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Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-free <t>7H9</t> ( a ), Fe-free Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation
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1) Product Images from "Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro"

Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro

Journal: BMC Research Notes

doi: 10.1186/s13104-017-2752-0

Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-free 7H9 ( a ), Fe-free Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation
Figure Legend Snippet: Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-free 7H9 ( a ), Fe-free Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation

Techniques Used: Mutagenesis, Mass Spectrometry, Standard Deviation

The comparison of the intracellular iron levels in WT ms , ΔMycP3 ms , ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms strains under 7H9, Fe-depleted 7H9 and Fe rescued 7H9 media. The error bars show standard error of the mean (n = 4). The p values obtained using two-way ANOVA statistical analysis between different culturing conditions for all four strains are smaller than 0.0001 ( *** ), an example is shown for the WT ms
Figure Legend Snippet: The comparison of the intracellular iron levels in WT ms , ΔMycP3 ms , ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms strains under 7H9, Fe-depleted 7H9 and Fe rescued 7H9 media. The error bars show standard error of the mean (n = 4). The p values obtained using two-way ANOVA statistical analysis between different culturing conditions for all four strains are smaller than 0.0001 ( *** ), an example is shown for the WT ms

Techniques Used: Mass Spectrometry

Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-depleted 7H9 ( a ), and Fe-depleted Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation
Figure Legend Snippet: Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-depleted 7H9 ( a ), and Fe-depleted Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation

Techniques Used: Mutagenesis, Mass Spectrometry, Standard Deviation

Gene expression analysis of mycP 3 in WT ms , ΔMycP 3ms , ΔMycP 3ms ::Pr1MycP 3 , and ΔMycP 3ms ::Pr2MycP 3 strains under normal 7H9 (iron rich), Fe-free 7H9, and Fe-rescued 7H9 media. The results were normalized against the RNA copy number of sigA . The p values obtained using two-way ANOVA statistical analysis (n = 3) (*p
Figure Legend Snippet: Gene expression analysis of mycP 3 in WT ms , ΔMycP 3ms , ΔMycP 3ms ::Pr1MycP 3 , and ΔMycP 3ms ::Pr2MycP 3 strains under normal 7H9 (iron rich), Fe-free 7H9, and Fe-rescued 7H9 media. The results were normalized against the RNA copy number of sigA . The p values obtained using two-way ANOVA statistical analysis (n = 3) (*p

Techniques Used: Expressing, Mass Spectrometry

Related Articles

Clone Assay:

Article Title: MspA Nanopores from Subunit Dimers
Article Snippet: Bacterial Strains and Growth Conditions M. smegmatis ML16, which lacks the porin genes mspA , mspC , and mspD was grown at 37°C in 7H9 liquid medium (BD Biosciences) supplemented with 0.2% glycerol and 0.05% Tween 80 or on 7H10 agar (BD Biosciences) supplemented with 0.2% glycerol, unless otherwise indicated. .. Escherichia coli DH5α was used for cloning experiments and was routinely grown in Luria-Bertani broth (LB) at 37°C.

Article Title: Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis
Article Snippet: Bacterial Strains and Growth Conditions — M. smegmatis ML16, which lacks the porin genes mspA, mspC , and mspD ( ) was grown at 37 °C in 7H9 liquid medium (BD Biosciences) supplemented with 0.2% glycerol and 0.05% Tween 80 or on 7H10 agar (BD Biosciences) supplemented with 0.2% glycerol, unless otherwise indicated. .. E. coli DH5α was used for cloning experiments and was routinely grown in Luria-Bertani broth at 37 °C.

Centrifugation:

Article Title: Increasing Prevalence of Nontuberculous Mycobacteria in Respiratory Specimens from US-Affiliated Pacific Island Jurisdictions 1
Article Snippet: Specimens were digested by using the standard NALC-NaOH method, followed by centrifugation ( ). .. The remaining sediment was resuspended and used to inoculate 7H9 liquid medium (BACTEC MGIT 960; Becton Dickinson, Franklin Lakes, NJ, USA), broth medium, and solid medium (Middlebrook 7H10 agar slant; Becton Dickinson).

Construct:

Article Title: Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis
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Real-time Polymerase Chain Reaction:

Article Title: Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans
Article Snippet: Identification was confirmed by using a multiplex real-time PCR as previously described [ ]. .. To produce a large enough inoculum, both isolates were cultured at 32°C in Middlebrook 7H9 liquid medium (Becton Dickinson, Le Pont de Claix, France) complemented with oleic acid, bovine albumin, dextrose, and catalase enrichment (OADC, Becton Dickinson).

Incubation:

Article Title: Increasing Prevalence of Nontuberculous Mycobacteria in Respiratory Specimens from US-Affiliated Pacific Island Jurisdictions 1
Article Snippet: The remaining sediment was resuspended and used to inoculate 7H9 liquid medium (BACTEC MGIT 960; Becton Dickinson, Franklin Lakes, NJ, USA), broth medium, and solid medium (Middlebrook 7H10 agar slant; Becton Dickinson). .. The broth medium was incubated in a MGIT 960 semiautomated system (Becton Dickinson) and the 7H10 agar slant was incubated at 35°C in an incubator containing 5% CO2 .

Article Title: Glutathione deficiency in type 2 diabetes impairs cytokine responses and control of intracellular bacteria
Article Snippet: Cells were incubated for at 37°C at 5% CO2 for 24 hours before the supernatants were collected for cytokine analysis. .. Frozen stocks of M. tuberculosis H37Rv wild-type strain were prepared by resuspending a log-phase bacterial culture in Middlebrook 7H9 Liquid Medium (BD Biosciences) supplemented with 25% glycerol and stored as 1 ml single-use aliquots at –80°C.

Article Title: A Nonribosomal Peptide Synthase Gene Driving Virulence in Mycobacterium tuberculosis
Article Snippet: Bacteria were grown in Middlebrook 7H9 liquid medium (BD Biosciences) supplemented with 0.05% Tween 80, 0.2% glycerol, and 10% oleic acid-albumin-dextrose-catalase (OADC) for 7 to 10 days and then subcultured in Proskauer Beck (PB) medium supplemented with 0.05% Tween 80 and 2% glycerol to the mid-log phase. .. Viable bacteria were determined by CFU enumeration after 21 to 28 days of incubation at 37°C.

Article Title: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases
Article Snippet: .. Bacterial strains and growth conditions Mycobacterium marinum CECT 7091 reference strain (Colección Española de Cultivos Tipo, Valencia, Spain) was grown in sterile 250 ml flasks with 7H9 liquid medium supplemented with ADC (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 31 °C with continuous shaking for 7 days in the dark. .. Spectrophotometric absorbance was measured at 600 nm (OD600 ) and the concentration was adjusted to 108 –109 CFU ml−1 .

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Article Snippet: The Middlebrook 7H9 liquid medium (BD Diagnostic Systems) used in the BioSampler studies was supplemented with 10% ADC (BD Diagnostic Systems) and 0.05% Tween 80 (Sigma-Aldrich, St. Louis, MO). .. BCG organisms were inoculated into Middlebrook 7H9 liquid medium and incubated at 37 ± 2°C with gentle agitation for 15 to 25 days or until a pellicle was formed.

Infection:

Article Title: Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle
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Article Title: Glutathione deficiency in type 2 diabetes impairs cytokine responses and control of intracellular bacteria
Article Snippet: Paragraph title: Bacterial infection. ... Frozen stocks of M. tuberculosis H37Rv wild-type strain were prepared by resuspending a log-phase bacterial culture in Middlebrook 7H9 Liquid Medium (BD Biosciences) supplemented with 25% glycerol and stored as 1 ml single-use aliquots at –80°C.

Article Title: Interleukin-17A as a Biomarker for Bovine Tuberculosis
Article Snippet: Paragraph title: Study overview and aerosol infection with Mycobacterium bovis . ... Challenge inocula from frozen stocks were prepared in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10% oleic acid-albumin-dextrose complex (OADC) plus 0.05% Tween 80 (Sigma, St. Louis, MO), following standard techniques ( ).

Expressing:

Article Title: Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells
Article Snippet: Both bacteria, ATCC 19977 and CRM0019, were transformed with the Escherichia coli -mycobacterium expression vector pMV262 containing the green fluorescent protein (GFP) gene (pMV262-gfp ). .. All mycobacterial strains were grown in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ), supplemented with oleic acid, albumin, dextrose and catalase (OADC – Becton Dickinson), 0.5% glycerol and 0.05% Tween 80 at 37 °C, for 72 h. Transformed bacteria were grown in medium containing kanamycin (50 μg/ml) and sub-cultured in fresh medium for 24 h before the experiments.

Transformation Assay:

Article Title: Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells
Article Snippet: .. All mycobacterial strains were grown in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ), supplemented with oleic acid, albumin, dextrose and catalase (OADC – Becton Dickinson), 0.5% glycerol and 0.05% Tween 80 at 37 °C, for 72 h. Transformed bacteria were grown in medium containing kanamycin (50 μg/ml) and sub-cultured in fresh medium for 24 h before the experiments. .. Mycobacteria-cells infection A549 cells and macrophages (5 × 104 per well) previously grown in 24-well plates in medium without antibiotics were infected simultaneously with single-cell bacterial suspensions.

Derivative Assay:

Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro
Article Snippet: Mycobacterium smegmatis mc2 155 (a gift from Rob Warren, South Africa) was used as the parent wildtype strain (WTms ) from which the MycP3 deletion mutant (ΔMycP3ms ) was derived, and as template to generate two complementation strains (ΔMycP3ms ::pr1MycP3ms and ΔMycP3ms ::pr2MycP3ms ). .. M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516).

Cell Culture:

Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro
Article Snippet: .. M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516). .. Fe-free 7H9 (omitting ferric ammonium citrate) and Fe-free Sauton’s medium (3.5 mM KH2 PO4 (Sigma-Aldrich, USA, Catalogue No. NIST200B), 25 mM l -asparagine (Sigma-Aldrich, USA, Catalogue No. A0884), 10 mM citric acid (Sigma-Aldrich, USA, Catalogue No. 791725), 4 mM MgSO4 ·6H2 O (Sigma-Aldrich, USA, Catalogue No. 746452), 5% (v/v) glycerol, and 0.05% (v/v) Tween-80) were also used to monitor bacterial growth under iron-limiting condition.

Article Title: Glutathione deficiency in type 2 diabetes impairs cytokine responses and control of intracellular bacteria
Article Snippet: Frozen stocks of M. tuberculosis H37Rv wild-type strain were prepared by resuspending a log-phase bacterial culture in Middlebrook 7H9 Liquid Medium (BD Biosciences) supplemented with 25% glycerol and stored as 1 ml single-use aliquots at –80°C. .. For each infection assay, a frozen stock of H37Rv mycobacteria was defrosted and immediately diluted at the appropriate concentration in antibiotic-free cell culture medium; 500 μl of inoculum was added directly to the cells, which were incubated at 37°C and 5% CO2 for 24 hours before the supernatants were collected for cytokine analysis.

Article Title: Containment of Bioaerosol Infection Risk by the Xpert MTB/RIF Assay and Its Applicability to Point-of-Care Settings ▿
Article Snippet: M. tuberculosis strain H37Rv was obtained from the American Tissue Culture Collection (ATCC), Manassas, VA. Mycobacteria were cultured on 7H10 solid medium (BD Diagnostic Systems, Sparks, MD) supplemented with 0.5% glycerol and 10% albumin-dextrose-catalase with sodium chloride (Middlebrook ADC Enrichment; BD Diagnostic Systems). .. The Middlebrook 7H9 liquid medium (BD Diagnostic Systems) used in the BioSampler studies was supplemented with 10% ADC (BD Diagnostic Systems) and 0.05% Tween 80 (Sigma-Aldrich, St. Louis, MO).

Article Title: Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans
Article Snippet: .. To produce a large enough inoculum, both isolates were cultured at 32°C in Middlebrook 7H9 liquid medium (Becton Dickinson, Le Pont de Claix, France) complemented with oleic acid, bovine albumin, dextrose, and catalase enrichment (OADC, Becton Dickinson). .. The M . ulcerans isolate was manipulated in a Biosafety Level 3 laboratory, Medical School, Marseilles, France.

Inhibition:

Article Title: Host-Mediated Bioactivation of Pyrazinamide: Implications for Efficacy, Resistance, and Therapeutic Alternatives
Article Snippet: M. tuberculosis strains H37Rv and H37Ra (ATCC 25177) were used to measure growth inhibition in the presence of increasing doses of PZA and POA using a standardized turbidometric assay. .. M. tuberculosis was grown in Middlebrook 7H9 liquid medium (BD Difco) supplemented with 10% albumin-dextrose-catalase (ADC), 0.5% glycerol, and 0.05% Tween-80 (Sigma) in T25 culture flasks at 37 °C at 80 rpm.

Sequencing:

Article Title: Host-Mediated Bioactivation of Pyrazinamide: Implications for Efficacy, Resistance, and Therapeutic Alternatives
Article Snippet: In addition, the pncA DNA sequence is 100% conserved in H37Ra (MRA_2058) and H37Rv (Rv2043c). .. M. tuberculosis was grown in Middlebrook 7H9 liquid medium (BD Difco) supplemented with 10% albumin-dextrose-catalase (ADC), 0.5% glycerol, and 0.05% Tween-80 (Sigma) in T25 culture flasks at 37 °C at 80 rpm.

Article Title: Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans
Article Snippet: Mycobacterial strains An M . marinum clinical isolate [ ] was identified by rpoB gene sequence analysis [ ]. .. To produce a large enough inoculum, both isolates were cultured at 32°C in Middlebrook 7H9 liquid medium (Becton Dickinson, Le Pont de Claix, France) complemented with oleic acid, bovine albumin, dextrose, and catalase enrichment (OADC, Becton Dickinson).

Recombinant:

Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro
Article Snippet: Bacterial strains, culture media and plasmid DNA Escherichia coli XL-1 blue (Stratagene, USA, Catalogue No. 200249) was used for manipulating and propagating recombinant plasmid DNA. .. M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516).

Ziehl-Neelsen Stain:

Article Title: Increasing Prevalence of Nontuberculous Mycobacteria in Respiratory Specimens from US-Affiliated Pacific Island Jurisdictions 1
Article Snippet: A portion of the sediment was then used to prepare an AFB smear. .. The remaining sediment was resuspended and used to inoculate 7H9 liquid medium (BACTEC MGIT 960; Becton Dickinson, Franklin Lakes, NJ, USA), broth medium, and solid medium (Middlebrook 7H10 agar slant; Becton Dickinson).

RNA Sequencing Assay:

Article Title: Interleukin-17A as a Biomarker for Bovine Tuberculosis
Article Snippet: Samples from four independent studies were included, i.e., one study that compared the virulence of two field strains of M. bovis in calves, two vaccine efficacy studies, and a M. bovis infection-only study (for RNA-Seq samples). .. Challenge inocula from frozen stocks were prepared in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10% oleic acid-albumin-dextrose complex (OADC) plus 0.05% Tween 80 (Sigma, St. Louis, MO), following standard techniques ( ).

Mutagenesis:

Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro
Article Snippet: Mycobacterium smegmatis mc2 155 (a gift from Rob Warren, South Africa) was used as the parent wildtype strain (WTms ) from which the MycP3 deletion mutant (ΔMycP3ms ) was derived, and as template to generate two complementation strains (ΔMycP3ms ::pr1MycP3ms and ΔMycP3ms ::pr2MycP3ms ). .. M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516).

Isolation:

Article Title: Lysosomal killing of Mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy
Article Snippet: Mycobacterial strains were maintained in Middlebrook 7H9 liquid medium (BD Biosciences, San Jose, CA) or on Middlebrook 7H11 agar (BD Biosciences) plates supplemented with oleic acid, albumin, dextrose, catalase (OADC; BD Biosciences). .. BMMΦ were isolated from BALB/c mice or from LC3-GFP transgenic mice (LC3-GFP 54) and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Invitrogen)/5% horse serum (Invitrogen)/1.5 g/liter sodium pyruvate (Invitrogen)/20% L cell-conditioned medium.

Article Title: Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells
Article Snippet: M. abscessus subsp. abscessus smooth variant was isolated from the sputum of CF patients [ ], and kindly provided by Prof. Dr. John Perry, Newcastle University. .. All mycobacterial strains were grown in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ), supplemented with oleic acid, albumin, dextrose and catalase (OADC – Becton Dickinson), 0.5% glycerol and 0.05% Tween 80 at 37 °C, for 72 h. Transformed bacteria were grown in medium containing kanamycin (50 μg/ml) and sub-cultured in fresh medium for 24 h before the experiments.

Article Title: Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans
Article Snippet: M . ulcerans ATCC19423T strain Agy99 (classical lineage) [ ] was isolated in 1999 in Ghana (Ga District, Great Accra Region) from the biopsy of an ulcerative lesion on the right elbow of a female patient. .. To produce a large enough inoculum, both isolates were cultured at 32°C in Middlebrook 7H9 liquid medium (Becton Dickinson, Le Pont de Claix, France) complemented with oleic acid, bovine albumin, dextrose, and catalase enrichment (OADC, Becton Dickinson).

Microscopy:

Article Title: Increasing Prevalence of Nontuberculous Mycobacteria in Respiratory Specimens from US-Affiliated Pacific Island Jurisdictions 1
Article Snippet: The remaining sediment was resuspended and used to inoculate 7H9 liquid medium (BACTEC MGIT 960; Becton Dickinson, Franklin Lakes, NJ, USA), broth medium, and solid medium (Middlebrook 7H10 agar slant; Becton Dickinson). .. The prepared smear was heat-fixed at 70°C–80°C for 2 h. The smear was then stained by using the fluorochrome stain method, which used auramine O, a decolorizing solution, and a potassium permanganate counterstain and read by using a fluorescent microscope.

Mouse Assay:

Article Title: Lysosomal killing of Mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy
Article Snippet: Mycobacterial strains were maintained in Middlebrook 7H9 liquid medium (BD Biosciences, San Jose, CA) or on Middlebrook 7H11 agar (BD Biosciences) plates supplemented with oleic acid, albumin, dextrose, catalase (OADC; BD Biosciences). .. BMMΦ were isolated from BALB/c mice or from LC3-GFP transgenic mice (LC3-GFP 54) and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Invitrogen)/5% horse serum (Invitrogen)/1.5 g/liter sodium pyruvate (Invitrogen)/20% L cell-conditioned medium.

Transgenic Assay:

Article Title: Lysosomal killing of Mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy
Article Snippet: Mycobacterial strains were maintained in Middlebrook 7H9 liquid medium (BD Biosciences, San Jose, CA) or on Middlebrook 7H11 agar (BD Biosciences) plates supplemented with oleic acid, albumin, dextrose, catalase (OADC; BD Biosciences). .. BMMΦ were isolated from BALB/c mice or from LC3-GFP transgenic mice (LC3-GFP 54) and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Invitrogen)/5% horse serum (Invitrogen)/1.5 g/liter sodium pyruvate (Invitrogen)/20% L cell-conditioned medium.

IA:

Article Title: Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle
Article Snippet: For the challenge inoculum, low-passage-number cultures (≤3 passages) of M. bovis were prepared, using standard techniques , in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10% oleic acid-albumin-dextrose complex (OADC) plus 0.05% Tween 80. .. Holstein steers were obtained from tuberculosis-free and paratuberculosis-monitored herds in Iowa and were housed in a biosafety level 3 (BSL-3) facility at the National Animal Disease Center (Ames, IA), according to institutional biosafety (permit IBC-0004RA) and animal care and use committee guidelines (with ethical approval via animal care and use protocol ACUP-3859).

Article Title: Interleukin-17A as a Biomarker for Bovine Tuberculosis
Article Snippet: Challenge inocula from frozen stocks were prepared in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10% oleic acid-albumin-dextrose complex (OADC) plus 0.05% Tween 80 (Sigma, St. Louis, MO), following standard techniques ( ). .. Holstein steers were obtained from TB-free herds in Iowa and housed in a biosafety level 3 (BSL3) facility at the National Animal Disease Center (Ames, IA), according to institutional biosafety and animal care and use committee guidelines and oversight (i.e., formal review and approval of studies).

Plasmid Preparation:

Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro
Article Snippet: Paragraph title: Bacterial strains, culture media and plasmid DNA ... M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516).

Article Title: Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells
Article Snippet: In some experiments, M. smegmatis mc2 155, wild type or transformed with the same plasmid, was used. .. All mycobacterial strains were grown in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ), supplemented with oleic acid, albumin, dextrose and catalase (OADC – Becton Dickinson), 0.5% glycerol and 0.05% Tween 80 at 37 °C, for 72 h. Transformed bacteria were grown in medium containing kanamycin (50 μg/ml) and sub-cultured in fresh medium for 24 h before the experiments.

Article Title: Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis
Article Snippet: Bacterial Strains and Growth Conditions — M. smegmatis ML16, which lacks the porin genes mspA, mspC , and mspD ( ) was grown at 37 °C in 7H9 liquid medium (BD Biosciences) supplemented with 0.2% glycerol and 0.05% Tween 80 or on 7H10 agar (BD Biosciences) supplemented with 0.2% glycerol, unless otherwise indicated. .. Construction of MspA Loop 6 Deletion Mutants —The L6 deletion mutants were constructed using the plasmid pMN016, which carries the psmyc -mspA transcriptional fusion ( ) as a template.

Multiplex Assay:

Article Title: Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans
Article Snippet: Identification was confirmed by using a multiplex real-time PCR as previously described [ ]. .. To produce a large enough inoculum, both isolates were cultured at 32°C in Middlebrook 7H9 liquid medium (Becton Dickinson, Le Pont de Claix, France) complemented with oleic acid, bovine albumin, dextrose, and catalase enrichment (OADC, Becton Dickinson).

In Vitro:

Article Title: Host-Mediated Bioactivation of Pyrazinamide: Implications for Efficacy, Resistance, and Therapeutic Alternatives
Article Snippet: Paragraph title: In Vitro Growth Inhibitory Assay ... M. tuberculosis was grown in Middlebrook 7H9 liquid medium (BD Difco) supplemented with 10% albumin-dextrose-catalase (ADC), 0.5% glycerol, and 0.05% Tween-80 (Sigma) in T25 culture flasks at 37 °C at 80 rpm.

Spectrophotometry:

Article Title: Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans
Article Snippet: To produce a large enough inoculum, both isolates were cultured at 32°C in Middlebrook 7H9 liquid medium (Becton Dickinson, Le Pont de Claix, France) complemented with oleic acid, bovine albumin, dextrose, and catalase enrichment (OADC, Becton Dickinson). .. The suspension was calibrated at 105 mycobacteria/mL by observing and counting dispersed bacteria after Ziehl-Neelsen staining and by measuring the optical density of the suspension (Cell Density Meter Model 40 spectrophotometer, Fischer Scientific, Illkirch, France).

Concentration Assay:

Article Title: Host-Mediated Bioactivation of Pyrazinamide: Implications for Efficacy, Resistance, and Therapeutic Alternatives
Article Snippet: M. tuberculosis was grown in Middlebrook 7H9 liquid medium (BD Difco) supplemented with 10% albumin-dextrose-catalase (ADC), 0.5% glycerol, and 0.05% Tween-80 (Sigma) in T25 culture flasks at 37 °C at 80 rpm. .. PZA and POA (pyrazinecarboxylic acid, Sigma-Aldrich) were freshly dissolved in 90% DMSO at a concentration of 500 mM and sterilized using a 0.2 µm PTFE membrane filter (Acrodisc PALL).

Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro
Article Snippet: M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516). .. Additionally, the iron deprivation rescuing of the M. smegmatis cultured in iron-deprived 7H9 or Sauton’s media was achieved by supplementation of ferric ammonium citrate in the same concentration of normal 7H9 or Sauton’s media.

Article Title: Glutathione deficiency in type 2 diabetes impairs cytokine responses and control of intracellular bacteria
Article Snippet: Frozen stocks of M. tuberculosis H37Rv wild-type strain were prepared by resuspending a log-phase bacterial culture in Middlebrook 7H9 Liquid Medium (BD Biosciences) supplemented with 25% glycerol and stored as 1 ml single-use aliquots at –80°C. .. Bacterial concentration was determined by plating appropriate dilutions of a representative vial of stock on Middlebrook 7H11 Agar supplemented with 10% Middlebrook OADC Enrichment (BD Biosciences) and quantifying the number of viable colonies after incubating the plates for 16 days at 37°C and 5% CO2 .

Article Title: A Nonribosomal Peptide Synthase Gene Driving Virulence in Mycobacterium tuberculosis
Article Snippet: Bacteria were grown in Middlebrook 7H9 liquid medium (BD Biosciences) supplemented with 0.05% Tween 80, 0.2% glycerol, and 10% oleic acid-albumin-dextrose-catalase (OADC) for 7 to 10 days and then subcultured in Proskauer Beck (PB) medium supplemented with 0.05% Tween 80 and 2% glycerol to the mid-log phase. .. To determine the concentration of M. tuberculosis in aliquots, 6 frozen aliquots were serial diluted and plated on Middlebrook 7H11 (BD Biosciences) agar plates supplemented with 10% OADC and 0.5% glycerol.

Article Title: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases
Article Snippet: Bacterial strains and growth conditions Mycobacterium marinum CECT 7091 reference strain (Colección Española de Cultivos Tipo, Valencia, Spain) was grown in sterile 250 ml flasks with 7H9 liquid medium supplemented with ADC (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated at 31 °C with continuous shaking for 7 days in the dark. .. Spectrophotometric absorbance was measured at 600 nm (OD600 ) and the concentration was adjusted to 108 –109 CFU ml−1 .

Staining:

Article Title: Increasing Prevalence of Nontuberculous Mycobacteria in Respiratory Specimens from US-Affiliated Pacific Island Jurisdictions 1
Article Snippet: The remaining sediment was resuspended and used to inoculate 7H9 liquid medium (BACTEC MGIT 960; Becton Dickinson, Franklin Lakes, NJ, USA), broth medium, and solid medium (Middlebrook 7H10 agar slant; Becton Dickinson). .. The prepared smear was heat-fixed at 70°C–80°C for 2 h. The smear was then stained by using the fluorochrome stain method, which used auramine O, a decolorizing solution, and a potassium permanganate counterstain and read by using a fluorescent microscope.

Article Title: Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans
Article Snippet: To produce a large enough inoculum, both isolates were cultured at 32°C in Middlebrook 7H9 liquid medium (Becton Dickinson, Le Pont de Claix, France) complemented with oleic acid, bovine albumin, dextrose, and catalase enrichment (OADC, Becton Dickinson). .. The suspension was calibrated at 105 mycobacteria/mL by observing and counting dispersed bacteria after Ziehl-Neelsen staining and by measuring the optical density of the suspension (Cell Density Meter Model 40 spectrophotometer, Fischer Scientific, Illkirch, France).

Variant Assay:

Article Title: Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells
Article Snippet: M. abscessus subsp. abscessus smooth variant was isolated from the sputum of CF patients [ ], and kindly provided by Prof. Dr. John Perry, Newcastle University. .. All mycobacterial strains were grown in Middlebrook 7H9 liquid medium (Becton Dickinson, Franklin Lakes, NJ), supplemented with oleic acid, albumin, dextrose and catalase (OADC – Becton Dickinson), 0.5% glycerol and 0.05% Tween 80 at 37 °C, for 72 h. Transformed bacteria were grown in medium containing kanamycin (50 μg/ml) and sub-cultured in fresh medium for 24 h before the experiments.

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    Becton Dickinson 7h9 liquid medium
    Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-free <t>7H9</t> ( a ), Fe-free Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation
    7h9 Liquid Medium, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-free 7H9 ( a ), Fe-free Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation

    Journal: BMC Research Notes

    Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro

    doi: 10.1186/s13104-017-2752-0

    Figure Lengend Snippet: Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-free 7H9 ( a ), Fe-free Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation

    Article Snippet: M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516).

    Techniques: Mutagenesis, Mass Spectrometry, Standard Deviation

    The comparison of the intracellular iron levels in WT ms , ΔMycP3 ms , ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms strains under 7H9, Fe-depleted 7H9 and Fe rescued 7H9 media. The error bars show standard error of the mean (n = 4). The p values obtained using two-way ANOVA statistical analysis between different culturing conditions for all four strains are smaller than 0.0001 ( *** ), an example is shown for the WT ms

    Journal: BMC Research Notes

    Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro

    doi: 10.1186/s13104-017-2752-0

    Figure Lengend Snippet: The comparison of the intracellular iron levels in WT ms , ΔMycP3 ms , ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms strains under 7H9, Fe-depleted 7H9 and Fe rescued 7H9 media. The error bars show standard error of the mean (n = 4). The p values obtained using two-way ANOVA statistical analysis between different culturing conditions for all four strains are smaller than 0.0001 ( *** ), an example is shown for the WT ms

    Article Snippet: M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516).

    Techniques: Mass Spectrometry

    Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-depleted 7H9 ( a ), and Fe-depleted Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation

    Journal: BMC Research Notes

    Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro

    doi: 10.1186/s13104-017-2752-0

    Figure Lengend Snippet: Growth curves of the WT, ΔMycP 3 mutant and the two complementation strains, ΔMycP3 ms ::pr1MycP3 ms and ΔMycP3 ms ::pr2MycP3 ms under Fe-depleted 7H9 ( a ), and Fe-depleted Sauton’s media ( b ). The growth curves were done in triplicate, error bars show standard deviation

    Article Snippet: M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516).

    Techniques: Mutagenesis, Mass Spectrometry, Standard Deviation

    Gene expression analysis of mycP 3 in WT ms , ΔMycP 3ms , ΔMycP 3ms ::Pr1MycP 3 , and ΔMycP 3ms ::Pr2MycP 3 strains under normal 7H9 (iron rich), Fe-free 7H9, and Fe-rescued 7H9 media. The results were normalized against the RNA copy number of sigA . The p values obtained using two-way ANOVA statistical analysis (n = 3) (*p

    Journal: BMC Research Notes

    Article Title: Two promoters in the esx-3 gene cluster of Mycobacterium smegmatis respond inversely to different iron concentrations in vitro

    doi: 10.1186/s13104-017-2752-0

    Figure Lengend Snippet: Gene expression analysis of mycP 3 in WT ms , ΔMycP 3ms , ΔMycP 3ms ::Pr1MycP 3 , and ΔMycP 3ms ::Pr2MycP 3 strains under normal 7H9 (iron rich), Fe-free 7H9, and Fe-rescued 7H9 media. The results were normalized against the RNA copy number of sigA . The p values obtained using two-way ANOVA statistical analysis (n = 3) (*p

    Article Snippet: M. smegmatis was cultured using both Middlebrook 7H9 liquid medium (Becton–Dickinson, USA, Catalogue No. 221832) and Difco 7H11 solid medium (Becton–Dickinson, USA, Catalogue No. 212304) both supplemented with 0.05% (v/v) Tween 80 (Sigma Aldrich, USA, Catalogue No. P1754), 0.5% (w/v) glucose (Sigma Aldrich, USA, Catalogue No. 47829), and 0.5% (v/v) glycerol (Sigma-Aldrich, USA, Catalogue No. G5516).

    Techniques: Expressing, Mass Spectrometry

    Mutation of fadA5 does not cause cholesterol toxicity. Strains were grown to exponential phase in 7H9 medium supplemented with glycerol and dextrose. Cultures were diluted into 7H9 medium containing glycerol and dextrose with or without cholesterol (1 mg ml −1 in 20% Tween). Values are for the wild-type H37Rv strain without cholesterol, wild-type H37Rv with cholesterol, the fadA5 mutant without cholesterol, and fadA5 mutant with cholesterol, as indicated. Data represent the results of the experiment done in triplicate.

    Journal: Infection and Immunity

    Article Title: A Thiolase of Mycobacterium tuberculosis Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol ▿ Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol ▿ †

    doi: 10.1128/IAI.00893-09

    Figure Lengend Snippet: Mutation of fadA5 does not cause cholesterol toxicity. Strains were grown to exponential phase in 7H9 medium supplemented with glycerol and dextrose. Cultures were diluted into 7H9 medium containing glycerol and dextrose with or without cholesterol (1 mg ml −1 in 20% Tween). Values are for the wild-type H37Rv strain without cholesterol, wild-type H37Rv with cholesterol, the fadA5 mutant without cholesterol, and fadA5 mutant with cholesterol, as indicated. Data represent the results of the experiment done in triplicate.

    Article Snippet: M. tuberculosis cultures were grown at 37°C in Middlebrook 7H9 liquid medium (Becton Dickinson), supplemented with 0.05% Tween-80, 10% albumin-dextrose-NaCl complex (ADN) , and 0.2% glycerol, or on Middlebrook 7H10 plates supplemented the same way.

    Techniques: Mutagenesis

    fadA5 is required for growth on cholesterol as the sole carbon source. The strains were grown in 7H9 medium containing 1 mg ml −1 (2.6 mM) cholesterol in tyloxapol or 7H9 medium containing only tyloxapol at 37°C. The wild-type H37Rv (Rv), fadA5 mutant, and complemented fadA5 (fadA5comp) strains were grown with and without cholesterol as indicated on the graph. Data represent results of each experiment run in duplicate.

    Journal: Infection and Immunity

    Article Title: A Thiolase of Mycobacterium tuberculosis Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol ▿ Is Required for Virulence and Production of Androstenedione and Androstadienedione from Cholesterol ▿ †

    doi: 10.1128/IAI.00893-09

    Figure Lengend Snippet: fadA5 is required for growth on cholesterol as the sole carbon source. The strains were grown in 7H9 medium containing 1 mg ml −1 (2.6 mM) cholesterol in tyloxapol or 7H9 medium containing only tyloxapol at 37°C. The wild-type H37Rv (Rv), fadA5 mutant, and complemented fadA5 (fadA5comp) strains were grown with and without cholesterol as indicated on the graph. Data represent results of each experiment run in duplicate.

    Article Snippet: M. tuberculosis cultures were grown at 37°C in Middlebrook 7H9 liquid medium (Becton Dickinson), supplemented with 0.05% Tween-80, 10% albumin-dextrose-NaCl complex (ADN) , and 0.2% glycerol, or on Middlebrook 7H10 plates supplemented the same way.

    Techniques: Mutagenesis

    RaaS is important for persistence of M. tuberculosis in prolonged stationary phase and in human macrophages. (A) Wild-type, Δ raaS Mtb , and Δ raaS Mtb com strains were incubated in 7H9 medium without shaking at 37°C for 7 months. **, Δ raaS Mtb had a significant survival defect ( P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions

    doi: 10.1128/AAC.02774-13

    Figure Lengend Snippet: RaaS is important for persistence of M. tuberculosis in prolonged stationary phase and in human macrophages. (A) Wild-type, Δ raaS Mtb , and Δ raaS Mtb com strains were incubated in 7H9 medium without shaking at 37°C for 7 months. **, Δ raaS Mtb had a significant survival defect ( P

    Article Snippet: Mycobacterium bovis BCG Glaxo strain and Mycobacterium tuberculosis H37Rv were grown in Sauton's or Middlebrook 7H9 liquid medium (Becton, Dickinson and Company) supplemented with albumin-dextrose complex.

    Techniques: Incubation

    Volatiles released by M. tuberculosis grown in a 7H9 broth liquid medium.

    Journal: Beilstein Journal of Organic Chemistry

    Article Title: The volatiles of pathogenic and nonpathogenic mycobacteria and related bacteria

    doi: 10.3762/bjoc.8.31

    Figure Lengend Snippet: Volatiles released by M. tuberculosis grown in a 7H9 broth liquid medium.

    Article Snippet: Bacterial colonies from a 7H11 solid medium (Becton, Dickinson & Co., Sparks, USA, see ) or bacteria culture (100 µL) from a 7H9 liquid medium (Becton, Dickinson & Co., Sparks, USA, see ) were inoculated on a 7H11 solid medium and were spread out with sterile disposable loops to cover the entire plate/medium surface.

    Techniques: