All mycobacterial strains (Supplementary file 2 – Key Reagents) were grown in liquid culture containing Difco Middlebrook 7H9 Broth (BD Biosciences, San Jose, CA) and supplemented with 0.2% (vol/vol) glycerol (Sigma-Aldrich, St. Louis, MO), 0.005% (vol/vol) Tween 80 (Sigma-Aldrich, St. Louis, MO), and 10% (vol/vol) BBL Middlebrook OADC Enrichment (BD Biosciences, San Jose, CA). For M. smegmatis, liquid cultures were incubated at 37°C with orbital shaking at 100 rpm, until the desired growth density was attained – measured by spectrophotometry at a wavelength of 600 nm – before further experimentation. Solid media comprised Difco Middlebrook 7H10 Agar (BD Biosciences, San Jose, CA) supplemented with 0.5% (vol/vol) glycerol (Sigma-Aldrich, St. Louis, MO), and 10% (vol/vol) BBL Middlebrook OADC Enrichment (BD Biosciences, San Jose, CA). Solid media plates were incubated at 37°C for 3–4 days or until colonies had formed.
Difco middlebrook 7h10 agar
Manufactured by BD
Sourced in United States
Difco Middlebrook 7H10 Agar is a solid culture medium used for the cultivation and isolation of Mycobacterium species, including the causative agent of tuberculosis. It provides the necessary nutrients and growth factors for the optimal growth of these slow-growing bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Difco middlebrook 7h9 broth, by BD (2 mentions)
Glycerol, by Merck Group (2 mentions)
Tween 80, by Merck Group (2 mentions)
Bbl middlebrook oadc enrichment, by BD (1 mentions)
Middlebrook adc enrichment, by BD (1 mentions)
Middlebrook oadc enrichment, by BD (1 mentions)
Oadc enrichment, by BD (1 mentions)
Difco middlebrook 7h9, by BD (1 mentions)
Oleic albumin dextrose catalase oadc, by BD (1 mentions)
8 protocols using difco middlebrook 7h10 agar
1
Cultivating Mycobacterial Strains in Liquid and Solid Media
2
Mycobacterial Culture on Middlebrook 7H10 Agar
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The strains tested were cultured using Difco Middlebrook 7H10 Agar (BD company). The medium was prepared as follows. First, 19 g of 7H10 (powder) was suspended in 900 mL of purified water containing 5 mL of glycerol and then mixed thoroughly. Next, the powder was completely dissolved by heating with frequent agitation for 1 min and then sterilized at 121°C for 10 min. Last, 100 mL of Middlebrook OADC enrichment solution (BD, Franklin Lakes, NJ, USA) was added aseptically to the medium after cooling to 50–55°C.
3
Vaccination against Tuberculosis in Mice
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Groups of C57BL/6 male mice (n = 5) were immunized s.c. with either Qβ-mIL-1b(D143K) or Qβ VLPs three times before (weeks −5, −3, and −1) and once after (week 10) aerosol infection with 100 colony-forming units/lung of M. tuberculosis H37Rv (week 0). Body weights of infected mice were measured weekly over a period of 36 weeks. At the end of the experiment, lungs and spleens of infected mice were removed and homogenized in 0.04% Tween 80. Ten-fold dilutions of the homogenized tissues were plated in duplicate onto Difco Middlebrook 7H10 Agar (BD Biosciences) plates supplemented with 10% Dubos oleic albumin complex medium (BD Biosciences) and 0.5% glycerol and incubated at 37 °C. After 21 days, bacterial colonies were counted.
4
Recombinant BCG Vaccine with MyHCα Epitope
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Oligonucleotides that encode a 16-amino-acid MyHCα CD4+ T-cell epitope (aa 614–629; SLKLMATLFSTYASAD) were chemically synthesized. The sequences were 5′-TCGA GTCTGAAGCTGATGGCGACCCTGTTCTCGACCTACGCGTCGGCGGAT-3′ for the upper strand and 5′-TCGA ATCCGCCGACGCGTAGGTCGAGAACAGGGTCGCCATCAGCTTCAGAC-3′ for the lower strand with cohesive ends of the XhoI recognition site (underlined) at both terminals of each DNA. The DNA oligomers were cloned into an XhoI site in the coding region of the Ag85B gene from M. kansasii cloned into pSO246 to be fused with the gene, and the generated plasmid was named pSO246-MyHCα. pSO246-MyHCα and pSO246 (empty plasmid: control) vectors were introduced into M. bovis BCG Tokyo 172 strain by electroporation, giving rise to rBCG-MyHCα and rBCG-pSO246, respectively. These transformants were selected on a Difco Middlebrook 7H10-agar (BD) plates containing Middlebrook OADC enrichment (BD) and 30 μg/mL kanamycin and grown in Difco Middlebrook 7H9-broth (BD) containing Middlebrook ADC enrichment (BD) and 0.05 Tween 80 at 37 °C for 2 weeks. After harvesting, rBCG was washed twice with PBS and then used for immunization.
5
Determining Antimicrobial Susceptibility of M. avium
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M. avium isolates were incubated in Difco Middlebrook 7H10 agar from BD (Franklin Lakes, NJ, USA) with 5% oleic acid–albumin–dextrose–catalase.12 ,15 (link) The microdilution method was performed for fluoroquinolone and aminoglycoside susceptibility testing against M. avium using cation-adjusted Mueller-Hinton broth with the addition of 5% oleic acid–albumin–dextrose–catalase.12 ,15 (link) The experiments were conducted in 96-well microplates. First, 0.5 McFarland standard bacterial suspensions were prepared. Second, the bacterial solutions and drug dilution mixtures were added to wells with a blank control included.12 ,15 (link) Finally, the 96-well microplates were incubated at 37°C with 5% carbon dioxide. The minimum inhibitory concentration (MIC), MIC50, and MIC90 values for each antimicrobial agent were determined according to the previous method.12 Fluoroquinolone and aminoglycoside resistance were identified using the MIC breakpoints.12 ,16
6
Quantifying Viable Mycobacterium tuberculosis
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Cultured M. tuberculosis complex cells were suspended either in phosphate buffered saline (PBS) or in human sputum mixtures as described above, at estimated densities of approximately 1×107 CFU/mL sputum [calculated based on optical density (A600) measurement of cultures]. After the various treatments described in this report, 0.1 mL samples of the suspensions were plated in triplicate onto Difco Middlebrook 7H10 agar with 10% (v/v) OADC enrichment (BD Diagnostics, Sparks, MD). Plates were incubated at 37°C for up to 35 days. Untreated sputum samples rapidly overgrew plates due to the natural flora of sputum. Therefore, no-treatment controls consisted of untreated suspensions of M. tuberculosis complex cells in PBS. This strategy enabled us to quantify viable M. tuberculosis complex cells in untreated controls.
7
Hypoxic M. marinum Growth Protocol
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To replicate the experimental paradigm of the M. tuberculosis natural growth,12 the two strains of M. marinum that are most commonly utilized in the zebrafish as a disease model are grown for an extended period of time in hypoxic conditions. To remain hypoxic, individual culture tubes can only be used for a single time point, as opening the tube for sampling introduces oxygen to the culture. M. marinum strains E1119 and MUSA20 were cultured in DifcoTM Middlebrook 7H9 broth medium (BD Biosciences, Franklin Lakes, NJ) containing 0.05% Tween80 (Merck KGaA, Darmstadt, Germany) and 10% BBLTM Middlebrook Albumin Dextrose Catalase Enrichment (BD Biosciences) at 30°C in closed tubes without disturbance up to 221 days. For each strain, three biological replicates from individually grown colonies were collected in individual tubes per time point, with a start inoculum at OD600 of 0.05, approximating 1× 106 CFU/mL. At different time points (0, 8, 11, 18, 23, 28, 36, 42, 49, 56, 64, 69, 77, 85, 92, 112, 114, and 221 days), the viability of the cultures, defined as CFUs per mL, was determined by plating a series of 10‐fold dilutions (ranging from 1:10 to 1:1,000,000) in triplicate on Difco Middlebrook 7H10 agar (BD Biosciences) containing 5% glycerol (Sigma‐Aldrich, Saint Louis, MO) and 10% BBLTM Middlebrook Oleic Albumin Dextrose Catalase Enrichment (BD Biosciences).
8
Mycobacterial and E. coli Culture Protocols
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M. tuberculosis H37Rv, M. bovis BCG China (purchased from China National Institutes Food and Drug Control), and rBCG strains were cultured on Difco™ Middlebrook 7H9 (BD, NJ, USA) or Difco™ Middlebrook 7H10 agar (BD, NJ, USA), supplemented with 10% oleic-albumin-dextrose-catalase (OADC) (BD, NJ, USA), 0.5% glycerol, and 0.05% Tween 80. Escherichia coli DH5α, and BL21(DE3) were cultured in Luria-Bertani medium or agar and used for cloning and expression. Kanamycin was used at a concentration of 25 μg/mL and ampicillin used at a concentration of 100 μg/mL.
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