Structured Review

Difco 7h10 agar
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 <t>7H10</t> 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
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1) Product Images from "Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp"

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

Journal: Journal of Bacteriology

doi: 10.1128/JB.00759-13

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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

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

2) Product Images from "Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis"

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00879-12

Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of
Figure Legend Snippet: Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of

Techniques Used: Expressing

3) Product Images from "CD8+ T Cells Participate in the Memory Immune Response to Mycobacterium tuberculosis"

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

Journal: Infection and Immunity

doi: 10.1128/IAI.69.7.4320-4328.2001

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.
Figure Legend 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.

Techniques Used: Mouse Assay, Infection

4) Product Images from "Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146658

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.
Figure Legend 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.

Techniques Used: Isolation, Sampling, Microscopy

5) Product Images from "A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice"

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

Journal: Infection and Immunity

doi: 10.1128/IAI.00229-12

Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)
Figure Legend Snippet: Sensitivity of M. tuberculosis strains to oxidative stress. (A and B) Diameters of the zones of inhibition produced in the presence of 500 mM H 2 O 2 (A) or 40 mM menadione (B). KO, knockout. (C) Diameters of the zone of inhibition on 7H10 agar (white columns)

Techniques Used: Inhibition, Produced, Knock-Out

6) Product Images from "A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis"

Article Title: A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.9.2856-2861.2004

Diagnostic nitrate reductase activity of M. tuberculosis , M. bovis , M. bovis BCG, and various T215C mutants of M. tuberculosis. All strains were subcultured on 7H10 agar plates for 3 weeks. One loop of bacilli was used for inoculation of phosphate buffer supplemented with nitrate. Following incubation for 2 h at 37°C, production of nitrite was tested. The presence of nitrite was visualized by adding naphthylamide and sulfanilic acid reagents, which form a red diazonium dye when reacting with nitrite.
Figure Legend Snippet: Diagnostic nitrate reductase activity of M. tuberculosis , M. bovis , M. bovis BCG, and various T215C mutants of M. tuberculosis. All strains were subcultured on 7H10 agar plates for 3 weeks. One loop of bacilli was used for inoculation of phosphate buffer supplemented with nitrate. Following incubation for 2 h at 37°C, production of nitrite was tested. The presence of nitrite was visualized by adding naphthylamide and sulfanilic acid reagents, which form a red diazonium dye when reacting with nitrite.

Techniques Used: Diagnostic Assay, Activity Assay, Incubation

7) Product Images from "Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis"

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

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.41.7.3252-3259.2003

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.
Figure Legend 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.

Techniques Used: Diagnostic Assay, Activity Assay, Mutagenesis

8) Product Images from "Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis"

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00879-12

Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of
Figure Legend Snippet: Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of

Techniques Used: Expressing

9) Product Images from "Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis"

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00879-12

Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of
Figure Legend Snippet: Weakening the interaction between CarD and the RNAP compromises the survival of mycobacteria during oxidative stress. (A and B) Survival of M. smegmatis strains during oxidative stress. The log-phase M. smegmatis Δ carD attB ∷ tet-carD strains expressing CarD WT , CarD R25E , or CarD R47E in LB were treated for 1 h with either 10 mM or 25 mM H 2 O 2 . After treatment, dilutions were plated on LB. Panel A shows one such experiment, and B graphically represents survival as a ratio of CFU in treated cultures to that in untreated cultures. (C) Survival of the M. tuberculosis strains during oxidative stress. The log-phase M. tuberculosis Δ carD attB ∷ tet-carD strains expressing CarD WT or CarD R47E growing in 7H9 broth were treated for 75 h with 25 mM H 2 O 2 . After treatment, dilutions were plated on 7H10 agar, and survival is graphically represented as the ratio of CFU in treated cultures to that in untreated cultures. The graphs in panels B and C show the mean± standard error of the mean (SEM), and each sample is represented by a black circle. The significance levels in panels B and C were determined by calculating P values by Student's t test; an asterisk indicates significance with a P value of

Techniques Used: Expressing

Related Articles

Diagnostic Assay:

Article Title: Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis
Article Snippet: .. Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco) supplemented with 0.2% glycerol and 10% ADS. ..

Article Title: A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis
Article Snippet: .. Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco Laboratories, Inc.) supplemented with 0.2% glycerol, and 10% ADS. .. For the experiment in Fig. , bacilli were inoculated into phosphate buffer supplemented with 10 mM nitrate.

Clone Assay:

Article Title: A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice
Article Snippet: Escherichia coli strains JM109 and XL-10 (Stratagene) were used for cloning and were grown in Luria-Bertani (LB) broth. .. M. tuberculosis strains were maintained in 7H10 agar (Difco) supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% ADN supplement (0.5% albumin, 0.2% dextrose, 0.085% NaCl).

Construct:

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis
Article Snippet: All M. tuberculosis strains were derived from the Erdman strain and were grown at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 60 μl/liter oleic acid, 5 g/liter bovine serum albumin (BSA), 2 g/liter dextrose, 0.003 g/liter catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Gene switching was used to construct strains of mycobacteria expressing different carD alleles and to test for their viability ( ).

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: Bacteriological, Molecular and Imaging Analyses The M . smegmatis ::gfp reporter mutant expressing green fluorescent protein (GFP) was constructed by introduction of the pMSP12GFP plasmid [ ] into M . smegmatis mc2 155. .. For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex.

Incubation:

Article Title: Mycobacterium tuberculosis Protein Rv3841 Activates Dendritic Cells and Contributes to a T Helper 1 Immune Response
Article Snippet: After that, a prepared T-cell mixture was added to each well and incubated for 3 d. The T-cell mixture was CD4+ T-cells cocultured for 3 d with antigen-activated DCs (DC : T-cell ratio = 1 : 10). .. The bacterial counts were inspected by serial dilution on 7H10 agar (Difco Laboratories) supplemented with 0.05% glycerol and 10% OADC at 37°C.

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp
Article Snippet: All M. tuberculosis strains were derived from Erdman and were grown planktonically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. The 24-well dish was placed in a tightly sealed Tupperware dish for 3 weeks, at which point the Tupperware was opened and incubated for another week before photographing.

Article Title: Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis
Article Snippet: According to the recommendations of the American Society for Microbiology, the diagnostic nitrate reductase activity test must be performed with actively growing cultures that are inoculated directly into phosphate buffer supplemented with nitrate, followed by incubation for 2 h at 37°C. .. Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco) supplemented with 0.2% glycerol and 10% ADS.

Article Title: Unique role for ATG5 in PMN-mediated immunopathology during M. tuberculosis infection
Article Snippet: Cells and Media Mycobacterium tuberculosis Erdman was cultured at 37 C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Lavage cells were treated with ACK lysis buffer (0.15M NH4 Cl, 10mM KHCO3 , 0.1mM EDTA) to lyse red blood cells, plated in tissue culture treated plates, and incubated at 37°C in 5% CO2 for at least 4 hours to allow adherence of MΦ .

Article Title: AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non‐coding small RNA Mcr11
Article Snippet: Lysates were sonicated briefly as described (McDonough and Kress, ) and serially diluted in DPBS with 0.05% Tween‐80 before plating for enumeration of CFUs on 7H10 agar (Difco) supplemented with 10% OADC and 0.01% cyclohexamide. .. CFUs were recorded after 4 weeks of incubation at 37°C.

Article Title: A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis
Article Snippet: According to recommendations by the American Society for Microbiology, the diagnostic nitrate reductase activity test must be performed with actively growing cultures which are inoculated directly into phosphate buffer supplemented with nitrate and incubated for 2 h at 37°C. .. Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco Laboratories, Inc.) supplemented with 0.2% glycerol, and 10% ADS.

Activity Assay:

Article Title: Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis
Article Snippet: .. Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco) supplemented with 0.2% glycerol and 10% ADS. ..

Article Title: A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis
Article Snippet: .. Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco Laboratories, Inc.) supplemented with 0.2% glycerol, and 10% ADS. .. For the experiment in Fig. , bacilli were inoculated into phosphate buffer supplemented with 10 mM nitrate.

Infection:

Article Title: Mycobacterium tuberculosis Protein Rv3841 Activates Dendritic Cells and Contributes to a T Helper 1 Immune Response
Article Snippet: The number of internalized mycobacteria within the BMDM was measured by lysing the infected cells. .. The bacterial counts were inspected by serial dilution on 7H10 agar (Difco Laboratories) supplemented with 0.05% glycerol and 10% OADC at 37°C.

Article Title: AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non‐coding small RNA Mcr11
Article Snippet: Infected macrophages were maintained in fresh DMEM with 20% FBS at 37°C. .. Lysates were sonicated briefly as described (McDonough and Kress, ) and serially diluted in DPBS with 0.05% Tween‐80 before plating for enumeration of CFUs on 7H10 agar (Difco) supplemented with 10% OADC and 0.01% cyclohexamide.

Expressing:

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis
Article Snippet: All M. tuberculosis strains were derived from the Erdman strain and were grown at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 60 μl/liter oleic acid, 5 g/liter bovine serum albumin (BSA), 2 g/liter dextrose, 0.003 g/liter catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Gene switching was used to construct strains of mycobacteria expressing different carD alleles and to test for their viability ( ).

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: Bacteriological, Molecular and Imaging Analyses The M . smegmatis ::gfp reporter mutant expressing green fluorescent protein (GFP) was constructed by introduction of the pMSP12GFP plasmid [ ] into M . smegmatis mc2 155. .. For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex.

Ex Vivo:

Article Title: Unique role for ATG5 in PMN-mediated immunopathology during M. tuberculosis infection
Article Snippet: Cells and Media Mycobacterium tuberculosis Erdman was cultured at 37 C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Ex vivo MΦ were enriched from mice via bronchoalveolar lavage or peritoneal lavage with DMEM + 10% FBS + 1% MEM non-essential amino acids (Cellgro 25–025-CI) + 100 U/mL Penicillin + 100 mg/mL Streptomycin (Sigma P4333).

Western Blot:

Article Title: Unique role for ATG5 in PMN-mediated immunopathology during M. tuberculosis infection
Article Snippet: Cells and Media Mycobacterium tuberculosis Erdman was cultured at 37 C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Wells were washed vigorously with PBS to remove non-adherent cells and lysed in 2X Laemmli buffer for western blot analysis.

Transformation Assay:

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis
Article Snippet: All M. tuberculosis strains were derived from the Erdman strain and were grown at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 60 μl/liter oleic acid, 5 g/liter bovine serum albumin (BSA), 2 g/liter dextrose, 0.003 g/liter catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Specifically, the M. tuberculosis Δ carD attB ∷ tet-carD merodiploid strain (as described previously [ ]) was transformed with pDB19- Rv3583c WT , pDB19- Rv3583c R25E , pDB19- Rv3583c R47E , or pDB19- Rv3583c R25E,R47E (expresses wild-type M. tuberculosis CarD [CarDWT ], CarDR25E , CarDR47E , or CarDR25E,R47E , respectively, from a constitutive P myc1-tetO promoter, zeocin resistant) to replace the pMSG430 Rv3583c construct (expresses M. tuberculosis CarD from a constitutive P myc1-tetO promoter, kanamycin resistant) at the attB site of M. tuberculosis Δ carD attB ∷ tet-carD ( ).

Derivative Assay:

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis
Article Snippet: .. All M. tuberculosis strains were derived from the Erdman strain and were grown at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 60 μl/liter oleic acid, 5 g/liter bovine serum albumin (BSA), 2 g/liter dextrose, 0.003 g/liter catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Gene switching was used to construct strains of mycobacteria expressing different carD alleles and to test for their viability ( ).

Article Title: Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp
Article Snippet: .. All M. tuberculosis strains were derived from Erdman and were grown planktonically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). ..

Serial Dilution:

Article Title: Mycobacterium tuberculosis Protein Rv3841 Activates Dendritic Cells and Contributes to a T Helper 1 Immune Response
Article Snippet: .. The bacterial counts were inspected by serial dilution on 7H10 agar (Difco Laboratories) supplemented with 0.05% glycerol and 10% OADC at 37°C. ..

Cell Culture:

Article Title: The impact of ISGylation during Mycobacterium tuberculosis infection in mice
Article Snippet: .. Mycobacterium tuberculosis Erdman was cultured at 37 °C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Mice were bred and housed at Washington University School of Medicine in accordance with all federal and university guidelines, under specific-pathogen-free conditions (8).

Article Title: Polymorphic Nucleotide within the Promoter of Nitrate Reductase (NarGHJI) Is Specific for Mycobacterium tuberculosis
Article Snippet: .. Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco) supplemented with 0.2% glycerol and 10% ADS. ..

Article Title: Unique role for ATG5 in PMN-mediated immunopathology during M. tuberculosis infection
Article Snippet: .. Cells and Media Mycobacterium tuberculosis Erdman was cultured at 37 C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Ex vivo MΦ were enriched from mice via bronchoalveolar lavage or peritoneal lavage with DMEM + 10% FBS + 1% MEM non-essential amino acids (Cellgro 25–025-CI) + 100 U/mL Penicillin + 100 mg/mL Streptomycin (Sigma P4333).

Article Title: A Promoter Mutation Causes Differential Nitrate Reductase Activity of Mycobacterium tuberculosis and Mycobacterium bovis
Article Snippet: .. Thus, to test for diagnostic nitrate reductase activity, mycobacteria were cultured on 7H10 agar (Difco Laboratories, Inc.) supplemented with 0.2% glycerol, and 10% ADS. .. For the experiment in Fig. , bacilli were inoculated into phosphate buffer supplemented with 10 mM nitrate.

Inhibition:

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis
Article Snippet: .. For the zone of inhibition assays, 500 μl of a log-phase culture was plated on 7H10 agar. .. A single 6-mm disk (Sigma) was placed in the middle of the freshly plated bacterial lawn, and 5 μl of either 200 mg/ml streptomycin or 50 mg/ml isoniazid (INH) was spotted on the disk.

Article Title: Interaction of CarD with RNA Polymerase Mediates Mycobacterium tuberculosis Viability, Rifampin Resistance, and Pathogenesis
Article Snippet: .. The depletion of CarD or weakening of the CarD/RNAP β interaction had no effect on sensitivity to up to 8 mg/ml PZA in media at pH 5.2 in 2 h of transient-treatment liquid culture assays and no reproducible effect on INH sensitivity in the disk zone of inhibition assays, transient-treatment liquid culture assays with up to 500 μg/ml INH, or MIC determination assays done by plating strains on 7H10 agar containing INH (as done previously [ ]) (data not shown). .. However, we found that the M. smegmatis CarDR25E strain, as well as a strain depleted for CarDWT , were significantly more sensitive to streptomycin treatment than control strains in both the disk zone of inhibition assays ( and , CarDR25E only) and transient-treatment liquid assays ( to , CarDR25E and CarD depletion).

Imaging:

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: Paragraph title: Bacteriological, Molecular and Imaging Analyses ... For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex.

Polymerase Chain Reaction:

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex. .. PCR genotyping of the Mtb RD9 region was performed by amplifying genomic DNA from putative Mtb CFU using the primer set RD9/qRTF ( 5’-tgagtggcgatggtcaacac-3’ ) and RD9/qRTR ( 5’-gatggcgttcggaaagaaac-3’ ).

Sonication:

Article Title: AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non‐coding small RNA Mcr11
Article Snippet: .. Lysates were sonicated briefly as described (McDonough and Kress, ) and serially diluted in DPBS with 0.05% Tween‐80 before plating for enumeration of CFUs on 7H10 agar (Difco) supplemented with 10% OADC and 0.01% cyclohexamide. .. CFUs were recorded after 4 weeks of incubation at 37°C.

Mutagenesis:

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: Bacteriological, Molecular and Imaging Analyses The M . smegmatis ::gfp reporter mutant expressing green fluorescent protein (GFP) was constructed by introduction of the pMSP12GFP plasmid [ ] into M . smegmatis mc2 155. .. For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex.

Isolation:

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: .. For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex. .. Owing to the selectivity provided by the aph cassette on pMSP12GFP, all media contained kanamycin (Sigma) at a final concentration of 20 μg/mL.

Mouse Assay:

Article Title: Unique role for ATG5 in PMN-mediated immunopathology during M. tuberculosis infection
Article Snippet: Cells and Media Mycobacterium tuberculosis Erdman was cultured at 37 C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Ex vivo MΦ were enriched from mice via bronchoalveolar lavage or peritoneal lavage with DMEM + 10% FBS + 1% MEM non-essential amino acids (Cellgro 25–025-CI) + 100 U/mL Penicillin + 100 mg/mL Streptomycin (Sigma P4333).

Article Title: Protective Effect of a Lipid-Based Preparation from Mycobacterium smegmatis in a Murine Model of Progressive Pulmonary Tuberculosis
Article Snippet: Bacilli Load Two months after challenge, the mice were euthanized and one lung from each animal was aseptically removed and homogenized with a Polytron in sterile isotonic saline solution containing 0.05% Tween 80 (Sigma). .. Homogenized lungs were serially diluted and plated in duplicates on 7H10 agar (Difco Lab cod) plates.

Plasmid Preparation:

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: Bacteriological, Molecular and Imaging Analyses The M . smegmatis ::gfp reporter mutant expressing green fluorescent protein (GFP) was constructed by introduction of the pMSP12GFP plasmid [ ] into M . smegmatis mc2 155. .. For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex.

Concentration Assay:

Article Title: Real-Time Investigation of Tuberculosis Transmission: Developing the Respiratory Aerosol Sampling Chamber (RASC)
Article Snippet: For determination of CFU in controlled aerosol release experiments, bacilli were propagated in liquid 7H9 medium (Difco) supplemented with 0.05% Tween-80, 0.2% glycerol and albumin/NaCl/ glucose (ADC) complex and/or isolated on solid 7H10 agar (Difco) supplemented with 0.2% glycerol and oleic acid/albumin/dextrose/catalase (OADC) complex. .. Owing to the selectivity provided by the aph cassette on pMSP12GFP, all media contained kanamycin (Sigma) at a final concentration of 20 μg/mL.

Lysis:

Article Title: Unique role for ATG5 in PMN-mediated immunopathology during M. tuberculosis infection
Article Snippet: Cells and Media Mycobacterium tuberculosis Erdman was cultured at 37 C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth). .. Lavage cells were treated with ACK lysis buffer (0.15M NH4 Cl, 10mM KHCO3 , 0.1mM EDTA) to lyse red blood cells, plated in tissue culture treated plates, and incubated at 37°C in 5% CO2 for at least 4 hours to allow adherence of MΦ .

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  • 99
    Difco 7h10 agar
    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 <t>7H10</t> 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
    7h10 Agar, supplied by Difco, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 19 article reviews
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    7h10 agar - by Bioz Stars, 2020-01
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    97
    Difco 7h10 agar medium
    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 <t>7H10</t> medium. Data are means ± standard deviations for three independent cultures. **, P
    7h10 Agar Medium, supplied by Difco, used in various techniques. Bioz Stars score: 97/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/7h10 agar medium/product/Difco
    Average 97 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    7h10 agar medium - by Bioz Stars, 2020-01
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    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: All M. tuberculosis strains were derived from Erdman and were grown planktonically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth).

    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: All M. tuberculosis strains were derived from Erdman and were grown planktonically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth).

    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: All M. tuberculosis strains were derived from Erdman and were grown planktonically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth).

    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: All M. tuberculosis strains were derived from Erdman and were grown planktonically at 37°C in 7H9 (broth) or 7H10 (agar) (Difco) medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, and 0.05% Tween 80 (broth).

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

    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: