Structured Review

Difco middlebrook 7h9 medium
M. smegmatis Δ cpa shows normal growth behavior under standard culture conditions but displays a slight growth defect during carbon starvation. ( A ) Msm parent and knockout cells were cultured in <t>Middlebrook</t> <t>7H9</t> medium at 37°C and cell density was measured at 600 nm at the indicated time points. Both strains showed identical growth behavior indicating that Cpa is dispensable during standard cell culture conditions. ( B ) cpa -knockout cells show impaired growth in the same medium devoid of glycerol as a main carbon source. Both strains were cultured in a minimal medium in absence of glycerol at 37°C. Both growth curves are representative of three or more independent experiments with the mean values ± SD plotted.
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Images

1) Product Images from "Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms"

Article Title: Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms

Journal: eLife

doi: 10.7554/eLife.34055

M. smegmatis Δ cpa shows normal growth behavior under standard culture conditions but displays a slight growth defect during carbon starvation. ( A ) Msm parent and knockout cells were cultured in Middlebrook 7H9 medium at 37°C and cell density was measured at 600 nm at the indicated time points. Both strains showed identical growth behavior indicating that Cpa is dispensable during standard cell culture conditions. ( B ) cpa -knockout cells show impaired growth in the same medium devoid of glycerol as a main carbon source. Both strains were cultured in a minimal medium in absence of glycerol at 37°C. Both growth curves are representative of three or more independent experiments with the mean values ± SD plotted.
Figure Legend Snippet: M. smegmatis Δ cpa shows normal growth behavior under standard culture conditions but displays a slight growth defect during carbon starvation. ( A ) Msm parent and knockout cells were cultured in Middlebrook 7H9 medium at 37°C and cell density was measured at 600 nm at the indicated time points. Both strains showed identical growth behavior indicating that Cpa is dispensable during standard cell culture conditions. ( B ) cpa -knockout cells show impaired growth in the same medium devoid of glycerol as a main carbon source. Both strains were cultured in a minimal medium in absence of glycerol at 37°C. Both growth curves are representative of three or more independent experiments with the mean values ± SD plotted.

Techniques Used: Knock-Out, Cell Culture

2) Product Images from "Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation"

Article Title: Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation

Journal: Microbiology

doi: 10.1099/mic.0.040675-0

TEM analysis of M. tuberculosis H37Rv (a) and the LIX11 mutant strain (b), showing differences in the staining pattern of the cell wall. Wild-type H37Rv and the LIX11 mutant were cultivated in Middlebrook 7H9-Tw-ADS medium to exponential phase (OD 600 0.3–0.4) and processed for electron microscopy in two separate experiments. One representative picture is shown. Scale bars, 0.2 μm. Arrows indicate dense granular staining present in the cell wall of the wild-type, but absent in LIX11.
Figure Legend Snippet: TEM analysis of M. tuberculosis H37Rv (a) and the LIX11 mutant strain (b), showing differences in the staining pattern of the cell wall. Wild-type H37Rv and the LIX11 mutant were cultivated in Middlebrook 7H9-Tw-ADS medium to exponential phase (OD 600 0.3–0.4) and processed for electron microscopy in two separate experiments. One representative picture is shown. Scale bars, 0.2 μm. Arrows indicate dense granular staining present in the cell wall of the wild-type, but absent in LIX11.

Techniques Used: Transmission Electron Microscopy, Mutagenesis, Staining, Electron Microscopy

3) Product Images from "A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival"

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021941

Knock-down of gre in mycobacteria is deleterious to the cell growth. ( A ) The pMV261-vector control, Mtb Gre anti-sense (pMV greAS ) and Mtb Gre over-expressing (pMV greOE ) M. smegmatis and M. tuberculosis H37Ra cells were grown for 18 hrs or 7 days respectively in liquid cultures at 37°C under shaking condition, serially diluted (10 −7 for vector control and over-expression and 10 −5 for anti-sense) and plated to determine the cell viability. ( B ) Growth curve of M. smegmatis cells with vector control, Mtb Gre anti-sense and Mtb Gre over-expression constructs. Cultures were diluted in Middlebrook 7H9 broth to give an initial OD 600 of 0.02 to 0.04 and incubated for 36 hours. The growth curves were plotted by measuring cell viability by dilution plating at different time points. ( C ) Western blot of the M. smegmatis cell lysates from vector control (lane 1) and anti-sense Mtb Gre construct (lane 2) using a polyclonal antibody against Mtb Gre. Purified Mtb Gre has been used as a positive control (lane 3).
Figure Legend Snippet: Knock-down of gre in mycobacteria is deleterious to the cell growth. ( A ) The pMV261-vector control, Mtb Gre anti-sense (pMV greAS ) and Mtb Gre over-expressing (pMV greOE ) M. smegmatis and M. tuberculosis H37Ra cells were grown for 18 hrs or 7 days respectively in liquid cultures at 37°C under shaking condition, serially diluted (10 −7 for vector control and over-expression and 10 −5 for anti-sense) and plated to determine the cell viability. ( B ) Growth curve of M. smegmatis cells with vector control, Mtb Gre anti-sense and Mtb Gre over-expression constructs. Cultures were diluted in Middlebrook 7H9 broth to give an initial OD 600 of 0.02 to 0.04 and incubated for 36 hours. The growth curves were plotted by measuring cell viability by dilution plating at different time points. ( C ) Western blot of the M. smegmatis cell lysates from vector control (lane 1) and anti-sense Mtb Gre construct (lane 2) using a polyclonal antibody against Mtb Gre. Purified Mtb Gre has been used as a positive control (lane 3).

Techniques Used: Plasmid Preparation, Expressing, Over Expression, Construct, Incubation, Western Blot, Purification, Positive Control

Related Articles

Diagnostic Assay:

Article Title: In vitro culture medium influences the vaccine efficacy of Mycobacterium bovis BCG
Article Snippet: .. Middlebrook 7H9 medium (M7H9; Difco Laboratories/BD Diagnostic Systems, Sparks, MD) was supplemented with oleic acid-albumin-dextrose-catalase (OADC Enrichment; Difco Laboratories/BD Diagnostic Systems, Detroit, MI) and 0.05% tyloxapol (Sigma–Aldrich, St. Louis, MO). ..

Clone Assay:

Article Title: Immunopathologic Effects of Tumor Necrosis Factor Alpha in Murine Mycobacterial Infection Are Dose Dependent
Article Snippet: Murine cDNA for TNF-α was cloned into the plasmids pRBD3 and pRBD4, as described previously ( ). .. The BCG-TNF and BCG-vector cells were grown to mid-log phase in Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) containing kanamycin (18 μg/ml) (Sigma, St. Louis, Mo.) with minimal agitation for 7 days and kept frozen in aliquots until use ( ).

Centrifugation:

Article Title: Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase
Article Snippet: The mycobacteria were grown in Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with 10 % OADC supplement (BD Pharmingen, San Diego, CA, USA), 0.5 % glycerol, and 0.05 % Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C. .. Mmass and Mabc were collected by centrifugation, homogenization, and filtration.

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival
Article Snippet: Bacterial strains, plasmids and the growth conditions M. smegmatis mc2 155 and M. smegmatis SM07sigA , were cultured in Middlebrook 7H9 medium (Difco) containing 0.05% Tween-80 (Sigma) and 0.4% glucose (Sigma) under shaking conditions at 37°C. .. To check the expression pattern of Gre at different growth phases in M. smegmatis and M. tuberculosis , cells were grown for 12, 18, 24, 30, 36 and 48 hrs (for M. smegmatis ) or 3, 5, 7, 12 and 20 days (for M. tuberculosis ), pelleted down by centrifugation, lysed by sonication and cell extracts were prepared.

Article Title: Cloning and Expression of Immunoreactive Antigens from Mycobacterium tuberculosis
Article Snippet: Confluent cells from six culture flasks were harvested by adding 3 ml of Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) into each flask and gently flushing the surface of the flask. .. Dislocated cells were placed into sterile plastic tubes (Falcon), and cells were pelleted by centrifugation at 1,100 × g for 5 min.

Article Title: Key Residues in Mycobacterium tuberculosis Protein Kinase G Play a Role in Regulating Kinase Activity and Survival in the Host *
Article Snippet: M. smegmatis harboring pVV16-PknG, pVV16-PknG-K181M, or pVV16-PknG-T63A were grown in 200 ml of Middlebrook 7H9 medium (Difco) supplemented with albumin, dextrose, and catalase and 25 μg/ml kanamycin until an O.D. of 1.5 was reached. .. Cells were harvested by centrifugation at 3,000 × g for 10 min at room temperature and washed once with phosphate-free 7H9 medium, followed by phosphate starvation in 200 ml of 7H9 phosphate-free medium for 6 h. Cells were harvested as described, and resuspended in 50 ml of phosphate-free medium containing phosphatase inhibitors (sodium orthovanadate, β-glycerophosphate, sodium fluoride, and sodium pyrophosphate) and 20 mCi of [32 P]orthophosphoric acid and incubated at 37 °C for 8 h at 100 × g .

Filtration:

Article Title: Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase
Article Snippet: The mycobacteria were grown in Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with 10 % OADC supplement (BD Pharmingen, San Diego, CA, USA), 0.5 % glycerol, and 0.05 % Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C. .. Mmass and Mabc were collected by centrifugation, homogenization, and filtration.

Construct:

Article Title: The phosphatase PPM1A controls monocyte-to-macrophage differentiation
Article Snippet: The M. tuberculosis H37Rv derived auxotroph strain mc2 6206 was grown in Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol, 0.02% Tyloxapol and 10% OADC (Remel). .. The PPM1A promoter (transcript 1) was constructed using the entire exon 1 sequence (410 bp) flanked by 1355 bp upstream of it and the first 20 bp of Exon 2, resulting in a final sequence of 1785 bp.

Incubation:

Article Title: Cloning and Expression of Immunoreactive Antigens from Mycobacterium tuberculosis
Article Snippet: M. tuberculosis cells (ATCC 27294) were cultured in MycoFlasks (Gibco, BRL) containing Lowenstein-Jensen medium at 37°C with 10% CO2 in a humidified incubation chamber (Jouan IG/50 model). .. Confluent cells from six culture flasks were harvested by adding 3 ml of Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) into each flask and gently flushing the surface of the flask.

Article Title: Key Residues in Mycobacterium tuberculosis Protein Kinase G Play a Role in Regulating Kinase Activity and Survival in the Host *
Article Snippet: M. smegmatis harboring pVV16-PknG, pVV16-PknG-K181M, or pVV16-PknG-T63A were grown in 200 ml of Middlebrook 7H9 medium (Difco) supplemented with albumin, dextrose, and catalase and 25 μg/ml kanamycin until an O.D. of 1.5 was reached. .. Cells were harvested by centrifugation at 3,000 × g for 10 min at room temperature and washed once with phosphate-free 7H9 medium, followed by phosphate starvation in 200 ml of 7H9 phosphate-free medium for 6 h. Cells were harvested as described, and resuspended in 50 ml of phosphate-free medium containing phosphatase inhibitors (sodium orthovanadate, β-glycerophosphate, sodium fluoride, and sodium pyrophosphate) and 20 mCi of [32 P]orthophosphoric acid and incubated at 37 °C for 8 h at 100 × g .

Infection:

Article Title: Mycobacterial infection induces eosinophilia and production of α-defensin by eosinophils in mice
Article Snippet: In addition, it has been reported that eosinophil infiltration into tissues occurs also in experimental mycobacterial infection in mice [ , ]. .. M. avium strain 104 was grown at 37°C in Middlebrook 7H9 medium (Difco, Detroit, MI, U.S.A.) supplemented with 10.0% albumin-dextrose-catalase (ADC) and 0.05% Tween80, and intraperitoneally administered with following three different doses: 1.0 × 109 (high), 1.0 × 108 (middle), and 1.0 × 107 cells (low) in 0.5 ml .

Article Title: Mycobacterium tuberculosis Interferes with the Response to Infection by Inducing the Host EphA2 Receptor
Article Snippet: Paragraph title: Bacterial culture and aerosol infection ... M. tuberculosis Erdman cultures were grown at 37°C in Middlebrook 7H9 medium (Difco) supplemented with 10% albumin-dextrose complex and 0.25% Tween-80 (USB) to an optical density at 600 nm of ∼0.8–1.

Expressing:

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival
Article Snippet: Bacterial strains, plasmids and the growth conditions M. smegmatis mc2 155 and M. smegmatis SM07sigA , were cultured in Middlebrook 7H9 medium (Difco) containing 0.05% Tween-80 (Sigma) and 0.4% glucose (Sigma) under shaking conditions at 37°C. .. To check the expression pattern of Gre at different growth phases in M. smegmatis and M. tuberculosis , cells were grown for 12, 18, 24, 30, 36 and 48 hrs (for M. smegmatis ) or 3, 5, 7, 12 and 20 days (for M. tuberculosis ), pelleted down by centrifugation, lysed by sonication and cell extracts were prepared.

Article Title: Immunopathologic Effects of Tumor Necrosis Factor Alpha in Murine Mycobacterial Infection Are Dose Dependent
Article Snippet: The expression vectors contained a kanamycin resistance gene. .. The BCG-TNF and BCG-vector cells were grown to mid-log phase in Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) containing kanamycin (18 μg/ml) (Sigma, St. Louis, Mo.) with minimal agitation for 7 days and kept frozen in aliquots until use ( ).

Article Title: Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation
Article Snippet: M. tuberculosis H37Rv and H37Ra strains were analysed for differences in STPK gene expression. .. M. tuberculosis cultures were cultivated in Middlebrook 7H9 medium (Difco) supplemented with 0.05 % Tween 80 (Tw) and ADS [0.5 % albumin/0.2 % dextrose (glucose)/0.085 % saline] (henceforth referred to as 7H9-Tw-ADS), or on Middlebrook 7H10 agar plates at 37 °C.

Western Blot:

Article Title: Prolonged Toll-Like Receptor Signaling by Mycobacterium tuberculosis and Its 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Induced Regulation of Selected Genes in Macrophages
Article Snippet: M. tuberculosis (American Type Culture Collection) was grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, Mich.) with albumin, dextrose, and catalase enrichments (Difco) and then harvested and frozen at −80°C ( ). .. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining or Western analysis by using a polyclonal rabbit anti-BCG serum (that detects many mycobacterial Ags) revealed a single major band of appropriate size that stained with monoclonal Ab to 19-kDa lipoprotein.

Over Expression:

Article Title: Anti-tubercular Activity of Pyrazinamide is Independent of trans-Translation and RpsA
Article Snippet: Bacterial strains and growth conditions Mycobacterium tuberculosis strain H37Ra was grown in Middlebrook 7H9 medium (Difco) supplemented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC) (Difco), 0.2% (vol/vol) glycerol, and 0.05% (vol/vol) tyloxapol. .. Escherichia coli strains DH5α used for the propagation of phasmids and plasmids and BL21(DE3) used for overexpression and purification of protein were grown in Lysogeny Broth (LB).

Derivative Assay:

Article Title: The phosphatase PPM1A controls monocyte-to-macrophage differentiation
Article Snippet: .. The M. tuberculosis H37Rv derived auxotroph strain mc2 6206 was grown in Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol, 0.02% Tyloxapol and 10% OADC (Remel). ..

Silver Staining:

Article Title: Prolonged Toll-Like Receptor Signaling by Mycobacterium tuberculosis and Its 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Induced Regulation of Selected Genes in Macrophages
Article Snippet: M. tuberculosis (American Type Culture Collection) was grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, Mich.) with albumin, dextrose, and catalase enrichments (Difco) and then harvested and frozen at −80°C ( ). .. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining or Western analysis by using a polyclonal rabbit anti-BCG serum (that detects many mycobacterial Ags) revealed a single major band of appropriate size that stained with monoclonal Ab to 19-kDa lipoprotein.

Cell Culture:

Article Title: Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase
Article Snippet: The Mabc type strain ATCC 19977 and all Mass strains were cultured as described previously [ ]. .. The mycobacteria were grown in Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with 10 % OADC supplement (BD Pharmingen, San Diego, CA, USA), 0.5 % glycerol, and 0.05 % Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C.

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival
Article Snippet: .. Bacterial strains, plasmids and the growth conditions M. smegmatis mc2 155 and M. smegmatis SM07sigA , were cultured in Middlebrook 7H9 medium (Difco) containing 0.05% Tween-80 (Sigma) and 0.4% glucose (Sigma) under shaking conditions at 37°C. .. M. tuberculosis H37Ra cells were cultured in Middlebrook 7H9 medium supplemented with ADC consisting of 0.2% glycerol (Sigma) and 0.05% Tween-80 at 37°C.

Article Title: Cloning and Expression of Immunoreactive Antigens from Mycobacterium tuberculosis
Article Snippet: M. tuberculosis cells (ATCC 27294) were cultured in MycoFlasks (Gibco, BRL) containing Lowenstein-Jensen medium at 37°C with 10% CO2 in a humidified incubation chamber (Jouan IG/50 model). .. Confluent cells from six culture flasks were harvested by adding 3 ml of Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) into each flask and gently flushing the surface of the flask.

Article Title: Identification of a Stress-Induced Factor of Corynebacterineae That Is Involved in the Regulation of the Outer Membrane Lipid Composition ▿
Article Snippet: C. glutamicum RES167 ( ) was cultured on brain heart infusion (BHI) medium (Difco). .. M. smegmatis mc2 155 was grown on Middlebrook 7H9 medium (Difco) supplemented with 0.05% Tween 80 to prevent aggregation.

Article Title: Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms
Article Snippet: Mycobacterium smegmatis mc(2) 155 ( Msm ) was grown in liquid Middlebrook 7H9 medium (Difco) supplemented with 0.025% (w/v) Tween 80 and 0.2% (w/v) glycerol, unless otherwise stated. .. For carbon starvation, the cells were first cultured small-scale in minimal medium (adapted from ( ) containing: 40 mM K2 HPO4 , 22 mM KH2 PO4 , 15 mM (NH4 )2 SO4 , 1.7 mM sodium citrate, 0.4 mM MgSO4 , 0.05% (w/v) Tween 80 and 0.4% (w/v) glycerol.

Sequencing:

Article Title: The phosphatase PPM1A controls monocyte-to-macrophage differentiation
Article Snippet: The M. tuberculosis H37Rv derived auxotroph strain mc2 6206 was grown in Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol, 0.02% Tyloxapol and 10% OADC (Remel). .. The PPM1A promoter (transcript 1) was constructed using the entire exon 1 sequence (410 bp) flanked by 1355 bp upstream of it and the first 20 bp of Exon 2, resulting in a final sequence of 1785 bp.

Sonication:

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival
Article Snippet: Bacterial strains, plasmids and the growth conditions M. smegmatis mc2 155 and M. smegmatis SM07sigA , were cultured in Middlebrook 7H9 medium (Difco) containing 0.05% Tween-80 (Sigma) and 0.4% glucose (Sigma) under shaking conditions at 37°C. .. To check the expression pattern of Gre at different growth phases in M. smegmatis and M. tuberculosis , cells were grown for 12, 18, 24, 30, 36 and 48 hrs (for M. smegmatis ) or 3, 5, 7, 12 and 20 days (for M. tuberculosis ), pelleted down by centrifugation, lysed by sonication and cell extracts were prepared.

Recombinant:

Article Title: Immunopathologic Effects of Tumor Necrosis Factor Alpha in Murine Mycobacterial Infection Are Dose Dependent
Article Snippet: Paragraph title: Recombinant BCG. ... The BCG-TNF and BCG-vector cells were grown to mid-log phase in Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) containing kanamycin (18 μg/ml) (Sigma, St. Louis, Mo.) with minimal agitation for 7 days and kept frozen in aliquots until use ( ).

Article Title: Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation
Article Snippet: Mycobacterium smegmatis mc2 155 was used for the isolation and propagation of recombinant mycobacteriophage for mutagenesis of pknK . .. M. tuberculosis cultures were cultivated in Middlebrook 7H9 medium (Difco) supplemented with 0.05 % Tween 80 (Tw) and ADS [0.5 % albumin/0.2 % dextrose (glucose)/0.085 % saline] (henceforth referred to as 7H9-Tw-ADS), or on Middlebrook 7H10 agar plates at 37 °C.

Nucleic Acid Electrophoresis:

Article Title: Prolonged Toll-Like Receptor Signaling by Mycobacterium tuberculosis and Its 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Induced Regulation of Selected Genes in Macrophages
Article Snippet: M. tuberculosis (American Type Culture Collection) was grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, Mich.) with albumin, dextrose, and catalase enrichments (Difco) and then harvested and frozen at −80°C ( ). .. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining or Western analysis by using a polyclonal rabbit anti-BCG serum (that detects many mycobacterial Ags) revealed a single major band of appropriate size that stained with monoclonal Ab to 19-kDa lipoprotein.

In Vivo:

Article Title: Key Residues in Mycobacterium tuberculosis Protein Kinase G Play a Role in Regulating Kinase Activity and Survival in the Host *
Article Snippet: Paragraph title: In Vivo Labeling of M. smegmatis ... M. smegmatis harboring pVV16-PknG, pVV16-PknG-K181M, or pVV16-PknG-T63A were grown in 200 ml of Middlebrook 7H9 medium (Difco) supplemented with albumin, dextrose, and catalase and 25 μg/ml kanamycin until an O.D. of 1.5 was reached.

Mutagenesis:

Article Title: Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation
Article Snippet: Mycobacterium smegmatis mc2 155 was used for the isolation and propagation of recombinant mycobacteriophage for mutagenesis of pknK . .. M. tuberculosis cultures were cultivated in Middlebrook 7H9 medium (Difco) supplemented with 0.05 % Tween 80 (Tw) and ADS [0.5 % albumin/0.2 % dextrose (glucose)/0.085 % saline] (henceforth referred to as 7H9-Tw-ADS), or on Middlebrook 7H10 agar plates at 37 °C.

Isolation:

Article Title: Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer
Article Snippet: M. ulcerans strains were isolated from BU lesions of patients from Cameroon (strains S1013, S1213 and S1298), Cote d’Ivoire (Cu001), Australia (strains S1251, S1273 and S1231) or Japan (strains S1324, S1325 and S1326). .. For in vitro compound testing, bacteria were grown either in Middlebrook 7H9 medium (Difco) or in Middlebrook 7H10 agar (Difco), supplemented with 10% (vol/vol) Middlebrook OADC supplement.

Article Title: Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation
Article Snippet: Mycobacterium smegmatis mc2 155 was used for the isolation and propagation of recombinant mycobacteriophage for mutagenesis of pknK . .. M. tuberculosis cultures were cultivated in Middlebrook 7H9 medium (Difco) supplemented with 0.05 % Tween 80 (Tw) and ADS [0.5 % albumin/0.2 % dextrose (glucose)/0.085 % saline] (henceforth referred to as 7H9-Tw-ADS), or on Middlebrook 7H10 agar plates at 37 °C.

Labeling:

Article Title: Key Residues in Mycobacterium tuberculosis Protein Kinase G Play a Role in Regulating Kinase Activity and Survival in the Host *
Article Snippet: Paragraph title: In Vivo Labeling of M. smegmatis ... M. smegmatis harboring pVV16-PknG, pVV16-PknG-K181M, or pVV16-PknG-T63A were grown in 200 ml of Middlebrook 7H9 medium (Difco) supplemented with albumin, dextrose, and catalase and 25 μg/ml kanamycin until an O.D. of 1.5 was reached.

Mouse Assay:

Article Title: Mycobacterial infection induces eosinophilia and production of α-defensin by eosinophils in mice
Article Snippet: Forty pathogen free female 8 weeks old C57BL/6JJc1 mice were purchased from Clea Japan (Tokyo, Japan). .. M. avium strain 104 was grown at 37°C in Middlebrook 7H9 medium (Difco, Detroit, MI, U.S.A.) supplemented with 10.0% albumin-dextrose-catalase (ADC) and 0.05% Tween80, and intraperitoneally administered with following three different doses: 1.0 × 109 (high), 1.0 × 108 (middle), and 1.0 × 107 cells (low) in 0.5 ml .

Article Title: Mycobacterium tuberculosis Interferes with the Response to Infection by Inducing the Host EphA2 Receptor
Article Snippet: M. tuberculosis Erdman cultures were grown at 37°C in Middlebrook 7H9 medium (Difco) supplemented with 10% albumin-dextrose complex and 0.25% Tween-80 (USB) to an optical density at 600 nm of ∼0.8–1. .. Mice ( n = 5 per group) were infected with M. tuberculosis by the aerosol method, using a Madison aerosol chamber delivering ∼100 bacilli [ ].

Article Title: In vitro culture medium influences the vaccine efficacy of Mycobacterium bovis BCG
Article Snippet: Middlebrook 7H9 medium (M7H9; Difco Laboratories/BD Diagnostic Systems, Sparks, MD) was supplemented with oleic acid-albumin-dextrose-catalase (OADC Enrichment; Difco Laboratories/BD Diagnostic Systems, Detroit, MI) and 0.05% tyloxapol (Sigma–Aldrich, St. Louis, MO). .. For culturing M. tuberculosis from tissues of previously BCG immunized mice, 2 µg/ml of thiophene-2-carboxylic acid hydrazide (TCH, Sigma–Aldrich) was added to the plates to selectively inhibit the growth of residual BCG organisms.

Staining:

Article Title: Prolonged Toll-Like Receptor Signaling by Mycobacterium tuberculosis and Its 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Induced Regulation of Selected Genes in Macrophages
Article Snippet: M. tuberculosis (American Type Culture Collection) was grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, Mich.) with albumin, dextrose, and catalase enrichments (Difco) and then harvested and frozen at −80°C ( ). .. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining or Western analysis by using a polyclonal rabbit anti-BCG serum (that detects many mycobacterial Ags) revealed a single major band of appropriate size that stained with monoclonal Ab to 19-kDa lipoprotein.

Purification:

Article Title: Prolonged Toll-Like Receptor Signaling by Mycobacterium tuberculosis and Its 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Induced Regulation of Selected Genes in Macrophages
Article Snippet: Paragraph title: M. tuberculosis culture and purification of 19-kDa lipoprotein. ... M. tuberculosis (American Type Culture Collection) was grown to log phase in Middlebrook 7H9 medium (Difco, Detroit, Mich.) with albumin, dextrose, and catalase enrichments (Difco) and then harvested and frozen at −80°C ( ).

Article Title: Anti-tubercular Activity of Pyrazinamide is Independent of trans-Translation and RpsA
Article Snippet: Bacterial strains and growth conditions Mycobacterium tuberculosis strain H37Ra was grown in Middlebrook 7H9 medium (Difco) supplemented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC) (Difco), 0.2% (vol/vol) glycerol, and 0.05% (vol/vol) tyloxapol. .. Escherichia coli strains DH5α used for the propagation of phasmids and plasmids and BL21(DE3) used for overexpression and purification of protein were grown in Lysogeny Broth (LB).

Plasmid Preparation:

Article Title: A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival
Article Snippet: Bacterial strains, plasmids and the growth conditions M. smegmatis mc2 155 and M. smegmatis SM07sigA , were cultured in Middlebrook 7H9 medium (Difco) containing 0.05% Tween-80 (Sigma) and 0.4% glucose (Sigma) under shaking conditions at 37°C. .. Knock-down of gre expression in M. smegmatis mc2 155 and M. tuberculosis H37Ra was carried out by generating the plasmid pMVgreAS (Mtbgre in anti-sense orientation) in pMV261 .

Article Title: Immunopathologic Effects of Tumor Necrosis Factor Alpha in Murine Mycobacterial Infection Are Dose Dependent
Article Snippet: Cells of recombinant M. bovis BCG strain Montreal secreting murine TNF-α (BCG-TNF) or containing the vector only (BCG-vector) were a kind gift from Richard Young of the Whitehead Institute, Cambridge, Mass. .. The BCG-TNF and BCG-vector cells were grown to mid-log phase in Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) containing kanamycin (18 μg/ml) (Sigma, St. Louis, Mo.) with minimal agitation for 7 days and kept frozen in aliquots until use ( ).

Article Title: The phosphatase PPM1A controls monocyte-to-macrophage differentiation
Article Snippet: The M. tuberculosis H37Rv derived auxotroph strain mc2 6206 was grown in Middlebrook 7H9 medium (Difco) supplemented with 0.2% glycerol, 0.02% Tyloxapol and 10% OADC (Remel). .. Escherichia coli strain DH5α was used for plasmid propagation, and was routinely grown in Luria-Bertani broth at 37 °C.

In Vitro:

Article Title: Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer
Article Snippet: .. For in vitro compound testing, bacteria were grown either in Middlebrook 7H9 medium (Difco) or in Middlebrook 7H10 agar (Difco), supplemented with 10% (vol/vol) Middlebrook OADC supplement. ..

Homogenization:

Article Title: Mycobacterium massiliense Induces Inflammatory Responses in Macrophages Through Toll-Like Receptor 2 and c-Jun N-Terminal Kinase
Article Snippet: The mycobacteria were grown in Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with 10 % OADC supplement (BD Pharmingen, San Diego, CA, USA), 0.5 % glycerol, and 0.05 % Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C. .. Mmass and Mabc were collected by centrifugation, homogenization, and filtration.

Lysis:

Article Title: Key Residues in Mycobacterium tuberculosis Protein Kinase G Play a Role in Regulating Kinase Activity and Survival in the Host *
Article Snippet: M. smegmatis harboring pVV16-PknG, pVV16-PknG-K181M, or pVV16-PknG-T63A were grown in 200 ml of Middlebrook 7H9 medium (Difco) supplemented with albumin, dextrose, and catalase and 25 μg/ml kanamycin until an O.D. of 1.5 was reached. .. Cells were harvested and lysed using a bead beater in lysis buffer containing phosphate-buffered saline and protease inhibitors.

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  • 94
    Difco 7h9 liquid medium
    Growth of M. smegmatis in various nitrogen sources. Wild-type SMR5 and glnR deletion strain MH1 were grown in the presence of the indicated substances (10 mM final concentration) as sole nitrogen source. Standard <t>7H9</t> medium was used as positive control; 7H9 lacking any nitrogen source as negative control (7H9-N). (A) Putative nitrogen sources deduced from microarray results, (B) putative nitrogen sources used by closely related species.
    7h9 Liquid Medium, supplied by Difco, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/7h9 liquid medium/product/Difco
    Average 94 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    7h9 liquid medium - by Bioz Stars, 2020-02
    94/100 stars
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    97
    Difco middlebrook 7h9 medium
    Kinetics and inducibility of fructose, glucose, and glycerol uptake by M. smegmatis . (A) M. smegmatis mc 2 155 was grown in <t>Middlebrook</t> <t>7H9</t> medium in the presence of 2% glycerol (open circles) or 2% fructose (closed circles). Accumulation
    Middlebrook 7h9 Medium, supplied by Difco, used in various techniques. Bioz Stars score: 97/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/middlebrook 7h9 medium/product/Difco
    Average 97 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    middlebrook 7h9 medium - by Bioz Stars, 2020-02
    97/100 stars
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    95
    Difco middlebrook 7h9
    Toxic effect of the five toxins on the growth of M. smegmatis . (A) Transformation of pMV306AC-toxin plasmid into M. smegmatis mc 2 155. One microgram of DNA was electrophorated and cells were plated on <t>Middlebrook</t> 7H10 solid media. The plates were incubated for 3 days at 37°C. (B) Effect of toxin on the induction on colony formation of M. smegmatis mc 2 155. Each transformant from (A) was grown for 3 days at 37°C. The culture was diluted 100-fold into Middlebrook <t>7H9</t> liquid media and streaked on Middlebrook 7H10 solid media in the absence and presence of 0.2% acetamide. The numbers in the circle correspond to those in (A) .
    Middlebrook 7h9, supplied by Difco, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/middlebrook 7h9/product/Difco
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    middlebrook 7h9 - by Bioz Stars, 2020-02
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    Growth of M. smegmatis in various nitrogen sources. Wild-type SMR5 and glnR deletion strain MH1 were grown in the presence of the indicated substances (10 mM final concentration) as sole nitrogen source. Standard 7H9 medium was used as positive control; 7H9 lacking any nitrogen source as negative control (7H9-N). (A) Putative nitrogen sources deduced from microarray results, (B) putative nitrogen sources used by closely related species.

    Journal: BMC Research Notes

    Article Title: Nitrogen starvation-induced transcriptome alterations and influence of transcription regulator mutants in Mycobacterium smegmatis

    doi: 10.1186/1756-0500-6-482

    Figure Lengend Snippet: Growth of M. smegmatis in various nitrogen sources. Wild-type SMR5 and glnR deletion strain MH1 were grown in the presence of the indicated substances (10 mM final concentration) as sole nitrogen source. Standard 7H9 medium was used as positive control; 7H9 lacking any nitrogen source as negative control (7H9-N). (A) Putative nitrogen sources deduced from microarray results, (B) putative nitrogen sources used by closely related species.

    Article Snippet: Mycobacterial strains were grown in Middlebrook 7H9 liquid medium (Difco Laboratories; per 900 ml approx.

    Techniques: Concentration Assay, Positive Control, Negative Control, Microarray

    Effect of DETA-NO on growth of cultured M. bovis BCG. Bacteria growing exponentially in 7H9 medium were transferred to evacuated (Vacutainer) tubes at mid-log phase, and at time zero either the cells were left untreated (open circles) or DETA-NO was added

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: In Vitro Model of Mycobacterial Growth Arrest Using Nitric Oxide with Limited Air ▿

    doi: 10.1128/AAC.00442-08

    Figure Lengend Snippet: Effect of DETA-NO on growth of cultured M. bovis BCG. Bacteria growing exponentially in 7H9 medium were transferred to evacuated (Vacutainer) tubes at mid-log phase, and at time zero either the cells were left untreated (open circles) or DETA-NO was added

    Article Snippet: M. bovis BCG, substrain Pasteur, isolate KD1295 , was grown in Middlebrook 7H9 liquid medium (catalog no. 271310; Difco) or on 7H10 agar medium (catalog no. 262710; Difco) supplemented with 10% albumin dextrose complex, 0.2% glycerol, and 0.05% Tween 80 ( ).

    Techniques: Cell Culture

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

    Journal:

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

    doi: 10.1128/JB.00257-07

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

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

    Techniques:

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2016.02071

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

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

    Techniques: Transformation Assay, Plasmid Preparation, Incubation