Strains were grown to the mid-log phase (A600 ∼ 0.5) and diluted 1:100 to yield concentrations of ∼105-106 CFUs/ml in Middlebrook 7H9 medium. Antibiotics were added to each sample at defined concentrations, and the bacterial samples were collected before the addition of the antibiotic and at 24, 48, and 72 h after antibiotic challenge. To determine the number of viable cells, CFUs were determined through serial 10-fold dilutions using 20 μl of culture and 180 μl of 7H9 medium. Twenty microliters of each dilution were plated on 7H10 agar (Sigma–Aldrich). All the plates were incubated at 37 °C for 4 days before the colonies were counted.
7h10 agar
Manufactured by Merck Group
7H10 agar is a solid culture medium used for the growth and isolation of mycobacteria, particularly Mycobacterium tuberculosis. It provides a nutritious environment for the cultivation of these microorganisms. The formulation and composition of 7H10 agar support the specific growth requirements of mycobacteria.
Automatically generated - may contain errors
Lab products found in correlation
Albumin dextrose saline, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Glycerol, by Carl Roth (1 mentions)
Hygromycin b, by Carl Roth (1 mentions)
Tween 80, by Carl Roth (1 mentions)
Lb agar plates, by Carl Roth (1 mentions)
E coli dh5α t1r, by Thermo Fisher Scientific (1 mentions)
Middlebrook 7h9 medium, by BD (1 mentions)
2 protocols using 7h10 agar
1
Antibiotic Susceptibility Assay
2
Cloning and Propagation of Bacterial Strains
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All cloning and plasmid propagation were conducted in E. coli DH5α‐T1R (Life Technologies) using standard protocols. E. coli was transformed by conventional heat shock transformation and grown in Lennox Broth (LB) or LB agar plates (Carl Roth). The seamless ligation cloning extract (SLiCE) was produced from the E. coli DH10B‐PPY strain.27 In liquid cultures M. smegmatis mc2155 groEL1ΔC16 was grown in Middlebrook 7H9 medium (BD Biosciences) supplemented with 0.2% (w/v) glucose (Carl Roth), 342 mM NaCl), 0.05% (v/v) Tween‐80 (Carl Roth) and 0.2% (v/v) glycerol (Carl Roth). Alternatively, the solid growth media used was 7H10 agar (Sigma‐Aldrich) supplemented with 10% albumin–dextrose saline (ADS: 5% (w/v) BSA cold ethanol fraction, pH 5.2, ≥96% (Sigma‐Aldrich), 2% (w/v) glucose (Carl Roth), 342 mM NaCl), 0.05% (v/v) Tween‐80 (Carl Roth) and 0.2% (v/v) glycerol (Carl Roth). All bacterial strains were grown at 37° C. Where required the growth media was supplemented with 94 μM hygromycin B (Carl Roth) or 35 μM kanamycin (Sigma‐Aldrich).
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