Structured Review

Difco 7h9 liquid medium
Effects of pH on the rate of inactivation of M. gordonae . Experimental conditions were a temperature of 25°C and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook <t>7H9-Tween</t> medium. No, initial number of CFU; N, number of CFU at each time point. Experiments were conducted with different phosphate (0.05 M) buffers within the pH range of 6.0 to 8.0. Slopes were calculated as follows: pH 6, y = 0.11 x ; pH 7, y = 0.09 x ; and pH 8, y = 0.02 x . R 2 , correlation coefficient values.
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1) Product Images from "Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System"

Article Title: Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.68.3.1025-1032.2002

Effects of pH on the rate of inactivation of M. gordonae . Experimental conditions were a temperature of 25°C and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook 7H9-Tween medium. No, initial number of CFU; N, number of CFU at each time point. Experiments were conducted with different phosphate (0.05 M) buffers within the pH range of 6.0 to 8.0. Slopes were calculated as follows: pH 6, y = 0.11 x ; pH 7, y = 0.09 x ; and pH 8, y = 0.02 x . R 2 , correlation coefficient values.
Figure Legend Snippet: Effects of pH on the rate of inactivation of M. gordonae . Experimental conditions were a temperature of 25°C and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook 7H9-Tween medium. No, initial number of CFU; N, number of CFU at each time point. Experiments were conducted with different phosphate (0.05 M) buffers within the pH range of 6.0 to 8.0. Slopes were calculated as follows: pH 6, y = 0.11 x ; pH 7, y = 0.09 x ; and pH 8, y = 0.02 x . R 2 , correlation coefficient values.

Techniques Used: Concentration Assay

Effects of temperature on chlorine inactivation of M. gordonae . Experimental conditions were pH 7 and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook 7H9-Tween medium. The chlorine susceptibility of M. gordonae was analyzed at 4, 16, and 25°C. No, initial number of CFU; N, number of CFU at each time point. Symbols: ▪, 4°C ( y = 0.01 x ); ▴, 16°C ( y = 0.02 x ); ⧫, 25°C ( y = 0.09 x ). R 2 , correlation coefficient values.
Figure Legend Snippet: Effects of temperature on chlorine inactivation of M. gordonae . Experimental conditions were pH 7 and an initial chlorine concentration of 0.5 mg/liter. Cells were grown in Middlebrook 7H9-Tween medium. The chlorine susceptibility of M. gordonae was analyzed at 4, 16, and 25°C. No, initial number of CFU; N, number of CFU at each time point. Symbols: ▪, 4°C ( y = 0.01 x ); ▴, 16°C ( y = 0.02 x ); ⧫, 25°C ( y = 0.09 x ). R 2 , correlation coefficient values.

Techniques Used: Concentration Assay

2) Product Images from "Octahedral ruthenium (II) polypyridyl complexes as antimicrobial agents against mycobacterium"

Article Title: Octahedral ruthenium (II) polypyridyl complexes as antimicrobial agents against mycobacterium

Journal: PeerJ

doi: 10.7717/peerj.3252

Complex 2 (A) was bacteriostatic and complex 3 (B) was bactericidal against M. smegmatis . Bacterial cells were inoculated 7H9 medium, and cultured without any drug or in the presence of complex 2 or 3 at various concentrations. At the indicated time points, aliquots of cell suspension were transferred and plated on drug-free 7H9 medium after 24 more hours of incubation.
Figure Legend Snippet: Complex 2 (A) was bacteriostatic and complex 3 (B) was bactericidal against M. smegmatis . Bacterial cells were inoculated 7H9 medium, and cultured without any drug or in the presence of complex 2 or 3 at various concentrations. At the indicated time points, aliquots of cell suspension were transferred and plated on drug-free 7H9 medium after 24 more hours of incubation.

Techniques Used: Cell Culture, Incubation

3) Product Images from "Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro"

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro

Journal: Infection and Immunity

doi:

Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).
Figure Legend Snippet: Effect of exogenous H 2 O 2 on mycobacterial viability in cell-free Middlebrook 7H9 medium. H 2 O 2 (at the concentrations shown) was added to growing bacteria, and the number of CFU was determined as described in Materials and Methods. Results are expressed as percent survival relative to baseline ± SEM and are from one representative experiment with six replicate cultures. The numbers of CFU used for the experiment (and expressed as 100% in the figure) were 4.7 × 10 5 for H37Rv, 3.2 × 10 5 for H37Rv Inh r , and 2.8 × 10 5 for H37Rv(pMH59).

Techniques Used:

4) Product Images from "A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria"

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

Journal: Nature Communications

doi: 10.1038/ncomms12527

Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).
Figure Legend Snippet: Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).

Techniques Used: Concentration Assay, Transferring

5) Product Images from "The Mycobacterium tuberculosis MmpL11 Cell Wall Lipid Transporter Is Important for Biofilm Formation, Intracellular Growth, and Nonreplicating Persistence"

Article Title: The Mycobacterium tuberculosis MmpL11 Cell Wall Lipid Transporter Is Important for Biofilm Formation, Intracellular Growth, and Nonreplicating Persistence

Journal: Infection and Immunity

doi: 10.1128/IAI.00131-17

M. tuberculosis mmpL11 mutant bacteria are shorter than the wild type in biofilms. Shown are representative images (left) and quantification of the lengths (right) of M. tuberculosis strains grown in either Sauton's medium lacking Tween 80, to promote biofilm formation (A), Sauton's medium containing Tween 80 (B), or 7H9 medium containing Tween 80 (C). The differences between the wild type and the mutant were significant by Student's t test (***, P
Figure Legend Snippet: M. tuberculosis mmpL11 mutant bacteria are shorter than the wild type in biofilms. Shown are representative images (left) and quantification of the lengths (right) of M. tuberculosis strains grown in either Sauton's medium lacking Tween 80, to promote biofilm formation (A), Sauton's medium containing Tween 80 (B), or 7H9 medium containing Tween 80 (C). The differences between the wild type and the mutant were significant by Student's t test (***, P

Techniques Used: Mutagenesis

Transmission electron microscopy reveals differences in ultrastructure between the wild-type and mmpL11 mutant M. tuberculosis strains grown in 7H9 medium containing Tween 80 (left) (planktonic) or Sauton's medium without Tween 80 (right) (biofilm). Arrows indicate crystal inclusions in mmpL11 mutants grown planktonically. Asterisks highlight the extracellular material that is enriched in the mmpL11 mutant relative to wild-type M. tuberculosis .
Figure Legend Snippet: Transmission electron microscopy reveals differences in ultrastructure between the wild-type and mmpL11 mutant M. tuberculosis strains grown in 7H9 medium containing Tween 80 (left) (planktonic) or Sauton's medium without Tween 80 (right) (biofilm). Arrows indicate crystal inclusions in mmpL11 mutants grown planktonically. Asterisks highlight the extracellular material that is enriched in the mmpL11 mutant relative to wild-type M. tuberculosis .

Techniques Used: Transmission Assay, Electron Microscopy, Mutagenesis

The M. tuberculosis mmpL11 mutant exhibits impaired biofilm formation that does not result from reduced replication in Sauton's medium. (A) Growth of M. tuberculosis wild-type and mmpL11 mutant strains in 7H9 medium. Average values and standard deviations for four biological replicates are shown. (B) Biofilm formation by M. tuberculosis wild-type, mmpL11 mutant, and complemented strains. Biofilms were cultured in 6-well plates for a total of 4 weeks. (C) Quantification of crystal violet staining of M. tuberculosis biofilms. Average values and standard deviations for three independent assays are shown. The difference between the wild type and the mutant was significant ( P ,
Figure Legend Snippet: The M. tuberculosis mmpL11 mutant exhibits impaired biofilm formation that does not result from reduced replication in Sauton's medium. (A) Growth of M. tuberculosis wild-type and mmpL11 mutant strains in 7H9 medium. Average values and standard deviations for four biological replicates are shown. (B) Biofilm formation by M. tuberculosis wild-type, mmpL11 mutant, and complemented strains. Biofilms were cultured in 6-well plates for a total of 4 weeks. (C) Quantification of crystal violet staining of M. tuberculosis biofilms. Average values and standard deviations for three independent assays are shown. The difference between the wild type and the mutant was significant ( P ,

Techniques Used: Mutagenesis, Cell Culture, Staining

6) Product Images from "MmpL11 Protein Transports Mycolic Acid-containing Lipids to the Mycobacterial Cell Wall and Contributes to Biofilm Formation in Mycobacterium smegmatis *"

Article Title: MmpL11 Protein Transports Mycolic Acid-containing Lipids to the Mycobacterial Cell Wall and Contributes to Biofilm Formation in Mycobacterium smegmatis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.473371

The M. smegmatis mmpL11 mutant grows like the wild type in planktonic cultures but demonstrates impaired biofilm formation. A , growth of wild-type M. smegmatis and the mmpL11 mutant GP02 in complete 7H9 medium containing albumin/dextrose/saline supplements and 0.05% Tween 80 was followed over time. B , pellicle formation by the wild type (mc 2 155/pVV16), the mmpL11 mutant (GP02/pVV16), and the complemented mutant (GP02/pVV16 mmpL11 SM ). Bacteria were subcultured in glass tubes and allowed to grow at 37 °C for 5 days without shaking. At day 3, a thin film could be observed across the surface of the medium in the wild-type and complemented strains ( left ). This developed into a textured biofilm over the next 2 days. Attachment of bacteria to the glass tube and growth away from the surface could be observed from the side ( right ). C , bacteria in pellicle cultures depicted in A were enumerated. Biofilms were disrupted mechanically via syringing, and serial dilutions were plated. The mean ± S.D. of three independent experiments is shown. There was no significant difference in cfu/ml between strains. D , biofilm formation by the wild type (mc 2 155/pVV16), the mmpL11 mutant (GP02/pVV16), the complemented mutant (GP02/pVV16 mmpL11 SM ), and the heterologously complemented strain (GP02/pVV16 mmpL11 TB ). Equivalent numbers of bacteria were subcultured in liquid biofilm medium (Sauton's medium lacking Tween 80). Plates were incubated for 4 days at 30 °C. E , scanning electron microscopy of biofilms formed by wild-type, mmpL11 mutant, and complemented strains.
Figure Legend Snippet: The M. smegmatis mmpL11 mutant grows like the wild type in planktonic cultures but demonstrates impaired biofilm formation. A , growth of wild-type M. smegmatis and the mmpL11 mutant GP02 in complete 7H9 medium containing albumin/dextrose/saline supplements and 0.05% Tween 80 was followed over time. B , pellicle formation by the wild type (mc 2 155/pVV16), the mmpL11 mutant (GP02/pVV16), and the complemented mutant (GP02/pVV16 mmpL11 SM ). Bacteria were subcultured in glass tubes and allowed to grow at 37 °C for 5 days without shaking. At day 3, a thin film could be observed across the surface of the medium in the wild-type and complemented strains ( left ). This developed into a textured biofilm over the next 2 days. Attachment of bacteria to the glass tube and growth away from the surface could be observed from the side ( right ). C , bacteria in pellicle cultures depicted in A were enumerated. Biofilms were disrupted mechanically via syringing, and serial dilutions were plated. The mean ± S.D. of three independent experiments is shown. There was no significant difference in cfu/ml between strains. D , biofilm formation by the wild type (mc 2 155/pVV16), the mmpL11 mutant (GP02/pVV16), the complemented mutant (GP02/pVV16 mmpL11 SM ), and the heterologously complemented strain (GP02/pVV16 mmpL11 TB ). Equivalent numbers of bacteria were subcultured in liquid biofilm medium (Sauton's medium lacking Tween 80). Plates were incubated for 4 days at 30 °C. E , scanning electron microscopy of biofilms formed by wild-type, mmpL11 mutant, and complemented strains.

Techniques Used: Mutagenesis, Incubation, Electron Microscopy

7) Product Images from "Rapid Cytolysis of Mycobacterium tuberculosis by Faropenem, an Orally Bioavailable β-Lactam Antibiotic"

Article Title: Rapid Cytolysis of Mycobacterium tuberculosis by Faropenem, an Orally Bioavailable β-Lactam Antibiotic

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.03461-14

Real-time single-cell analysis of faropenem-mediated growth inhibition. An M. tuberculosis strain expressing GFP was cultured in a microfluidic device under a constant flow of 7H9 medium and imaged at 1-h intervals. (A and D) Faropenem (28 μg/ml; ∼21× MIC) was added to the flow medium at 96 to 264 h. (B and E) Meropenem plus clavulanate (MPC; 8 μg/ml and 2.5 μg/ml, respectively; ∼26× MIC) was added to the flow medium at 96 to 330 h. (C and F) Isoniazid (0.5 μg/ml; ∼20× MIC) was added to the flow medium at 96 to 340 h. (A to C) Images were recorded on fluorescence (green) and phase channels and merged. Numbers (lower right) indicate the elapsed times (in hours). Labels (upper right) indicate the presence or absence of antibiotic in the flow medium. (D to F) Single-cell growth rates were determined by measuring the projected areas of single cells ( n = 50) during the indicated time intervals and fitting exponential curves to the data.
Figure Legend Snippet: Real-time single-cell analysis of faropenem-mediated growth inhibition. An M. tuberculosis strain expressing GFP was cultured in a microfluidic device under a constant flow of 7H9 medium and imaged at 1-h intervals. (A and D) Faropenem (28 μg/ml; ∼21× MIC) was added to the flow medium at 96 to 264 h. (B and E) Meropenem plus clavulanate (MPC; 8 μg/ml and 2.5 μg/ml, respectively; ∼26× MIC) was added to the flow medium at 96 to 330 h. (C and F) Isoniazid (0.5 μg/ml; ∼20× MIC) was added to the flow medium at 96 to 340 h. (A to C) Images were recorded on fluorescence (green) and phase channels and merged. Numbers (lower right) indicate the elapsed times (in hours). Labels (upper right) indicate the presence or absence of antibiotic in the flow medium. (D to F) Single-cell growth rates were determined by measuring the projected areas of single cells ( n = 50) during the indicated time intervals and fitting exponential curves to the data.

Techniques Used: Single-cell Analysis, Inhibition, Expressing, Cell Culture, Flow Cytometry, Fluorescence

Activity of faropenem against extracellular and intracellular M. tuberculosis . (A) Bacteria expressing luciferase were incubated for 7 days in 7H9 medium containing different concentrations of faropenem (FPM) or faropenem plus clavulanate (FPMC). Growth was measured using a luciferase-based assay, and the data were normalized to the values obtained in the absence of antibiotic. Results are representative of those from four experiments. (B) Log-phase cultures were exposed for 6 days to different concentrations of faropenem and plated to measure the number of CFU. Values were normalized to those for the untreated controls. Results are representative of those from two experiments. (C) Log-phase cultures were exposed to antibiotics with (filled symbols) or without (empty symbols) 2.5 μg/ml clavulanate. Circles, meropenem (8 μg/ml; ∼26× MIC with clavulanate, ∼3× MIC without clavulanate); squares, faropenem (8 μg/ml; ∼6× MIC with or without clavulanate); triangles, faropenem (28 μg/ml; ∼21× MIC); crosses, 0.5 μg/ml isoniazid (∼20× MIC). At 0, 1, 2, 4, and 8 days after antibiotic addition, aliquots were washed and plated to measure the number of CFU. Results are means ± SEMs from three experiments. The rebound of the numbers of CFU at 8 days in the culture exposed to isoniazid was due to the outgrowth of drug-resistant variants. (D) The stability of compounds in 7H9 medium was measured by HPLC-UV analysis. Clav, clavulanic acid; MPM, meropenem. (E and F) Bacteria expressing GFP were grown in RAW macrophages and left untreated (closed circles) or treated with 56 μg/ml faropenem (open squares), 50 μg/ml meropenem plus 2.5 μg/ml clavulanate (open circles), or 0.75 μg/ml isoniazid (crosses). (E) Macrophage lysates were plated at 0 h and at 1, 3, and 6 days postinfection to measure the number of CFU. Data are plotted as percent survival normalized to the number of organisms at 0 h. (F) The fraction of macrophages (Mϕ) containing GFP-positive M. tuberculosis was measured by flow cytometry at 1, 3, and 6 days postinfection. INH, isoniazid; FPM, faropenem; MPMC, meropenem plus clavulanate. Results are means ± SEMs from three experiments.
Figure Legend Snippet: Activity of faropenem against extracellular and intracellular M. tuberculosis . (A) Bacteria expressing luciferase were incubated for 7 days in 7H9 medium containing different concentrations of faropenem (FPM) or faropenem plus clavulanate (FPMC). Growth was measured using a luciferase-based assay, and the data were normalized to the values obtained in the absence of antibiotic. Results are representative of those from four experiments. (B) Log-phase cultures were exposed for 6 days to different concentrations of faropenem and plated to measure the number of CFU. Values were normalized to those for the untreated controls. Results are representative of those from two experiments. (C) Log-phase cultures were exposed to antibiotics with (filled symbols) or without (empty symbols) 2.5 μg/ml clavulanate. Circles, meropenem (8 μg/ml; ∼26× MIC with clavulanate, ∼3× MIC without clavulanate); squares, faropenem (8 μg/ml; ∼6× MIC with or without clavulanate); triangles, faropenem (28 μg/ml; ∼21× MIC); crosses, 0.5 μg/ml isoniazid (∼20× MIC). At 0, 1, 2, 4, and 8 days after antibiotic addition, aliquots were washed and plated to measure the number of CFU. Results are means ± SEMs from three experiments. The rebound of the numbers of CFU at 8 days in the culture exposed to isoniazid was due to the outgrowth of drug-resistant variants. (D) The stability of compounds in 7H9 medium was measured by HPLC-UV analysis. Clav, clavulanic acid; MPM, meropenem. (E and F) Bacteria expressing GFP were grown in RAW macrophages and left untreated (closed circles) or treated with 56 μg/ml faropenem (open squares), 50 μg/ml meropenem plus 2.5 μg/ml clavulanate (open circles), or 0.75 μg/ml isoniazid (crosses). (E) Macrophage lysates were plated at 0 h and at 1, 3, and 6 days postinfection to measure the number of CFU. Data are plotted as percent survival normalized to the number of organisms at 0 h. (F) The fraction of macrophages (Mϕ) containing GFP-positive M. tuberculosis was measured by flow cytometry at 1, 3, and 6 days postinfection. INH, isoniazid; FPM, faropenem; MPMC, meropenem plus clavulanate. Results are means ± SEMs from three experiments.

Techniques Used: Activity Assay, Expressing, Luciferase, Incubation, High Performance Liquid Chromatography, Flow Cytometry, Cytometry

Detection of membrane potential in single cells of isoniazid-treated M. tuberculosis . Bacteria were cultured in a microfluidic device under a constant flow of 7H9 medium. Medium conditions were as follows: t = 0 to 96 h, no antibiotic; t = 96 to 340 h, addition of isoniazid (0.5 μg/ml; 20× MIC); t = 340 to 400 h, no antibiotic. As an endpoint assay, 3 μM DiOC 2 was added to the flow medium, and imaging was continued at 1-h intervals on the FITC and Texas Red fluorescence channels. Representative snapshots and the merged images from four random positions in the microfluidic device are shown in the four rows. In this assay, all cells exhibit green fluorescence, but higher cytosolic concentrations of the dye lead to self-association and a shift toward red fluorescence emission in cells with an intact membrane potential.
Figure Legend Snippet: Detection of membrane potential in single cells of isoniazid-treated M. tuberculosis . Bacteria were cultured in a microfluidic device under a constant flow of 7H9 medium. Medium conditions were as follows: t = 0 to 96 h, no antibiotic; t = 96 to 340 h, addition of isoniazid (0.5 μg/ml; 20× MIC); t = 340 to 400 h, no antibiotic. As an endpoint assay, 3 μM DiOC 2 was added to the flow medium, and imaging was continued at 1-h intervals on the FITC and Texas Red fluorescence channels. Representative snapshots and the merged images from four random positions in the microfluidic device are shown in the four rows. In this assay, all cells exhibit green fluorescence, but higher cytosolic concentrations of the dye lead to self-association and a shift toward red fluorescence emission in cells with an intact membrane potential.

Techniques Used: Cell Culture, Flow Cytometry, End Point Assay, Imaging, Fluorescence

8) Product Images from "Experimental selection of long-term intracellular mycobacteria"

Article Title: Experimental selection of long-term intracellular mycobacteria

Journal: Cellular Microbiology

doi: 10.1111/cmi.12303

Phenotypic and genetic changes of selected mycobacteria.A. In vitro growth of both ancestral and selected bacteria in complete 7H9 medium. Curves represent the mean ± S.E.M. of four independent experiments, (**) P ≤ 0.01 from two-tailed Student's t -test.B. Isotopic labelling of fatty acids according to the carbon length associated to neutral lipids after 1 day of incorporation of 13 C-acetate in complete 7H9 medium. The plot shows the incorporation of 13 C-acetate into neutral lipids in ANC and SEL bacteria ( x -axis).C. In vitro growth of ancestral and selected bacteria in minimal medium (Sauton) containing Tween 80, glycerol or glucose as a sole carbon source during 7 and 14 days. Data represent the mean ± S.E.M. of at least three independent experiments, (**) P ≤ 0.01 from two-tailed Student's t -test.D. Percentage of the fatty acid methyl esters (FAME) area associated to neutral lipids and corresponding to the indicated carbon length for ancestral and selected strains growing in minimal Sauton medium after 1 day of incorporation of 13 C-glucose as sole carbon source.E. Enrichment into fatty acids associated to neutral lipids after 9 days of growth in ancestral and selected strains growing in Sauton medium with 13 C-acetate as sole carbon source.F. Enrichment into fatty acids associated to neutral lipids after 9 days of growth in ancestral and selected strains growing in Sauton medium with 13 C-glucose as sole carbon source.
Figure Legend Snippet: Phenotypic and genetic changes of selected mycobacteria.A. In vitro growth of both ancestral and selected bacteria in complete 7H9 medium. Curves represent the mean ± S.E.M. of four independent experiments, (**) P ≤ 0.01 from two-tailed Student's t -test.B. Isotopic labelling of fatty acids according to the carbon length associated to neutral lipids after 1 day of incorporation of 13 C-acetate in complete 7H9 medium. The plot shows the incorporation of 13 C-acetate into neutral lipids in ANC and SEL bacteria ( x -axis).C. In vitro growth of ancestral and selected bacteria in minimal medium (Sauton) containing Tween 80, glycerol or glucose as a sole carbon source during 7 and 14 days. Data represent the mean ± S.E.M. of at least three independent experiments, (**) P ≤ 0.01 from two-tailed Student's t -test.D. Percentage of the fatty acid methyl esters (FAME) area associated to neutral lipids and corresponding to the indicated carbon length for ancestral and selected strains growing in minimal Sauton medium after 1 day of incorporation of 13 C-glucose as sole carbon source.E. Enrichment into fatty acids associated to neutral lipids after 9 days of growth in ancestral and selected strains growing in Sauton medium with 13 C-acetate as sole carbon source.F. Enrichment into fatty acids associated to neutral lipids after 9 days of growth in ancestral and selected strains growing in Sauton medium with 13 C-glucose as sole carbon source.

Techniques Used: In Vitro, Two Tailed Test, Isotopic Labeling

9) Product Images from "Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level"

Article Title: Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level

Journal: Scientific Reports

doi: 10.1038/srep19695

Construction of MSMEG_1764 knockout mutant and complement strains. ( A ) PCR verification of the construction MSMEG_1764 knockout strain. Lanes: 1.Wild-type MS; 2. MSMEG_1764 knockout strain. ( B ) Verify the transcription of MSMEG_1764 knockout strain by RT-PCR. Wild type and Δ lat Msm strains were grown at 37C in MB 7H9 liquid medium to an OD600 of 0.8–1.0. Total bacterial RNA was isolated and subjected to RT-PCR to detect the expression of the lat Msm gene. ( C ) Construction of Δ lat-Rv3290c strain; Lanes: 1. Knockout strain complement with pALACE- Rv3290 c; 2. Knockout strain complement with pALACE plasmid. ( D ) Western-blotting to confirm the expression of Rv3290c.Lysates were prepared from bacterial cells cultured as in ( B ), after 16 h induction and subjected to Western blotting to detect His-tagged Rv3290c protein using mouse anti-His antibody.
Figure Legend Snippet: Construction of MSMEG_1764 knockout mutant and complement strains. ( A ) PCR verification of the construction MSMEG_1764 knockout strain. Lanes: 1.Wild-type MS; 2. MSMEG_1764 knockout strain. ( B ) Verify the transcription of MSMEG_1764 knockout strain by RT-PCR. Wild type and Δ lat Msm strains were grown at 37C in MB 7H9 liquid medium to an OD600 of 0.8–1.0. Total bacterial RNA was isolated and subjected to RT-PCR to detect the expression of the lat Msm gene. ( C ) Construction of Δ lat-Rv3290c strain; Lanes: 1. Knockout strain complement with pALACE- Rv3290 c; 2. Knockout strain complement with pALACE plasmid. ( D ) Western-blotting to confirm the expression of Rv3290c.Lysates were prepared from bacterial cells cultured as in ( B ), after 16 h induction and subjected to Western blotting to detect His-tagged Rv3290c protein using mouse anti-His antibody.

Techniques Used: Knock-Out, Mutagenesis, Polymerase Chain Reaction, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Plasmid Preparation, Western Blot, Cell Culture

The expression of M. smegmatis lat under nutrient starvation. Cells of M. smegmatis were initially grown in 7H9 medium to log-phase and then washed by 1 × PBS and resuspended in 1 × PBS, incubate at 37 °C, 110 rpm. RNAs were extracted from bacteria harvested at indicated time points. RT-PCR was performed as described in Materials and Methods. Data are means ± s.d. of triplicates in one of at least three experiments.
Figure Legend Snippet: The expression of M. smegmatis lat under nutrient starvation. Cells of M. smegmatis were initially grown in 7H9 medium to log-phase and then washed by 1 × PBS and resuspended in 1 × PBS, incubate at 37 °C, 110 rpm. RNAs were extracted from bacteria harvested at indicated time points. RT-PCR was performed as described in Materials and Methods. Data are means ± s.d. of triplicates in one of at least three experiments.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

10) Product Images from "Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity"

Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity

Journal: mBio

doi: 10.1128/mBio.00778-18

Incomplete repression of eccD 5 transcription has moderate effects on growth. (A to D) Wild-type M. tuberculosis Erdman (WT) and the Δ pstA1 , eccD 5 Tet-OFF, and Δ pstA1 eccD 5 Tet-OFF strains were inoculated in 7H9 complete medium at an OD 600 of 0.05 and grown at 37°C with aeration. Anhydrotetracycline hydrochloride (ATc; 100 ng/ml) was added at day 0 and day 7 as indicated. Growth was monitored by daily OD 600 measurements (A and C) and by plating serially diluted cultures on 7H10 medium to determine numbers of viable CFU per milliliter on days 0, 3, 6, 9, 12, and 15 (B and D). The key in panel B applies to panels A and B; the key in panel D applies to panels C and D. **, P
Figure Legend Snippet: Incomplete repression of eccD 5 transcription has moderate effects on growth. (A to D) Wild-type M. tuberculosis Erdman (WT) and the Δ pstA1 , eccD 5 Tet-OFF, and Δ pstA1 eccD 5 Tet-OFF strains were inoculated in 7H9 complete medium at an OD 600 of 0.05 and grown at 37°C with aeration. Anhydrotetracycline hydrochloride (ATc; 100 ng/ml) was added at day 0 and day 7 as indicated. Growth was monitored by daily OD 600 measurements (A and C) and by plating serially diluted cultures on 7H10 medium to determine numbers of viable CFU per milliliter on days 0, 3, 6, 9, 12, and 15 (B and D). The key in panel B applies to panels A and B; the key in panel D applies to panels C and D. **, P

Techniques Used:

11) Product Images from "A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria"

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria

Journal: Nature Communications

doi: 10.1038/ncomms12527

Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).
Figure Legend Snippet: Loss of ICD2 results in glutamate auxotrophy and impaired viability. ( a ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in Middlebrook 7H9 medium. Solid lines, culture density (OD 600 ). Dashed lines, glutamate concentration in culture medium. ( b ) Growth (OD 600 ) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). ( c ) Intracellular metabolites in wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis 24 h after transferring cells into minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments). CIT/ICT, citrate/isocitrate; α-KG, alpha-ketoglutarate; GLU, glutamate. ( d , e ) Growth (OD 600 ) of wild-type, Δ icd1 , Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and glutamate ( d ) or without glutamate ( e ). ( f ) Survival (CFU) of wild-type, Δ icd and Δ icd attB ::P np icd -complemented strains of M. smegmatis in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate). ( g ) Survival (CFU) of wild-type, Δ icd2 and Δ icd2 attB ::P np icd2 -complemented strains of M. bovis BCG in minimal medium supplemented with glucose and devoid of glutamate. Data are means±s.d. ( n =3 independent experiments each performed in triplicate).

Techniques Used: Concentration Assay, Transferring

Related Articles

Clone Assay:

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: The KatG-overexpressing construct consists of a 2.9-kb Eco RI fragment carrying the katG gene of H37Rv cloned into the polylinker site of pMV306 ( ). .. Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation.

Amplification:

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria
Article Snippet: .. Individual colonies were picked and amplified in 7H9 liquid medium (no antibiotics) and then plated on LB agar or 7H10 containing 5% sucrose to select for cells in which plasmid excision had occurred (second crossover). .. Individual colonies were picked and the replacement of the gene by the fusion gene or gene deletion was confirmed by PCR.

Construct:

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: This construct contains about 85 bp upstream of the GTG start codon of katG . .. Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation.

Incubation:

Article Title: Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level
Article Snippet: E.coli strains were grown on LB broth agar or in LB broth, Mycobacterium smegmatis mc2 155 was grown in 7H9 liquid medium (Difco) supplemented with 0.05% w/v Tween 80, 0.5%glycerol and 0.5%glucose or were grown on 7H10 agar supplemented with 1% glycerol and 0.5% glucose. .. In brief, exponential phase cultures were pelleted and washed twice with 1 × PBS before being resuspended in 1 × PBS, transferred to standing flasks or microwell and incubated at 37 °C, 110 rpm.

Article Title: MmpL11 Protein Transports Mycolic Acid-containing Lipids to the Mycobacterial Cell Wall and Contributes to Biofilm Formation in Mycobacterium smegmatis *
Article Snippet: Mycobacterial strains were maintained in Middlebrook 7H9 liquid medium (Difco) or on Middlebrook 7H11 agar plates (Difco) supplemented with 10% albumin/dextrose/saline. .. When required, cultures were incubated with the antibiotics kanamycin (25 μg/ml) and hygromycin (75 μg/ml).

Diffusion-based Assay:

Article Title: Octahedral ruthenium (II) polypyridyl complexes as antimicrobial agents against mycobacterium
Article Snippet: Disc diffusion assays were performed according to CLSI guidelines. .. Bacteria were grown in TSB medium; C. neoformans and C. albicans were grown in Yeast Extract Peptone Dextrose (YPD) medium; M. smegmatis mc2 155 was grown in 7H9 liquid medium (Difco) supplemented with 0.05% w/v Tween 80, 0.5% glycerol and 0.5% glucose or were grown on 7H10 agar supplemented with 1% glycerol and 0.5% glucose.

Activity Assay:

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: The strains used include the laboratory strains H37Rv (Trudeau Institute, Saranac Lake, N.Y.), CDC 1551 (T. M. Shinnick, Centers for Disease Control and Prevention, Atlanta, Ga.), ATCC 35825, and H37Rv Inhr , which was selected from H37Rv by direct plating onto 10 μg of isoniazid per ml (loss of KatG was verified by [i] activity assay, [ii] Western blotting using anti-KatG antipeptide antisera, and [iii] Southern blot analysis using the katG probe and restriction digestion as described previously [ ]), as well as the recombinant strain H37Rv(pMH59) and the recombinant strain H37Rv(pMH91), which have been previously described ( ). .. Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation.

Infection:

Article Title: Microanatomic Distribution of Myeloid Heme Oxygenase-1 Protects against Free Radical-Mediated Immunopathology in Human Tuberculosis
Article Snippet: .. Infection of Mice Mtb H37Rv was grown to mid-log phase in Middlebrook 7H9 liquid medium (Difco, Detroit, MI, USA) containing 0.2% glycerol, and 0.02% Tyloxapol supplemented with ADS (Albumin, Dextrose, Saline). ..

Expressing:

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: The vector system also has a synthetic promoter element incorporated into the polylinker driving expression of KatG ( ). .. Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation.

Article Title: Experimental selection of long-term intracellular mycobacteria
Article Snippet: .. Pasteur 1173P2 expressing GFP (BCG-GFP, ancestral strain (ANC) kindly provided by Dr Brigitte Gicquel, Pasteur Institute, France) were grown in roller flasks at 37°C in Middlebrook 7H9 liquid medium (Difco Laboratories, USA) containing 0.2% glycerol, 0.05% Tween 80 and supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase supplement) (BD Biosciences, USA) or in Middlebrook 7H10 plates (Difco Laboratories, USA) supplemented with 10% OADC. .. Generation of long-term infected macrophages (LTI) RAW 264.7 macrophages were grown in T-75 flasks (Marienfeld GmbH & Co, Germany) at approximately 80% confluence and were infected with ANC at MOI of 10.

Article Title: AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non‐coding small RNA Mcr11
Article Snippet: Mycobacterium tuberculosis H37Rv (Mtb) (ATCC 25618) was grown on 7H10 agar (Difco) supplemented with 10% oleic acid‐albumin‐dextrose‐catalase (OADC) (Becton Dickson and Company) and 0.01% cycloheximide or in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% (vol/vol) OADC, 0.2% (vol/vol) glycerol, 10% (vol/vol) and 0.05% (vol/vol) Tween‐80 (Sigma‐Aldrich). .. Cultures were grown to various growth phases in gently shaking vented 25‐cm2 tissue culture flasks (Corning) in low oxygen (1.3% O2 , 5% CO2 ) for gene expression experiments (Florczyk et al ., ).

Modification:

Article Title: Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity
Article Snippet: M. smegmatis strains were grown in Middlebrook 7H9 liquid medium (Difco) supplemented with Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment (Difco), 0.2% glycerol and 0.05% Tween80, with shaking, or on Middlebrook 7H10 solid medium (Difco) supplemented with 0.085% NaCl, 0.2% glucose and 0.5% glycerol. .. Briefly, pre-cultures were washed and inoculated into modified Mycobacterium phlei minimal medium (MPLN), which was modified by excluding asparagine and substituting with 10 mM sodium nitrate, in a final volume of 10 ml to an optical density at 600 nm (OD600 ) of 0.05.

Article Title: Experimental selection of long-term intracellular mycobacteria
Article Snippet: Cells RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC, Cat# TIB-71) and maintained in complete Dulbecco's Modified Eagles Medium (D-MEM) with 4.5 g l−1 glucose, 10% (v/v) heat-inactivated fetal calf serum (FCS, PAA, Austria) and 2 mM l -glutamine (PAA, Austria) (complete D-MEM medium). .. Pasteur 1173P2 expressing GFP (BCG-GFP, ancestral strain (ANC) kindly provided by Dr Brigitte Gicquel, Pasteur Institute, France) were grown in roller flasks at 37°C in Middlebrook 7H9 liquid medium (Difco Laboratories, USA) containing 0.2% glycerol, 0.05% Tween 80 and supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase supplement) (BD Biosciences, USA) or in Middlebrook 7H10 plates (Difco Laboratories, USA) supplemented with 10% OADC.

Article Title: MmpL11 Protein Transports Mycolic Acid-containing Lipids to the Mycobacterial Cell Wall and Contributes to Biofilm Formation in Mycobacterium smegmatis *
Article Snippet: Mycobacterial strains were maintained in Middlebrook 7H9 liquid medium (Difco) or on Middlebrook 7H11 agar plates (Difco) supplemented with 10% albumin/dextrose/saline. .. For biofilm growth, M. smegmatis was grown in polystyrene Petri dishes at 30 °C in modified Sauton's medium without Tween 80.

Western Blot:

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: The strains used include the laboratory strains H37Rv (Trudeau Institute, Saranac Lake, N.Y.), CDC 1551 (T. M. Shinnick, Centers for Disease Control and Prevention, Atlanta, Ga.), ATCC 35825, and H37Rv Inhr , which was selected from H37Rv by direct plating onto 10 μg of isoniazid per ml (loss of KatG was verified by [i] activity assay, [ii] Western blotting using anti-KatG antipeptide antisera, and [iii] Southern blot analysis using the katG probe and restriction digestion as described previously [ ]), as well as the recombinant strain H37Rv(pMH59) and the recombinant strain H37Rv(pMH91), which have been previously described ( ). .. Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation.

Transformation Assay:

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria
Article Snippet: Yellow fluorescent protein (YFP)-expressing derivatives of both E. coli strains were made by transformation with a plasmid encoding YFP . .. M. tuberculosis Erdman, M. tuberculosis H37Rv, M. tuberculosis CDC1551, M. tuberculosis HN787, M. bovis BCG (Pasteur 1173P2), M. smegmatis mc2 155 and derivative strains ( ) were grown in Middlebrook 7H9 liquid medium (Difco) supplemented with 0.5% albumin, 0.2% glucose, 0.085% NaCl, 0.5% glycerol and 0.05% tyloxapol.

Over Expression:

Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity
Article Snippet: Construction of the ΔvirR , ΔpstA1 ΔvirR , virR overexpression, eccD 5 Tet-OFF, and ΔpstA1 eccD 5 Tet-OFF strains are described below. .. Bacteria were routinely cultured in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 or on Middlebrook 7H10 agar medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) and 0.5% glycerol.

Derivative Assay:

Article Title: Rapid Cytolysis of Mycobacterium tuberculosis by Faropenem, an Orally Bioavailable β-Lactam Antibiotic
Article Snippet: .. Mycobacterium tuberculosis Erdman and H37Rv and strains derived from strains Erdman and H37Rv were grown in Middlebrook 7H9 liquid medium (Difco) containing 0.5% albumin, 0.085% NaCl, 0.2% glucose, 0.5% glycerol, and 0.05% Tween 80 or on Middlebrook 7H10 solid medium (Difco) containing 10% oleic acid-albumin-dextrose-catalase (Difco) and 0.5% glycerol. .. Uropathogenic Escherichia coli CFT073 and strains derived from strain CFT073 were grown in LB medium (Sigma).

Southern Blot:

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: The strains used include the laboratory strains H37Rv (Trudeau Institute, Saranac Lake, N.Y.), CDC 1551 (T. M. Shinnick, Centers for Disease Control and Prevention, Atlanta, Ga.), ATCC 35825, and H37Rv Inhr , which was selected from H37Rv by direct plating onto 10 μg of isoniazid per ml (loss of KatG was verified by [i] activity assay, [ii] Western blotting using anti-KatG antipeptide antisera, and [iii] Southern blot analysis using the katG probe and restriction digestion as described previously [ ]), as well as the recombinant strain H37Rv(pMH59) and the recombinant strain H37Rv(pMH91), which have been previously described ( ). .. Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation.

Cell Culture:

Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity
Article Snippet: .. Bacteria were routinely cultured in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 or on Middlebrook 7H10 agar medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) and 0.5% glycerol. .. Strains containing the pvirR plasmid were grown in the presence of 50 µg/ml hygromycin B (Sigma).

Article Title: The Mycobacterium tuberculosis MmpL11 Cell Wall Lipid Transporter Is Important for Biofilm Formation, Intracellular Growth, and Nonreplicating Persistence
Article Snippet: Mycobacterial strains were routinely maintained in Middlebrook 7H9 liquid medium (Difco) or on Middlebrook 7H11 agar (Difco) plates supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC; Difco) or albumin dextrose salts (ADS) containing 8.1 mg ml−1 NaCl, 50 mg ml−1 bovine serum albumin (BSA), and 20 mg ml−1 dextrose. .. Where indicated, bacteria were cultured in Sauton's medium containing 0.5 g liter−1 K2 HPO4 , 0.5 g liter−1 MgSO4 , 4.0 g liter−1 l -asparagine, 0.05 g liter−1 ferric ammonium citrate, 4.76% glycerol, and 1.0 mg liter−1 ZnSO4 , with a final pH of 7.0.

Article Title: Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level
Article Snippet: E.coli strains were grown on LB broth agar or in LB broth, Mycobacterium smegmatis mc2 155 was grown in 7H9 liquid medium (Difco) supplemented with 0.05% w/v Tween 80, 0.5%glycerol and 0.5%glucose or were grown on 7H10 agar supplemented with 1% glycerol and 0.5% glucose. .. For viability determination during starvation, bacteria were cultured in 50 ml volumes in 250 ml bottles (Shuniu), and the number of cfu/ml was determined by plating serial dilutions onto 7H10 agar from triplicate cultures at several time points (0 h, 24 h, and 72 h).

Generated:

Article Title: Mycobacterium smegmatis RoxY Is a Repressor of oxyS and Contributes to Resistance to Oxidative Stress and Bactericidal Ubiquitin-Derived Peptides ▿ and Contributes to Resistance to Oxidative Stress and Bactericidal Ubiquitin-Derived Peptides ▿ †
Article Snippet: The M. smegmatis transposon mutant library was generated in M. smegmatis mc2 155 using the mariner -based transposon mutagenesis vector pM272B as described previously ( , ). .. Mycobacterial strains were maintained in Middlebrook 7H9 liquid medium (Difco) or on Middlebrook 7H11 agar (Difco) plates supplemented with oleic acid-albumin-dextrose-catalase (OADC) (BD).

Recombinant:

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: The strains used include the laboratory strains H37Rv (Trudeau Institute, Saranac Lake, N.Y.), CDC 1551 (T. M. Shinnick, Centers for Disease Control and Prevention, Atlanta, Ga.), ATCC 35825, and H37Rv Inhr , which was selected from H37Rv by direct plating onto 10 μg of isoniazid per ml (loss of KatG was verified by [i] activity assay, [ii] Western blotting using anti-KatG antipeptide antisera, and [iii] Southern blot analysis using the katG probe and restriction digestion as described previously [ ]), as well as the recombinant strain H37Rv(pMH59) and the recombinant strain H37Rv(pMH91), which have been previously described ( ). .. Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation.

Mutagenesis:

Article Title: Mycobacterium smegmatis RoxY Is a Repressor of oxyS and Contributes to Resistance to Oxidative Stress and Bactericidal Ubiquitin-Derived Peptides ▿ and Contributes to Resistance to Oxidative Stress and Bactericidal Ubiquitin-Derived Peptides ▿ †
Article Snippet: The M. smegmatis transposon mutant library was generated in M. smegmatis mc2 155 using the mariner -based transposon mutagenesis vector pM272B as described previously ( , ). .. Mycobacterial strains were maintained in Middlebrook 7H9 liquid medium (Difco) or on Middlebrook 7H11 agar (Difco) plates supplemented with oleic acid-albumin-dextrose-catalase (OADC) (BD).

Isolation:

Article Title: Chlorine Disinfection of Atypical Mycobacteria Isolated from a Water Distribution System
Article Snippet: The other mycobacterial strains used were isolated from cold public water supplies in Paris. .. Cells were grown in Middlebrook 7H9 liquid medium (Difco Laboratories, West Molesley, Surrey, United Kingdom) containing 10% (vol/vol) oleic acid-albumin enrichment and 0.05% (vol/vol) Tween 80 at their optimal growth temperature (30 or 37°C, depending on the strain) in a rotary shaker (120 rpm).

Article Title: Experimental selection of long-term intracellular mycobacteria
Article Snippet: Bone marrow macrophages (BMMs) were isolated and maintained as described previously (Kasmapour et al ., ). .. Pasteur 1173P2 expressing GFP (BCG-GFP, ancestral strain (ANC) kindly provided by Dr Brigitte Gicquel, Pasteur Institute, France) were grown in roller flasks at 37°C in Middlebrook 7H9 liquid medium (Difco Laboratories, USA) containing 0.2% glycerol, 0.05% Tween 80 and supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase supplement) (BD Biosciences, USA) or in Middlebrook 7H10 plates (Difco Laboratories, USA) supplemented with 10% OADC.

Mouse Assay:

Article Title: Microanatomic Distribution of Myeloid Heme Oxygenase-1 Protects against Free Radical-Mediated Immunopathology in Human Tuberculosis
Article Snippet: .. Infection of Mice Mtb H37Rv was grown to mid-log phase in Middlebrook 7H9 liquid medium (Difco, Detroit, MI, USA) containing 0.2% glycerol, and 0.02% Tyloxapol supplemented with ADS (Albumin, Dextrose, Saline). ..

Plasmid Preparation:

Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity
Article Snippet: Bacteria were routinely cultured in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 or on Middlebrook 7H10 agar medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) and 0.5% glycerol. .. Strains containing the pvirR plasmid were grown in the presence of 50 µg/ml hygromycin B (Sigma).

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: The vector system also has a synthetic promoter element incorporated into the polylinker driving expression of KatG ( ). .. Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation.

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria
Article Snippet: Yellow fluorescent protein (YFP)-expressing derivatives of both E. coli strains were made by transformation with a plasmid encoding YFP . .. M. tuberculosis Erdman, M. tuberculosis H37Rv, M. tuberculosis CDC1551, M. tuberculosis HN787, M. bovis BCG (Pasteur 1173P2), M. smegmatis mc2 155 and derivative strains ( ) were grown in Middlebrook 7H9 liquid medium (Difco) supplemented with 0.5% albumin, 0.2% glucose, 0.085% NaCl, 0.5% glycerol and 0.05% tyloxapol.

Article Title: A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria
Article Snippet: .. Individual colonies were picked and amplified in 7H9 liquid medium (no antibiotics) and then plated on LB agar or 7H10 containing 5% sucrose to select for cells in which plasmid excision had occurred (second crossover). .. Individual colonies were picked and the replacement of the gene by the fusion gene or gene deletion was confirmed by PCR.

Article Title: Mycobacterium smegmatis RoxY Is a Repressor of oxyS and Contributes to Resistance to Oxidative Stress and Bactericidal Ubiquitin-Derived Peptides ▿ and Contributes to Resistance to Oxidative Stress and Bactericidal Ubiquitin-Derived Peptides ▿ †
Article Snippet: The M. smegmatis transposon mutant library was generated in M. smegmatis mc2 155 using the mariner -based transposon mutagenesis vector pM272B as described previously ( , ). .. Mycobacterial strains were maintained in Middlebrook 7H9 liquid medium (Difco) or on Middlebrook 7H11 agar (Difco) plates supplemented with oleic acid-albumin-dextrose-catalase (OADC) (BD).

Concentration Assay:

Article Title: Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity
Article Snippet: Bacteria were routinely cultured in Middlebrook 7H9 liquid medium (Difco) supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 or on Middlebrook 7H10 agar medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC; BD Biosciences) and 0.5% glycerol. .. Frozen stocks were prepared by growing cultures to mid-logarithmic phase, adding glycerol to a 15% final concentration, and aliquoting for storage at −80°C.

Article Title: The Mycobacterium tuberculosis MmpL11 Cell Wall Lipid Transporter Is Important for Biofilm Formation, Intracellular Growth, and Nonreplicating Persistence
Article Snippet: Mycobacterial strains were routinely maintained in Middlebrook 7H9 liquid medium (Difco) or on Middlebrook 7H11 agar (Difco) plates supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC; Difco) or albumin dextrose salts (ADS) containing 8.1 mg ml−1 NaCl, 50 mg ml−1 bovine serum albumin (BSA), and 20 mg ml−1 dextrose. .. Glycerol was added to 7H9 and 7H11 media at a final concentration of 0.5%.

Lysis:

Article Title: Mycobacterium tuberculosis Catalase and Peroxidase Activities and Resistance to Oxidative Killing in Human Monocytes In Vitro
Article Snippet: Mycobacterial strains were grown for 7 days in Middlebrook 7H9 liquid medium (Difco, Detroit, Mich.) containing 0.05% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) at 37°C with daily agitation. .. The culture medium was lipopolysaccharide free and without reactivity in the Limulus amoebocyte lysis assay (Whittaker Bioproducts, Walkersville, Md.).

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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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    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
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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
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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