agar 7h10 medium  (MiddleBrook Pharmaceuticals)

Journal: Human Vaccines & Immunotherapeutics

Article Title: Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity

doi: 10.1080/21645515.2017.1316911

Figure Lengend Snippet: Phenotypic characterization of BCG.HIVA 2auxo.int strain. We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA 2auxo.int strain (A) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (B) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (C) BCG.HIVA 2auxo.int plated on 7H10 without lysine and kanamycin supplementation; (D) BCG.HIVA 2auxo.int plated on 7H10 without lysine supplementation and with kanamycin.

Article Snippet: Bloom, and T. Hsu was transformed with the p2auxo.HIVAint plasmid DNA without the kanamycin resistance gene by electroporation.. Mycobacterial cultures were grown in Middlebrook 7H9 broth medium or on Middlebrook agar 7H10 medium supplemented with albumin-dextrose-catalase (ADC; Difco) containing 0.05% Tween 80.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness

doi: 10.3389/fmicb.2020.00413

Figure Lengend Snippet: GreA is required for resistance to adverse stress. (A–E, H–J) Survival of M. smegmatis strains during adverse stress. The strain symbols listed in the (J) . WT, Δ greA and comp- Δ greA strains were treated for at the indicated time with varying concentrations of SDS (A) , 5mM H 2 O 2 (B) , PBS + 0.05% Tween 80 (C) , low pH (pH4.5) (D) , heat shock (48°C) (E) , 1 μg/ml of streptomycin (H) , 100 μg/ml of rifampicin (I) , or 2.5 μg/ml of bedaquiline (J) . After treatment, dilutions were plated on 7H10 and survival is expressed as a ratio of CFUs compared to untreated cultures. (F) M. tuberculosis H37Rv strains were spotted on the 7H10 plates after they were treated under 0.5% SDS for 24 h. (G) WT, Δ greA , and comp- Δ greA strains from M. tuberculosis H37Ra were subjected to 0.5% SDS for 8 h. CFU enumeration was performed after incubation for the indicated time. These experiments were performed three times with similar results. Data are shown as mean ± SEM of three independent experiments. * p

Article Snippet: The comp- ΔgreA strains were selected on Middlebrook 7H10 agar medium (complemented with 10% OADC) containing 30 μg/ml kanamycin.

Techniques: Incubation

Journal: Frontiers in Microbiology

Article Title: Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness

doi: 10.3389/fmicb.2020.00413

Figure Lengend Snippet: Intracellular fitness of Δ greA mutant strain was evaluated in vivo and in vitro . RAW264.7 cells were seeded at 3 × 10 5 cells per well in 24-well culture plates. After adhesion, cells were infected with bacteria at MOI of 10. The extracellular bacteria were removed 2 h post infection by washing with PBS. The cells of M. smegmatis (A) , M. tuberculosis H37Ra (B) , and M. tuberculosis H37Rv (C) were further incubated for 24 and 48 h at 37°C. Then serially diluted (10-fold) cells lysates were spotted on a 7H10-OADC agar and cultured in 37°C. CFUs were counted after incubation for the indicated time for M. smegmatis and M. tuberculosis , respectively. (D) Mice were infected i.p. (1 × 10 7 ) with WT, Δ greA , or comp- Δ greA strains of M. tuberculosis H37Ra. The number of surviving bacilli by CFU counting at the indicated time. Data are shown as mean ± SEM of two independent experiments. * p

Article Snippet: The comp- ΔgreA strains were selected on Middlebrook 7H10 agar medium (complemented with 10% OADC) containing 30 μg/ml kanamycin.

Techniques: Mutagenesis, In Vivo, In Vitro, Infection, Incubation, Cell Culture, Mouse Assay

Journal: Scientific Reports

Article Title: Mycobacterium tuberculosis PE_PGRS41 Enhances the Intracellular Survival of M. smegmatis within Macrophages Via Blocking Innate Immunity and Inhibition of Host Defense

doi: 10.1038/srep46716

Figure Lengend Snippet: PE_PGRS41 expression in M. smegmatis results in increased cell wall permeability. ( A ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in 7H9 containing 2 μg/ml ethidium bromide for indicated time. ( B ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in 7H9 with 2 μM Nile red stain for indicated time. ( C ) The growth of Ms_Vec and Ms_PE_PGRS41 after treatment with different pH gradient for indicated time. The Ms_Vec and Ms_PE_PGRS41 strains were centrifuged, re-suspended to 5 ml MB 7H9 at an OD 600 of 0.5, 10-fold serial dilutions of Ms_Vec and Ms_PE_PGRS41 were spotted on MB 7H10. ( D ) Mid-log-phase cultures of Ms_Vec and Ms_PE_PGRS41 were incubated in MB 7H9 supplemented with 0.05% SDS for indicated time. And then the recombinant strains were plated onto 7H10 plates by serially ten-fold dilution, the bacterial numbers were counted after 3–4 days of cultivation.

Article Snippet: Reagents Middlebrook 7H10 agar and Middlebrook 7H9 broth medium were purchased from BD Difco Laboratories.

Techniques: Expressing, Permeability, Mass Spectrometry, Incubation, Staining, Recombinant

Journal: Scientific Reports

Article Title: Mycobacterium tuberculosis PE_PGRS41 Enhances the Intracellular Survival of M. smegmatis within Macrophages Via Blocking Innate Immunity and Inhibition of Host Defense

doi: 10.1038/srep46716

Figure Lengend Snippet: M. tuberculosis PE_PGRS41 expression in M. smegmatis decreased bacterial survival following exposure to various antibiotics, including vancomycin ( A ), norfloxacin ( B ), isoniazid ( C ), and ciprofloxacin ( D ). Ms_Vec and Ms_PE_PGRS41 were exposed to different concentration of vancomycin, norfloxacin, isoniazid, and ciprofloxacin for 5 h, then ten-fold dilution spotted the bacteria on MB 7H9 supplemented with 1% agar, the bacterial number of Ms_Vec and Ms_PE_PGRS41 were counted after 3 days cultivation.

Article Snippet: Reagents Middlebrook 7H10 agar and Middlebrook 7H9 broth medium were purchased from BD Difco Laboratories.

Techniques: Expressing, Mass Spectrometry, Concentration Assay

Journal: Scientific Reports

Article Title: Mycobacterium tuberculosis PE_PGRS41 Enhances the Intracellular Survival of M. smegmatis within Macrophages Via Blocking Innate Immunity and Inhibition of Host Defense

doi: 10.1038/srep46716

Figure Lengend Snippet: The effect of PE_PGRS41 on the growth of M. smegmatis . ( A ) PCR amplification of PE_PGRS41 encoding sequence from M. tuberculosis genome approximately 1086 bp. ( B ) M. tuberculosis PE_PGRS41 was expressed in M. smegmatis and detected using Western blotting. Cell lysates of Ms_Vec and Ms_PE_PGRS41 were subjected to Western blot to determine the expression of His-tagged PE_PGRS41 protein in M. smegmatis by anti-His antibody. ( C ) Cell fractionation experiments were performed to determine the sub-cellular localization of PE_PGRS41, GroEL2 protein serves as a cytoplasm marker of M. smegmatis , CW represents cell wall; CP represents cytoplasm. ( D ) The morphology of Ms_Vec and Ms_PE_PGRS41 were detected after induction by acetamide in the presence of hygromycin. ( E ) Ms_Vec and Ms_PE_PGRS41 were grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80, 1% acetamide and 0.2% glycerinum, with hygromycin (100 μg/ml). The OD 600 was determined at an interval of 4 h.

Article Snippet: Reagents Middlebrook 7H10 agar and Middlebrook 7H9 broth medium were purchased from BD Difco Laboratories.

Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Western Blot, Mass Spectrometry, Expressing, Cell Fractionation, Marker