate and altered colony morphology. (A) Growth kinetics of Mtb strains in supplemented Middlebrook 7H9 broth. ∗∗ p

    Article Snippet: One aliquot of the culture was incubated in a water bath at 42°C for 24 h. Another aliquot was pelleted and resuspended in supplemented 7H9 Middlebrook broth pH 4.5 at an OD600 of 0.1 and incubated for 7 days at 37°C.

    Techniques: Mutagenesis

    V-13–012725 and V-13–011503 inhibit cholesterol breakdown. (A) Growth of wild type Mtb is not inhibited in 7H9 OADC containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC 50 values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.

    Journal: PLoS Pathogens

    Article Title: Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

    doi: 10.1371/journal.ppat.1004679

    Figure Lengend Snippet: V-13–012725 and V-13–011503 inhibit cholesterol breakdown. (A) Growth of wild type Mtb is not inhibited in 7H9 OADC containing cholesterol (100 μM) and experimental compounds. (B) In 7H9 OADC containing cholesterol (100 μM) V-13–012725 and V-13–011503 specifically inhibit cholesterol turnover. (C) V-13–012725 and V-13–011503 directly inhibit the activity of the recombinant HsaAB enzyme complex with IC 50 values of 5.0 ± 0.8 and 11.0 ± 2.0 μM, respectively. (D) Chemical structures of V-13–012725, 2-(4-fluorophenyl)-5-methyl-1H-[1, 2, 4]triazolo[1, 5-a]pyrimidin-7-one and V-13–011503, 3,5-dimethyl-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,2-oxazole-4-carboxamide. Data are representative of at least two independent experiments and error bars represent s.d.

    Article Snippet: For inhibition assays conducted in Middlebrook 7H9 OADC the bacteria were cultured to mid-log phase (OD600 of 0.4) in 7H9 OADC and assayed in 96-well black clear bottom plates.

    Techniques: Activity Assay, Recombinant

    Distribution of hit compound IC 50 values in macrophages and in 7H9 OADC. Dot plot depicting the IC 50 values for the most potent 1,359 compounds in 7H9 OADC and in the macrophage infection assays. For both assays, compounds were tested across 8 separate 2-fold dilution series 50–0.4 μM. Universally active compounds with IC 50 values

    Journal: PLoS Pathogens

    Article Title: Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

    doi: 10.1371/journal.ppat.1004679

    Figure Lengend Snippet: Distribution of hit compound IC 50 values in macrophages and in 7H9 OADC. Dot plot depicting the IC 50 values for the most potent 1,359 compounds in 7H9 OADC and in the macrophage infection assays. For both assays, compounds were tested across 8 separate 2-fold dilution series 50–0.4 μM. Universally active compounds with IC 50 values

    Article Snippet: For inhibition assays conducted in Middlebrook 7H9 OADC the bacteria were cultured to mid-log phase (OD600 of 0.4) in 7H9 OADC and assayed in 96-well black clear bottom plates.

    Techniques: Infection

    Chemical rescue of Mtb ΔIcl1. (A) Growth of Mtb ΔIcl1 was monitored in 7H9 OADC containing cholesterol (100 μM) or propionate (100 μM) in the presence of V-13–012725 (25 μM) and V-13–011503 (25 μM). Growth rescue by the compounds V-13–012725 and V-13–011503 is specific to cholesterol with no growth is observed in media containing propionate. (B) The compound, V-13–009920 (25 μM) rescues Mtb ΔIcl1 growth in 7H9 OADC media containing cholesterol (100 μM) and propionate (100 μM). Chemical rescue by V-13–009920 is comparable to rescue by vitamin-B12 (10 μg/ml). The data are representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

    doi: 10.1371/journal.ppat.1004679

    Figure Lengend Snippet: Chemical rescue of Mtb ΔIcl1. (A) Growth of Mtb ΔIcl1 was monitored in 7H9 OADC containing cholesterol (100 μM) or propionate (100 μM) in the presence of V-13–012725 (25 μM) and V-13–011503 (25 μM). Growth rescue by the compounds V-13–012725 and V-13–011503 is specific to cholesterol with no growth is observed in media containing propionate. (B) The compound, V-13–009920 (25 μM) rescues Mtb ΔIcl1 growth in 7H9 OADC media containing cholesterol (100 μM) and propionate (100 μM). Chemical rescue by V-13–009920 is comparable to rescue by vitamin-B12 (10 μg/ml). The data are representative of two independent experiments.

    Article Snippet: For inhibition assays conducted in Middlebrook 7H9 OADC the bacteria were cultured to mid-log phase (OD600 of 0.4) in 7H9 OADC and assayed in 96-well black clear bottom plates.

    Techniques:

    Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in Middlebrook 7H9 medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation

    Journal:

    Article Title: A Genomic View of Sugar Transport in Mycobacterium smegmatis and Mycobacterium tuberculosis ▿

    doi: 10.1128/JB.00257-07

    Figure Lengend Snippet: Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in Middlebrook 7H9 medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation

    Article Snippet: M. smegmatis mc2 155 was grown in liquid cultures using Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol and 0.05% Tween 80 or minimal Hartmans-de Bont (HB) medium ( ) at 37°C.

    Techniques:

    Cell surface of Msmeg-PE11 is highly hydrophobic. ( a ) Pellicle formation of Msmeg-pVV or Msmeg-PE11 was monitored by growing standing cultures of the strains without shaking in Middlebrook 7H9 medium in absence of Tween 80 at 37 °C for various time points. ( b ) Biofilm formation was quantified by crystal violet staining for which Msmeg-pVV or Msmeg-PE11 cells were washed, stained with 1% crystal violet and ethanol extract was spectrophotometrically measured at 570 nm. ( c ) Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 medium with (upper panel) or without (lower panel) 0.05% Tween-80 at 37 °C with shaking for 24 h and 48 h. Cultures were then allowed to settle at room temperature for 30 min. ( d ) In another experiment, Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 broth with congo red (100 μg/ml) and 0.05% Tween 80 for 24 h, 48 h and 72 h at 37 °C. Cells were next washed and re-suspended in acetone. Congo red in the supernatant was spectrophotometrically measured at 490 nm. Data are representative of mean ± SD of three different experiments.

    Journal: Scientific Reports

    Article Title: PE11, a PE/PPE family protein of Mycobacterium tuberculosis is involved in cell wall remodeling and virulence

    doi: 10.1038/srep21624

    Figure Lengend Snippet: Cell surface of Msmeg-PE11 is highly hydrophobic. ( a ) Pellicle formation of Msmeg-pVV or Msmeg-PE11 was monitored by growing standing cultures of the strains without shaking in Middlebrook 7H9 medium in absence of Tween 80 at 37 °C for various time points. ( b ) Biofilm formation was quantified by crystal violet staining for which Msmeg-pVV or Msmeg-PE11 cells were washed, stained with 1% crystal violet and ethanol extract was spectrophotometrically measured at 570 nm. ( c ) Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 medium with (upper panel) or without (lower panel) 0.05% Tween-80 at 37 °C with shaking for 24 h and 48 h. Cultures were then allowed to settle at room temperature for 30 min. ( d ) In another experiment, Msmeg-pVV or Msmeg-PE11 were cultured in 7H9 broth with congo red (100 μg/ml) and 0.05% Tween 80 for 24 h, 48 h and 72 h at 37 °C. Cells were next washed and re-suspended in acetone. Congo red in the supernatant was spectrophotometrically measured at 490 nm. Data are representative of mean ± SD of three different experiments.

    Article Snippet: M. smegmatis culture and transformation M. smegmatis mc2 155 bacteria were grown in Middlebrook 7H9 medium (BD Difco, USA) supplemented with 10% OADC (HiMedia, India), 0.5% glycerol, and 0.05% Tween 80 (Fisher Scientific, USA).

    Techniques: Staining, Cell Culture

    FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

    doi: 10.3389/fcimb.2017.00089

    Figure Lengend Snippet: FAs of the K10 (Type C) and 2349/06-1 (Type S) isolates of Map that showed statistically significant differences in abundance between the two isolates . BOMAC and MOCL-4 cells were infected with the K10 and 2349/06-1 isolates of Map at MOI of 1:10. At 4 h p.i., the medium containing the extracellular bacteria was collected, centrifuged at 2000 g for 15 min and the FAMEs of the bacterial pellets extracted and analyzed by GC. FAs of the K10 and 2349/06-1 isolates of Map grown in Middlebrook 7H9 medium that showed statistically significant differences in abundance are also included in the figure. Relative amount of each FA for each experimental condition (7H9 medium or extracellular) is presented as the percentage of the total FAs content. Statistically significant differences are indicated with an asterisk.

    Article Snippet: The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium.

    Techniques: Infection

    Growth rates of wild-type M. chelonae ATCC 35752, its isogenic porin knock-out mutants and complemented MCH_4691 mutant strains in 7H9-OADC-Tween 80 broth at 30°C. Shown are representative results of two to three independent experiments using different culture batches.

    Journal: PLoS ONE

    Article Title: Gene Replacement in Mycobacterium chelonae: Application to the Construction of Porin Knock-Out Mutants

    doi: 10.1371/journal.pone.0094951

    Figure Lengend Snippet: Growth rates of wild-type M. chelonae ATCC 35752, its isogenic porin knock-out mutants and complemented MCH_4691 mutant strains in 7H9-OADC-Tween 80 broth at 30°C. Shown are representative results of two to three independent experiments using different culture batches.

    Article Snippet: M. chelonae strains ATCC 35752 and 9917 , and M. abscessus ATCC 19977 were grown in LB or Middlebrook 7H9-OADC broth (BD, Difco) supplemented with 0.05% Tween 80, 7H11-OADC agar (BD, Difco) or minimal Sauton's medium supplemented with 0.05% tyloxapol.

    Techniques: Knock-Out, Mutagenesis

    Gene replacement at the MCH_4689c , MCH_4690c and MCH_4691c porin loci of M. chelonae ATCC 35752 using the Ts-sacB and recombineering systems. (A) Porin gene cluster of M. chelonae ATCC 35752. The positions of the primers used to generate the allelic exchange substrates and analyze the candidate mutants are indicated. IGR1 and IGR2 represent the intergenic regions. (B) Candidate mutants obtained for each of the porin genes using the Ts-sacB or the recombineering systems were analyzed by PCR as described under Materials and Methods and confirmed by sequencing the regions flanking the resistance cassette. The expected size of the PCR fragments is 3.3 kb for the wild-type parent strain and 3.8 kb for the knock-out mutants. MWM, molecular weight marker. WT, wild-type. (C) Immunoblot analysis of porin production in the wild-type, mutant and complemented mutant strains. Strains were grown in 7H9-OADC-Tween 80 broth at 30°C to mid-log phase (OD600 = 1) and porins were selectively extracted from whole cells at 100°C using 0.5% n -octylpolyoxyethylene as a detergent as described [44] . Protein samples prepared from the same amount of cells for each strain were denatured by boiling in 80% DMSO followed by acetone precipitation [23] . Denatured proteins were loaded volume to volume, separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and porins were detected using rabbit antiserum to purified MspA [23] . Immune complexes were detected by chemiluminescence (Pierce, ELC) and semi-quantified using the Image Lab software (Biorad).

    Journal: PLoS ONE

    Article Title: Gene Replacement in Mycobacterium chelonae: Application to the Construction of Porin Knock-Out Mutants

    doi: 10.1371/journal.pone.0094951

    Figure Lengend Snippet: Gene replacement at the MCH_4689c , MCH_4690c and MCH_4691c porin loci of M. chelonae ATCC 35752 using the Ts-sacB and recombineering systems. (A) Porin gene cluster of M. chelonae ATCC 35752. The positions of the primers used to generate the allelic exchange substrates and analyze the candidate mutants are indicated. IGR1 and IGR2 represent the intergenic regions. (B) Candidate mutants obtained for each of the porin genes using the Ts-sacB or the recombineering systems were analyzed by PCR as described under Materials and Methods and confirmed by sequencing the regions flanking the resistance cassette. The expected size of the PCR fragments is 3.3 kb for the wild-type parent strain and 3.8 kb for the knock-out mutants. MWM, molecular weight marker. WT, wild-type. (C) Immunoblot analysis of porin production in the wild-type, mutant and complemented mutant strains. Strains were grown in 7H9-OADC-Tween 80 broth at 30°C to mid-log phase (OD600 = 1) and porins were selectively extracted from whole cells at 100°C using 0.5% n -octylpolyoxyethylene as a detergent as described [44] . Protein samples prepared from the same amount of cells for each strain were denatured by boiling in 80% DMSO followed by acetone precipitation [23] . Denatured proteins were loaded volume to volume, separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and porins were detected using rabbit antiserum to purified MspA [23] . Immune complexes were detected by chemiluminescence (Pierce, ELC) and semi-quantified using the Image Lab software (Biorad).

    Article Snippet: M. chelonae strains ATCC 35752 and 9917 , and M. abscessus ATCC 19977 were grown in LB or Middlebrook 7H9-OADC broth (BD, Difco) supplemented with 0.05% Tween 80, 7H11-OADC agar (BD, Difco) or minimal Sauton's medium supplemented with 0.05% tyloxapol.

    Techniques: Polymerase Chain Reaction, Sequencing, Knock-Out, Molecular Weight, Marker, Mutagenesis, SDS Page, Purification, Software