Structured Review

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) .
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Images

1) Product Images from "Functional Studies of Five Toxin-Antitoxin Modules in Mycobacterium tuberculosis H37Rv"

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

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02071

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) .
Figure Legend 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) .

Techniques Used: Transformation Assay, Plasmid Preparation, Incubation

2) Product Images from "Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy"

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

Journal: mBio

doi: 10.1128/mBio.00938-18

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

Techniques Used: In Vitro, Standard Deviation, Infection

mc 2 7901 and mc 2 7902 generate INH persisters in culture. (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100 and treated with INH (1 mg/liter). Samples were taken at the indicated time points, diluted, and plated for CFU. Mean with standard deviation is plotted ( n = 2). (B) mc 2 7901 and mc 2 7902 cultures treated or not with INH (1 mg/liter) for 2 days were infected with the phage Φ 2 DRM9 and analyzed by flow cytometry. Phage Φ 2 DRM9 contains both the L5 promoter driving GFP (mVenus) expression and the INH persister-specific dnaK promoter fused to the red fluorescent protein gene (RFP, tdTomato). The panels show the high-RFP population back-gated for GFP expression, representing the persister population (low GFP/high RFP).
Figure Legend Snippet: mc 2 7901 and mc 2 7902 generate INH persisters in culture. (A) Log-phase cultures of mc 2 7901 and mc 2 7902 grown in Middlebrook 7H9-OADC-glycerol-tyloxapol-PLAM were diluted 1/100 and treated with INH (1 mg/liter). Samples were taken at the indicated time points, diluted, and plated for CFU. Mean with standard deviation is plotted ( n = 2). (B) mc 2 7901 and mc 2 7902 cultures treated or not with INH (1 mg/liter) for 2 days were infected with the phage Φ 2 DRM9 and analyzed by flow cytometry. Phage Φ 2 DRM9 contains both the L5 promoter driving GFP (mVenus) expression and the INH persister-specific dnaK promoter fused to the red fluorescent protein gene (RFP, tdTomato). The panels show the high-RFP population back-gated for GFP expression, representing the persister population (low GFP/high RFP).

Techniques Used: Standard Deviation, Infection, Flow Cytometry, Cytometry, Expressing

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

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

3) Product Images from "Plasticity of Mycobacterium tuberculosis NADH dehydrogenases and their role in virulence"

Article Title: Plasticity of Mycobacterium tuberculosis NADH dehydrogenases and their role in virulence

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

doi: 10.1073/pnas.1721545115

Deletion of NADH dehydrogenase genes impacts NADH dehydrogenase expression levels and NADH/NAD + ratio. ( A ) Expression levels of ndh, ndhA , and nuoH in the NADH dehydrogenase mutants relative to their parental strain CDC1551. ( B ) NADH/NAD + ratio in NADH dehydrogenase mutants relative to their parental strain CDC1551. In these experiments, the strains were grown in Middlebrook 7H9, supplemented with OADC, glycerol, and tyloxapol to OD 600 nm ≈ 1. The complemented strains Δ ndh pMV361:: ndh and Δ ndhA pMV361:: ndhA are shown as Δ ndh c and Δ ndhA c, respectively. Δ nuo stands for Δ nuoAN . Average of three independent experiments is shown with SD.
Figure Legend Snippet: Deletion of NADH dehydrogenase genes impacts NADH dehydrogenase expression levels and NADH/NAD + ratio. ( A ) Expression levels of ndh, ndhA , and nuoH in the NADH dehydrogenase mutants relative to their parental strain CDC1551. ( B ) NADH/NAD + ratio in NADH dehydrogenase mutants relative to their parental strain CDC1551. In these experiments, the strains were grown in Middlebrook 7H9, supplemented with OADC, glycerol, and tyloxapol to OD 600 nm ≈ 1. The complemented strains Δ ndh pMV361:: ndh and Δ ndhA pMV361:: ndhA are shown as Δ ndh c and Δ ndhA c, respectively. Δ nuo stands for Δ nuoAN . Average of three independent experiments is shown with SD.

Techniques Used: Expressing

4) Product Images from "Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy"

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

Journal: mBio

doi: 10.1128/mBio.00938-18

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

Techniques Used: In Vitro, Standard Deviation, Infection

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

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

Related Articles

Transduction:

Article Title: Plasticity of Mycobacterium tuberculosis NADH dehydrogenases and their role in virulence
Article Snippet: The strains were grown at 37 °C in Middlebrook 7H9 (Difco), supplemented with 10% (vol/vol) OADC (oleic acid-albumin-dextrose-catalase; Difco), 0.2% (vol/vol) glycerol, 0.05% (vol/vol) tyloxapol. .. The ndh, ndhA , and the full operon nuoAN were deleted from Mtb CDC1551 and mc2 6230 using the specialized transduction system ( ).

Luciferase:

Article Title: The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model
Article Snippet: Nonopsonic binding studies were performed with live strains of Mycobacterium bovis bacilli Calmette-Guérin (BCG) expressing either Green Fluorescent Protein (BCG-GFP) or bacterial luciferase (BCG-lux) as well as Mycobacterium tuberculosis H37Rv expressing bacterial luciferase (Mtb-lux) and FITC-labeled zymosan (Molecular Probes). .. Mycobacteria were grown in Middlebrook 7H9 (Difco) with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment in the presence of 10µg/ml kanamycin (BCG-GFP) or 50µg/ml hygromycin (BCG-lux), respectively, and kept as 15% glycerol stocks at −80°C at a density of approximately 5×106 particles.

Construct:

Article Title: Mycobacterial SigA and SigB Cotranscribe Essential Housekeeping Genes during Exponential Growth
Article Snippet: Mycobacterium smegmatis was grown at 37°C in Middlebrook 7H9 (Difco) supplemented with 10% albumin-dextrose-catalase (ADC) and 0.05% Tween 20. .. Gene replacement mutants were constructed using recombineering as described previously ( ).

Incubation:

Article Title: Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria
Article Snippet: Western blotting Recombinant BCG colonies were grown at 37°C in Middlebrook 7H9 (Difco) medium supplemented with 10% ADC (5% bovine serum albumin fraction V, 2% dextrose, 0.003% beef catalase) and 0.05% Tween 80. .. Gagp26 was detected by incubation with a 1:500 dilution of polyclonal rabbit anti-Gag antibody followed by a 1:10000 dilution of anti-rabbit peroxidase-conjugated IgG (Amersham) and visualisation with an enhanced chemiluminescence kit (Amersham).

Expressing:

Article Title: The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model
Article Snippet: Nonopsonic binding studies were performed with live strains of Mycobacterium bovis bacilli Calmette-Guérin (BCG) expressing either Green Fluorescent Protein (BCG-GFP) or bacterial luciferase (BCG-lux) as well as Mycobacterium tuberculosis H37Rv expressing bacterial luciferase (Mtb-lux) and FITC-labeled zymosan (Molecular Probes). .. Mycobacteria were grown in Middlebrook 7H9 (Difco) with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment in the presence of 10µg/ml kanamycin (BCG-GFP) or 50µg/ml hygromycin (BCG-lux), respectively, and kept as 15% glycerol stocks at −80°C at a density of approximately 5×106 particles.

Article Title: Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria
Article Snippet: Western blotting Recombinant BCG colonies were grown at 37°C in Middlebrook 7H9 (Difco) medium supplemented with 10% ADC (5% bovine serum albumin fraction V, 2% dextrose, 0.003% beef catalase) and 0.05% Tween 80. .. We evaluated gag p26 expression with total protein extracts prepared as previously described [ ].

Western Blot:

Article Title: Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria
Article Snippet: .. Western blotting Recombinant BCG colonies were grown at 37°C in Middlebrook 7H9 (Difco) medium supplemented with 10% ADC (5% bovine serum albumin fraction V, 2% dextrose, 0.003% beef catalase) and 0.05% Tween 80. .. We evaluated gag p26 expression with total protein extracts prepared as previously described [ ].

Sterility:

Article Title: Sodium Hyaluronate Nanocomposite Respirable Microparticles to Tackle Antibiotic Resistance with Potential Application in Treatment of Mycobacterial Pulmonary Infections
Article Snippet: Strains were grown in Middlebrook 7H9 (MB7H9, Difco, Becton and Dickinson, Franklyn Lakes, NJ, USA) supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase; Becton and Dickinson) at 37 °C until reaching an optical density (OD) of 0.8 at 600 nm. .. Growth controls and a sterility control were included in each assay.

Ligand Binding Assay:

Article Title: The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model
Article Snippet: Paragraph title: Ligand Binding Assays ... Mycobacteria were grown in Middlebrook 7H9 (Difco) with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment in the presence of 10µg/ml kanamycin (BCG-GFP) or 50µg/ml hygromycin (BCG-lux), respectively, and kept as 15% glycerol stocks at −80°C at a density of approximately 5×106 particles.

Dissection:

Article Title: Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens
Article Snippet: Helminth burdens were determined by quantification of intestinal adult worms by examining faecal matter under a dissection microscope. .. Mycobacterium bovis BCG Pasteur (donated by Robin Warren, SU, South Africa) was propagated to logarithmic growth phase in Middlebrook 7H9 (Difco) liquid culture, supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% albumin-dextrose-catalase (ADC, Merck) at 37°C.

Infection:

Article Title: Mycobacteriumtuberculosis Exploits Human Interferon ? to Stimulate Macrophage Extracellular Trap Formation and Necrosis
Article Snippet: .. M. tuberculosis Infection M. tuberculosis H37Rv, ΔESX-1 (or ΔRD1 ), and complemented ΔESX-1 have been described elsewhere [ ] and were grown at 37°C in Middlebrook 7H9 (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (BBL, Becton Dickinson), 0.5% glycerol, and 0.05% Tween-80. ..

Article Title: Suppression of Protective Responses upon Activation of L-Type Voltage Gated Calcium Channel in Macrophages during Mycobacterium bovis BCG Infection
Article Snippet: Middlebrook 7H9 (DIFCO) broth was used to grow M . bovis BCG supplemented with 0.05% Tween 80 and 10% OADC (BD). .. For all experiments, M . bovis BCG and H37Rv were used at a Multiplicity of Infection (MOI) of 2.

Article Title: STAT3 expression by myeloid cells is detrimental for the T- cell-mediated control of infection with Mycobacterium tuberculosis
Article Snippet: .. Infection and infectivity assay BCG Montreal and M . tuberculosis Harlingen were grown in Middlebrook 7H9 (Difco, Detroit, MI) supplemented with albumin, dextrose, catalase and, for BCG cultures, 50 μg/ ml hygromycin (Sigma, St. Louis, MO). .. Mice were infected with 250 M . tuberculosis Harlingen strain by aerosol using a nose-only exposure unit (In-tox Products, Uppsala, Sweden)[ ].

Sonication:

Article Title: Mycobacteriumtuberculosis Exploits Human Interferon ? to Stimulate Macrophage Extracellular Trap Formation and Necrosis
Article Snippet: M. tuberculosis Infection M. tuberculosis H37Rv, ΔESX-1 (or ΔRD1 ), and complemented ΔESX-1 have been described elsewhere [ ] and were grown at 37°C in Middlebrook 7H9 (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (BBL, Becton Dickinson), 0.5% glycerol, and 0.05% Tween-80. .. Because the optical density of nonsonicated bacteria was typically twice that of sonicated bacteria, multiplicities of infection (MOIs) of 5 for nonsonicated bacteria and 10 for sonicated bacteria were used.

Article Title: The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model
Article Snippet: Mycobacteria were grown in Middlebrook 7H9 (Difco) with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment in the presence of 10µg/ml kanamycin (BCG-GFP) or 50µg/ml hygromycin (BCG-lux), respectively, and kept as 15% glycerol stocks at −80°C at a density of approximately 5×106 particles. .. For binding studies, BCG or Mtb aliquots were thawed, spun in a microcentrifuge for 1 min at 13000g , washed twice in DMEM followed by vigorous vortexing, and aggregates were dispersed by sonication for 30 sec at 50W in a cuphorn sonicator.

Binding Assay:

Article Title: The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model
Article Snippet: Nonopsonic binding studies were performed with live strains of Mycobacterium bovis bacilli Calmette-Guérin (BCG) expressing either Green Fluorescent Protein (BCG-GFP) or bacterial luciferase (BCG-lux) as well as Mycobacterium tuberculosis H37Rv expressing bacterial luciferase (Mtb-lux) and FITC-labeled zymosan (Molecular Probes). .. Mycobacteria were grown in Middlebrook 7H9 (Difco) with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment in the presence of 10µg/ml kanamycin (BCG-GFP) or 50µg/ml hygromycin (BCG-lux), respectively, and kept as 15% glycerol stocks at −80°C at a density of approximately 5×106 particles.

Mutagenesis:

Article Title: Assessment of Live Candidate Vaccines for Paratuberculosis in Animal Models and Macrophages ▿
Article Snippet: .. Both wild-type and mutant strains of M. paratuberculosis were grown in Middlebrook 7H9 (Difco) broth enriched with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.5% glycerol, 0.05% Tween 80, and 2 μg/ml mycobactin (Allied Monitor). ..

Size-exclusion Chromatography:

Article Title: The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model
Article Snippet: Mycobacteria were grown in Middlebrook 7H9 (Difco) with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment in the presence of 10µg/ml kanamycin (BCG-GFP) or 50µg/ml hygromycin (BCG-lux), respectively, and kept as 15% glycerol stocks at −80°C at a density of approximately 5×106 particles. .. For binding studies, BCG or Mtb aliquots were thawed, spun in a microcentrifuge for 1 min at 13000g , washed twice in DMEM followed by vigorous vortexing, and aggregates were dispersed by sonication for 30 sec at 50W in a cuphorn sonicator.

Microscopy:

Article Title: Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens
Article Snippet: Helminth burdens were determined by quantification of intestinal adult worms by examining faecal matter under a dissection microscope. .. Mycobacterium bovis BCG Pasteur (donated by Robin Warren, SU, South Africa) was propagated to logarithmic growth phase in Middlebrook 7H9 (Difco) liquid culture, supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% albumin-dextrose-catalase (ADC, Merck) at 37°C.

Mouse Assay:

Article Title: STAT3 expression by myeloid cells is detrimental for the T- cell-mediated control of infection with Mycobacterium tuberculosis
Article Snippet: Infection and infectivity assay BCG Montreal and M . tuberculosis Harlingen were grown in Middlebrook 7H9 (Difco, Detroit, MI) supplemented with albumin, dextrose, catalase and, for BCG cultures, 50 μg/ ml hygromycin (Sigma, St. Louis, MO). .. Mice were infected with 250 M . tuberculosis Harlingen strain by aerosol using a nose-only exposure unit (In-tox Products, Uppsala, Sweden)[ ].

Article Title: Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens
Article Snippet: Egg propagation in BALB/c IL-4 knock-out mice (gift from Frank Brombacher, University of Cape Town, South Africa), helminth collection, and excretory/secretory (E/S) antigen preparations, were performed as described previously [ , ]. .. Mycobacterium bovis BCG Pasteur (donated by Robin Warren, SU, South Africa) was propagated to logarithmic growth phase in Middlebrook 7H9 (Difco) liquid culture, supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% albumin-dextrose-catalase (ADC, Merck) at 37°C.

Tetrazolium Microplate Assay:

Article Title: Sodium Hyaluronate Nanocomposite Respirable Microparticles to Tackle Antibiotic Resistance with Potential Application in Treatment of Mycobacterial Pulmonary Infections
Article Snippet: Minimum Inhibitory Concentrations of the Compounds against M. tuberculosis Strains MIC determination of the compounds was conducted by the 96-well broth microdilution method using a tetrazolium microplate-based assay [ , ]. .. Strains were grown in Middlebrook 7H9 (MB7H9, Difco, Becton and Dickinson, Franklyn Lakes, NJ, USA) supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase; Becton and Dickinson) at 37 °C until reaching an optical density (OD) of 0.8 at 600 nm.

Plasmid Preparation:

Article Title: Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy
Article Snippet: The strains were grown in Middlebrook 7H9 (Difco, Sparks, MD) supplemented with 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC; Difco), 0.2% (vol/vol) glycerol, and 0.05% (vol/vol) tyloxapol at 37°C with shaking. .. The plasmid pYUB1471 , shuttle phasmid phAE159 , and phage phAE280 ( ) were obtained from laboratory stocks.

Article Title: Towards a new combination therapy for tuberculosis with next generation benzothiazinones
Article Snippet: Bacterial strains and culture conditions Bacterial strains, BTZ-resistant mycobacterial mutants and M. tuberculosis strain H37Rv were grown at 37°C with shaking in Middlebrook 7H9 (Difco) broth supplemented with 10% albumin-dextrose-catalase (ADC) enrichment, 0.2% glycerol, 0.05% Tween 80. .. The M. marinum strains E11 (Puttinaowarat et al , ) and M ((Stinear et al , )ATCC BAA-535) containing the plasmid pSMT3-mcherry (Meijer et al , ) to visualize bacteria, were routinely grown at 30°C in Middlebrook 7H9 broth (Difco) with 10% Middlebrook albumin-dextrose-catalase (ADC, BD Bioscience) and 0.05% Tween-80 by shaking at 90 rpm or on Middlebrook 7H10 agar (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, BD Bioscience) and 50 mg/ml hygromycin.

Recombinant:

Article Title: Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria
Article Snippet: .. Western blotting Recombinant BCG colonies were grown at 37°C in Middlebrook 7H9 (Difco) medium supplemented with 10% ADC (5% bovine serum albumin fraction V, 2% dextrose, 0.003% beef catalase) and 0.05% Tween 80. .. We evaluated gag p26 expression with total protein extracts prepared as previously described [ ].

In Vitro:

Article Title: Reprogramming of Small Noncoding RNA Populations in Peripheral Blood Reveals Host Biomarkers for Latent and Active Mycobacterium tuberculosis Infection
Article Snippet: Paragraph title: In vitro infections. ... M. tuberculosis strain H37Rv was grown to mid-log phase in 5 ml of Middlebrook 7H9 (Difco) liquid culture medium supplemented with 0.5% glycerol, 0.15% Tween 80, and 10% oleic acid-albumin-dextrose-catalase (BD Biosciences).

Knock-Out:

Article Title: Mycobacterium bovis BCG infection severely delays Trichuris muris expulsion and co-infection suppresses immune responsiveness to both pathogens
Article Snippet: Egg propagation in BALB/c IL-4 knock-out mice (gift from Frank Brombacher, University of Cape Town, South Africa), helminth collection, and excretory/secretory (E/S) antigen preparations, were performed as described previously [ , ]. .. Mycobacterium bovis BCG Pasteur (donated by Robin Warren, SU, South Africa) was propagated to logarithmic growth phase in Middlebrook 7H9 (Difco) liquid culture, supplemented with 0.2% glycerol, 0.05% Tween 80 and 10% albumin-dextrose-catalase (ADC, Merck) at 37°C.

Concentration Assay:

Article Title: Functional Studies of Five Toxin-Antitoxin Modules in Mycobacterium tuberculosis H37Rv
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. .. Chloramphenicol (Cm) or ampicillin (Amp) was added to a concentration of 50 μg/ml to cultures of E. coli , and kanamycin was added to a concentration of 50 μg/ml to cultures of E. coli and 25 μg/ml to cultures of M. smegmatis .

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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
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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
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    middlebrook 7h9 medium - by Bioz Stars, 2020-02
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    98
    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.
    Middlebrook 7h9 Broth, supplied by Difco, used in various techniques. Bioz Stars score: 98/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 4 article reviews
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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:

    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