Stock solution of pencillin G (1 mg/ml) was prepared by dissolving 1 mg reagent grade of penicillin G powder (Amresco, USA) in 1 ml sterile-distilled water.
The bone fragments were cultured in RPMI-1640 and F-12 HAMS (1 1) containing 10% fetal bovine serum (heat inactivated) (Invitrogen, Paisley, UK), 5 mg ml−1 of transferrin-sodium-selenite (Sigma-Aldrich), and antibiotics (10,000 units ml−1 of penicillin G, 10 mg ml−1 of streptomycin, and 25 mg ml−1 of amphotericin B) (Invitrogen), at 37°C in an atmosphere of 5% CO2.
After slow thawing, each individual swab was hydrated in phosphate-buffered saline (PBS) supplemented with antibiotics (100 U of penicillin with 100 mg of streptomycin/ml), incubated for 1 h at room temperature, clarified by centrifugation at 1500 g for 20 min and used for RNA extraction.
The concentration of penicillin measured 1 3 hours after the last consumption of a 250-mg penicillin V regimen taken four times daily for 3 4 days is 0.1 - 2 μg/ml [ 23].
Finally, NH4Ac buffer (100 mM, pH 6.0) was added to obtain a stock solution with a concentration of 1 mg ml−1 of penicillin base equivalent.
Stock standard solutions (1 mg ml−1) of penicillin references were prepared in MeOH, taking into account the purity of the reference standard material.
The coral was rinsed with autoclaved seawater and disinfected with 70% ethanol, and small pieces were placed on plates with Peptone Yeast Glucose (PYG) agar (1 L marine water, peptone (1.25 g), yeast extract (1.25 g), glucose (3.0 g), agar (20 g) supplemented with 100 mg mL 1 of penicillin and streptomycin).
Increasing frequencies of penicillin-nonsusceptible pneumococci (PSNP MICIC of penicillin >0.12 mg/L) became a worldwide problem in the 1980s (1, 2 ).
The MIC of penicillin against this organism tested by Etest was 0.5 mg/L.
