2.7. Bioactivity of the released antibiotics

The drug released from the microparticles (dual release study experiment) were tested for ability to inhibit the growth and kill S. epidermidis (strain was kindly given to us by Dr. Robert Blumenthal, Department of Microbiology and Immunology, University of Toledo Health Science Campus). The ability of the released drug to inhibit bacterial growth was determined by turbidity test. To grow S. epidermidis, bacterial stock previously stored and frozen at −70 °C was resuscitated on tryptic soy solid medium. Two colonies were selected and cultured overnight in 10 ml tryptic soy liquid media in two separate conical flasks in shaking water bath at 37 °C. After overnight incubation, a second transfer was made to fresh tryptic soy media (1:10 dilution) and was grown to attain an OD_{600 nm} = 0.05 for S. epidermidis (SPECTROstar Omega, USA). In the meantime, 100 μl of drug solution obtained for the release study were added to a 96-well plate (n = 3). As soon as an OD of 0.05 was obtained, 100 μl of the bacterial suspension was added to the wells and OD readings were taken at pre-determined time intervals for a period of 24 h. The growth curves plotted were compared against the growth of the control, which had only the bacterial culture in PBS.

where Ic and Is are the absorbance’s of control solution containing tryptic soy broth, and bacterial culture solution with and without drug release sample, respectively at 600 nm after 24 h [35].