7h9 broth Difco Search Results


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  • 92
    Difco 7h9 difco broth
    Characteristic serpentine structure of young M. tuberculosis colonies grown in Middlebrook <t>7H9</t> broth for MODS, as seen under an inverted-light microscope (original magnification, ×20).
    7h9 Difco Broth, supplied by Difco, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco bd difco middlebrook 7h9 broth
    Sources of RNA samples. ( A ) MTB H37Rv was grown in <t>Middlebrook</t> <t>7H9</t> 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 Samples were withdrawn for OD 600 measurements and RNA extraction; sigA mRNA and 16S rRNA were quantified by QRT-PCR.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.
    Bd Difco Middlebrook 7h9 Broth, supplied by Difco, used in various techniques. Bioz Stars score: 91/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Difco bd difco middlebrook 7h9 broth powder
    Sources of RNA samples. ( A ) MTB H37Rv was grown in <t>Middlebrook</t> <t>7H9</t> 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 Samples were withdrawn for OD 600 measurements and RNA extraction; sigA mRNA and 16S rRNA were quantified by QRT-PCR.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.
    Bd Difco Middlebrook 7h9 Broth Powder, supplied by Difco, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson middlebrook 7h9 broth
    Delayed resuscitation phenotype in the Mycobacterium tuberculosis RpoB H526D mutant. (A) Colony size of 28-day nutrient-starved cultures on <t>Middlebrook</t> 7H10 agar plates after plating for 26, 33 and 39 days. (B) Growth rates of 9-day nutrient-starved cultures in supplemented Middlebrook <t>7H9</t> broth, as measured by CFU on Middlebrook 7H10 agar plates. *p
    Middlebrook 7h9 Broth, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 773 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Difco 7h9 liquid broth
    Characteristics of the Mycobacterium tuberculosis P hsp60 TK strain. A. Equal numbers of M. tuberculosis H37Rv (wild-type) or M. tuberculosis P hsp60 TK strains were incubated with 1 µCi/ml of [ 125 I]FIAU at 37°C in Middlebrook <t>7H9</t> liquid broth for 6 and 24 hours. At each specified time-point equal aliquots were withdrawn from the cultures and washed 3 times to remove the media. Each pellet was resuspended in PBS in 1.5 ml Eppendorf tubes and disinfected with Lysol overnight. The activity for each Eppendorf was measured using a gamma counter. M. tuberculosis P hsp60 TK strain actively accumulates [ 125 I]FIAU in vitro . Mean uptake activity is 6086 (±536) and 8011 (±3233) counts per minute (cpm) at 6 and 24 hours for the M. tuberculosis P hsp60 TK strain which is significantly more than 549 (±50) and 615 (±260) cpm for the wild-type parent strain (p
    7h9 Liquid Broth, supplied by Difco, used in various techniques. Bioz Stars score: 88/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Difco 7h9 broth medium
    Genetic organization, growth analysis, bacterial survival, and Mtb Δ esxL mutant construction. A , schematic representation of e sxL in the M. tuberculosis H37Rv genome. RAW 264.7 ( B ) and THP-1 ( C ) were infected with M. smegmatis ( Msm ) pSMT3 and recombinant M. smegmatis esxL strains. The cells were lysed, and intracellular survival was determined 1, 8, and 24 h post-infection by a cfu assay. D , in vitro growth curve of the M. smegmatis WT, M. smegmatis pSMT3, and recombinant M. smegmatis esxL was determined by growing bacteria in <t>7H9</t> medium and measuring OD ( O.D 600 nm ) . E , extracellular expression of the esxL transcript was measured by qRT-PCR after growing M. smegmatis esxL in vitro for 4, 12, and 24 h. RNA was isolated at the respective time points. cDNA was synthesized, and the expression of esxL was determined using qRT-PCR. Transcript levels are represented relative to mRNA -fold change of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. F , intracellular expression of esxL transcript was measured by qRT-PCR. RNA was isolated from M. smegmatis esxL -infected macrophages at different time points. cDNA was synthesized, and the expression of esxL was determined using qRT PCR. Transcript levels are represented relative to mRNA level of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. G , schematic representation of construction of Mtb Δ esxL mutant by homologous recombination. The location of primers used for the confirmation of deletion mutant generation is depicted. H , confirmation of Mtb Δ esxL mutant generation. F1 and R2 primers were designed beyond the flanks, whereas R1 and F2 primers anneal to sacB-hyg r cassette. PCR using F1 and R1 is expected to give no product with the M. tuberculosis ( lane 1 ) and ∼1.3 kb with the Mtb Δ esxL ( lane 2 ); F2-R2 primer sets were expected to give no product with M. tuberculosis and ∼1.5 kb in Mtb Δ esxL mutant. Amplification of udgB with gene-specific primers was performed as a control. The experiments were performed in triplicate ( n = 3). Results are shown as mean ± S.D. ( error bars ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns , not significant.
    7h9 Broth Medium, supplied by Difco, used in various techniques. Bioz Stars score: 91/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Difco 7h9 bacto broth
    Genetic organization, growth analysis, bacterial survival, and Mtb Δ esxL mutant construction. A , schematic representation of e sxL in the M. tuberculosis H37Rv genome. RAW 264.7 ( B ) and THP-1 ( C ) were infected with M. smegmatis ( Msm ) pSMT3 and recombinant M. smegmatis esxL strains. The cells were lysed, and intracellular survival was determined 1, 8, and 24 h post-infection by a cfu assay. D , in vitro growth curve of the M. smegmatis WT, M. smegmatis pSMT3, and recombinant M. smegmatis esxL was determined by growing bacteria in <t>7H9</t> medium and measuring OD ( O.D 600 nm ) . E , extracellular expression of the esxL transcript was measured by qRT-PCR after growing M. smegmatis esxL in vitro for 4, 12, and 24 h. RNA was isolated at the respective time points. cDNA was synthesized, and the expression of esxL was determined using qRT-PCR. Transcript levels are represented relative to mRNA -fold change of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. F , intracellular expression of esxL transcript was measured by qRT-PCR. RNA was isolated from M. smegmatis esxL -infected macrophages at different time points. cDNA was synthesized, and the expression of esxL was determined using qRT PCR. Transcript levels are represented relative to mRNA level of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. G , schematic representation of construction of Mtb Δ esxL mutant by homologous recombination. The location of primers used for the confirmation of deletion mutant generation is depicted. H , confirmation of Mtb Δ esxL mutant generation. F1 and R2 primers were designed beyond the flanks, whereas R1 and F2 primers anneal to sacB-hyg r cassette. PCR using F1 and R1 is expected to give no product with the M. tuberculosis ( lane 1 ) and ∼1.3 kb with the Mtb Δ esxL ( lane 2 ); F2-R2 primer sets were expected to give no product with M. tuberculosis and ∼1.5 kb in Mtb Δ esxL mutant. Amplification of udgB with gene-specific primers was performed as a control. The experiments were performed in triplicate ( n = 3). Results are shown as mean ± S.D. ( error bars ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns , not significant.
    7h9 Bacto Broth, supplied by Difco, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    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: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson 7h9 broth medium
    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.
    7h9 Broth Medium, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebrook s 7h9 broth medium
    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 S 7h9 Broth Medium, supplied by Difco, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebroook 7h9 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.
    Middlebroook 7h9 Broth, supplied by Difco, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MiddleBrook Pharmaceuticals 7h9 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.
    7h9 Broth, supplied by MiddleBrook Pharmaceuticals, used in various techniques. Bioz Stars score: 93/100, based on 368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebrooke 7h9 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.
    Middlebrooke 7h9 Broth, supplied by Difco, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Difco middlebrock 7h9 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.
    Middlebrock 7h9 Broth, supplied by Difco, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Difco middlebrook 7h9 medium
    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 Medium, supplied by Difco, used in various techniques. Bioz Stars score: 93/100, based on 1065 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Characteristic serpentine structure of young M. tuberculosis colonies grown in Middlebrook 7H9 broth for MODS, as seen under an inverted-light microscope (original magnification, ×20).

    Journal:

    Article Title: Evaluation of Microscopic Observation Drug Susceptibility Assay for Detection of Multidrug-Resistant Mycobacterium tuberculosis ▿

    doi: 10.1128/JCM.01949-06

    Figure Lengend Snippet: Characteristic serpentine structure of young M. tuberculosis colonies grown in Middlebrook 7H9 broth for MODS, as seen under an inverted-light microscope (original magnification, ×20).

    Article Snippet: Middlebrook 7H9 broth was prepared by using a Middlebrook 7H9 broth base (Difco, Detroit, MI), 0.31% of glycerol (Sigma Chemical Co., St. Louis, MO), 0.125% of Bacto casitone, and 10% of OADC containing PANTA antibiotic (Becton, Dickinson and Company, Sparks, MD).

    Techniques: Light Microscopy

    Serial monitoring outcomes of RIF stability in L-J medium and 7H9 broth when kept at 37°C and 4°C. (a) Stability of RIF in 7H9 and L-J media when kept at 37°C. (b) Stability of RIF in 7H9 and L-J media when kept at 4°C.

    Journal: Journal of Clinical Microbiology

    Article Title: Rifampin Stability in 7H9 Broth and L?wenstein-Jensen Medium ▿

    doi: 10.1128/JCM.01951-10

    Figure Lengend Snippet: Serial monitoring outcomes of RIF stability in L-J medium and 7H9 broth when kept at 37°C and 4°C. (a) Stability of RIF in 7H9 and L-J media when kept at 37°C. (b) Stability of RIF in 7H9 and L-J media when kept at 4°C.

    Article Snippet: Rifampin was not stable in either L-J medium or 7H9 broth.

    Techniques:

    Detection limit of the SD MPT64 Rapid test for Mycobacterium (M.) bovis . The test was used to analyze a series of diluted suspensions of M. bovis AN5 cultured in Middlebrook 7H9 broth with enrichment. A representative image of three tests is shown. The results were interpreted as positive (+), negative (-), or weak (±). NC, negative control; 1, 2.1 × 10 3 CFU/mL of M. bovis ; 2, 4.2 × 10 3 CFU/mL of M. bovis ; 3, 8.5 × 10 3 CFU/mL of M. bovis ; 4, 1.7 × 10 4 CFU/mL of M. bovis ; 5, 3.4 × 10 4 CFU/mL of M. bovis ; 6, 6.8 × 10 4 CFU/mL of M. bovis ; 7, 1.3 × 10 5 CFU/mL of M. bovis ; 8, 2.7 × 10 5 CFU/mL of M. bovis ; 9, 5.4 × 10 5 CFU/mL of M. bovis ; 10, 1.0 × 10 6 CFU/mL of M. bovis ; 11, 2.0 × 10 6 CFU/mL of M. bovis .

    Journal: Journal of Veterinary Science

    Article Title: Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals

    doi: 10.4142/jvs.2015.16.1.31

    Figure Lengend Snippet: Detection limit of the SD MPT64 Rapid test for Mycobacterium (M.) bovis . The test was used to analyze a series of diluted suspensions of M. bovis AN5 cultured in Middlebrook 7H9 broth with enrichment. A representative image of three tests is shown. The results were interpreted as positive (+), negative (-), or weak (±). NC, negative control; 1, 2.1 × 10 3 CFU/mL of M. bovis ; 2, 4.2 × 10 3 CFU/mL of M. bovis ; 3, 8.5 × 10 3 CFU/mL of M. bovis ; 4, 1.7 × 10 4 CFU/mL of M. bovis ; 5, 3.4 × 10 4 CFU/mL of M. bovis ; 6, 6.8 × 10 4 CFU/mL of M. bovis ; 7, 1.3 × 10 5 CFU/mL of M. bovis ; 8, 2.7 × 10 5 CFU/mL of M. bovis ; 9, 5.4 × 10 5 CFU/mL of M. bovis ; 10, 1.0 × 10 6 CFU/mL of M. bovis ; 11, 2.0 × 10 6 CFU/mL of M. bovis .

    Article Snippet: M. bovis AN5 was grown in Middlebrook 7H9 broth media (Difco, USA) supplemented with 10% Middlebrook OADC enrichment medium (BBL, USA) to determine the detection limit of the test.

    Techniques: Cell Culture, Negative Control

    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

    Delayed resuscitation phenotype in the Mycobacterium tuberculosis RpoB H526D mutant. (A) Colony size of 28-day nutrient-starved cultures on Middlebrook 7H10 agar plates after plating for 26, 33 and 39 days. (B) Growth rates of 9-day nutrient-starved cultures in supplemented Middlebrook 7H9 broth, as measured by CFU on Middlebrook 7H10 agar plates. *p

    Journal: Future Microbiology

    Article Title: In vitro and in vivo fitness costs associated with Mycobacterium tuberculosis RpoB mutation H526D

    doi: 10.2217/fmb-2017-0022

    Figure Lengend Snippet: Delayed resuscitation phenotype in the Mycobacterium tuberculosis RpoB H526D mutant. (A) Colony size of 28-day nutrient-starved cultures on Middlebrook 7H10 agar plates after plating for 26, 33 and 39 days. (B) Growth rates of 9-day nutrient-starved cultures in supplemented Middlebrook 7H9 broth, as measured by CFU on Middlebrook 7H10 agar plates. *p

    Article Snippet: For growth kinetics assays in nutrient-rich broth, each strain was inoculated (final optical density [OD]600 nm ∼0.05) in 10 ml of supplemented Middlebrook 7H9 broth (BD Difco) containing 10% oleic acid-albumin-dextrose-catalase (BD), 0.1% glycerol and 0.05% Tween-80 (supplemented 7H9 broth), in 50 ml conical tubes in a roller incubator at 37°C for 13 days.

    Techniques: Mutagenesis

    Characteristics of the Mycobacterium tuberculosis P hsp60 TK strain. A. Equal numbers of M. tuberculosis H37Rv (wild-type) or M. tuberculosis P hsp60 TK strains were incubated with 1 µCi/ml of [ 125 I]FIAU at 37°C in Middlebrook 7H9 liquid broth for 6 and 24 hours. At each specified time-point equal aliquots were withdrawn from the cultures and washed 3 times to remove the media. Each pellet was resuspended in PBS in 1.5 ml Eppendorf tubes and disinfected with Lysol overnight. The activity for each Eppendorf was measured using a gamma counter. M. tuberculosis P hsp60 TK strain actively accumulates [ 125 I]FIAU in vitro . Mean uptake activity is 6086 (±536) and 8011 (±3233) counts per minute (cpm) at 6 and 24 hours for the M. tuberculosis P hsp60 TK strain which is significantly more than 549 (±50) and 615 (±260) cpm for the wild-type parent strain (p

    Journal: PLoS ONE

    Article Title: Bacterial Thymidine Kinase as a Non-Invasive Imaging Reporter for Mycobacterium tuberculosis in Live Animals

    doi: 10.1371/journal.pone.0006297

    Figure Lengend Snippet: Characteristics of the Mycobacterium tuberculosis P hsp60 TK strain. A. Equal numbers of M. tuberculosis H37Rv (wild-type) or M. tuberculosis P hsp60 TK strains were incubated with 1 µCi/ml of [ 125 I]FIAU at 37°C in Middlebrook 7H9 liquid broth for 6 and 24 hours. At each specified time-point equal aliquots were withdrawn from the cultures and washed 3 times to remove the media. Each pellet was resuspended in PBS in 1.5 ml Eppendorf tubes and disinfected with Lysol overnight. The activity for each Eppendorf was measured using a gamma counter. M. tuberculosis P hsp60 TK strain actively accumulates [ 125 I]FIAU in vitro . Mean uptake activity is 6086 (±536) and 8011 (±3233) counts per minute (cpm) at 6 and 24 hours for the M. tuberculosis P hsp60 TK strain which is significantly more than 549 (±50) and 615 (±260) cpm for the wild-type parent strain (p

    Article Snippet: M. tuberculosis strains and media M. tuberculosis H37Rv and M. tuberculosis Phsp60 TK strains were grown to mid-log phase in plastic roller bottles or as shaken cultures in plastic tubes at 37°C in Middlebrook 7H9 liquid broth (Difco Laboratories) supplemented with oleic acid albumin dextrose catalase (OADC) (Becton Dickinson), 0.5% glycerol, and 0.05% Tween 80.

    Techniques: Incubation, Activity Assay, In Vitro

    Genetic organization, growth analysis, bacterial survival, and Mtb Δ esxL mutant construction. A , schematic representation of e sxL in the M. tuberculosis H37Rv genome. RAW 264.7 ( B ) and THP-1 ( C ) were infected with M. smegmatis ( Msm ) pSMT3 and recombinant M. smegmatis esxL strains. The cells were lysed, and intracellular survival was determined 1, 8, and 24 h post-infection by a cfu assay. D , in vitro growth curve of the M. smegmatis WT, M. smegmatis pSMT3, and recombinant M. smegmatis esxL was determined by growing bacteria in 7H9 medium and measuring OD ( O.D 600 nm ) . E , extracellular expression of the esxL transcript was measured by qRT-PCR after growing M. smegmatis esxL in vitro for 4, 12, and 24 h. RNA was isolated at the respective time points. cDNA was synthesized, and the expression of esxL was determined using qRT-PCR. Transcript levels are represented relative to mRNA -fold change of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. F , intracellular expression of esxL transcript was measured by qRT-PCR. RNA was isolated from M. smegmatis esxL -infected macrophages at different time points. cDNA was synthesized, and the expression of esxL was determined using qRT PCR. Transcript levels are represented relative to mRNA level of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. G , schematic representation of construction of Mtb Δ esxL mutant by homologous recombination. The location of primers used for the confirmation of deletion mutant generation is depicted. H , confirmation of Mtb Δ esxL mutant generation. F1 and R2 primers were designed beyond the flanks, whereas R1 and F2 primers anneal to sacB-hyg r cassette. PCR using F1 and R1 is expected to give no product with the M. tuberculosis ( lane 1 ) and ∼1.3 kb with the Mtb Δ esxL ( lane 2 ); F2-R2 primer sets were expected to give no product with M. tuberculosis and ∼1.5 kb in Mtb Δ esxL mutant. Amplification of udgB with gene-specific primers was performed as a control. The experiments were performed in triplicate ( n = 3). Results are shown as mean ± S.D. ( error bars ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns , not significant.

    Journal: The Journal of Biological Chemistry

    Article Title: Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages

    doi: 10.1074/jbc.M117.775205

    Figure Lengend Snippet: Genetic organization, growth analysis, bacterial survival, and Mtb Δ esxL mutant construction. A , schematic representation of e sxL in the M. tuberculosis H37Rv genome. RAW 264.7 ( B ) and THP-1 ( C ) were infected with M. smegmatis ( Msm ) pSMT3 and recombinant M. smegmatis esxL strains. The cells were lysed, and intracellular survival was determined 1, 8, and 24 h post-infection by a cfu assay. D , in vitro growth curve of the M. smegmatis WT, M. smegmatis pSMT3, and recombinant M. smegmatis esxL was determined by growing bacteria in 7H9 medium and measuring OD ( O.D 600 nm ) . E , extracellular expression of the esxL transcript was measured by qRT-PCR after growing M. smegmatis esxL in vitro for 4, 12, and 24 h. RNA was isolated at the respective time points. cDNA was synthesized, and the expression of esxL was determined using qRT-PCR. Transcript levels are represented relative to mRNA -fold change of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. F , intracellular expression of esxL transcript was measured by qRT-PCR. RNA was isolated from M. smegmatis esxL -infected macrophages at different time points. cDNA was synthesized, and the expression of esxL was determined using qRT PCR. Transcript levels are represented relative to mRNA level of M. smegmatis esxL at 4 h, which is assigned a value of 1. The expression values were normalized with sigA. G , schematic representation of construction of Mtb Δ esxL mutant by homologous recombination. The location of primers used for the confirmation of deletion mutant generation is depicted. H , confirmation of Mtb Δ esxL mutant generation. F1 and R2 primers were designed beyond the flanks, whereas R1 and F2 primers anneal to sacB-hyg r cassette. PCR using F1 and R1 is expected to give no product with the M. tuberculosis ( lane 1 ) and ∼1.3 kb with the Mtb Δ esxL ( lane 2 ); F2-R2 primer sets were expected to give no product with M. tuberculosis and ∼1.5 kb in Mtb Δ esxL mutant. Amplification of udgB with gene-specific primers was performed as a control. The experiments were performed in triplicate ( n = 3). Results are shown as mean ± S.D. ( error bars ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns , not significant.

    Article Snippet: Mycobacterium smegmatis mc2 155 was grown in Middlebrook's 7H9 broth medium (Difco) containing 0.05% Tween 80, 0.5% glucose, and 0.5% albumin at 37 °C on a shaker at 120 rpm.

    Techniques: Mutagenesis, Infection, Recombinant, Colony-forming Unit Assay, In Vitro, Expressing, Quantitative RT-PCR, Isolation, Synthesized, Homologous Recombination, Polymerase Chain Reaction, Amplification

    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