S. aureus samples were obtained from The Charles T. Campbell Eye Microbiology Laboratory, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. A total of 13 samples were tested for the methicillin resistance using disk diffusion (24 (link)), thus determining their methicillin resistance before photoacoustic testing. Streaks of each S. aureus strain were grown on mannitol salt agar plates. Single colonies from each streak plate were used to regrow strains in mannitol salt broth for 2 h in a shaking water bath at 36.5 °C. This period ensured cells were growing and entering exponential growth phase. Oxacillin was added at a final concentration of 1 μg/ mlto half of each culture and grown for an additional 2 h (25 (link)). We cultured on agar plates solely for comparison to plate reader results. The photoacoustic method, in clinical implementation, will test samples directly taken from patients without the culture phase.
Before processing through PAFC system, 100 μLfrom each culture was removed and used for growth analysis in an H1 plate reader (Biotek, Winooski, Vermont). This procedure was done to verify the photoacoustic method and were not integral to photoacoustic testing. Growth curves were made for each culture by taking the optical density of each treated and untreated culture every minute over a 16 hour period. We determined that two hours of antibiotic treatment was sufficient to determine differential growth rates from prior experimentation. Prior to performing photoacoustic testing, treated and untreated samples were incubated side-by-side for two hours. Photoacoustic testing of treated and untreated samples for each isolate were alternated, so that both samples were tested within twenty minutes to allow for similar growth times. Thus, total bacteria number could be compared.
Before processing through PAFC system, 100 μLfrom each culture was removed and used for growth analysis in an H1 plate reader (Biotek, Winooski, Vermont). This procedure was done to verify the photoacoustic method and were not integral to photoacoustic testing. Growth curves were made for each culture by taking the optical density of each treated and untreated culture every minute over a 16 hour period. We determined that two hours of antibiotic treatment was sufficient to determine differential growth rates from prior experimentation. Prior to performing photoacoustic testing, treated and untreated samples were incubated side-by-side for two hours. Photoacoustic testing of treated and untreated samples for each isolate were alternated, so that both samples were tested within twenty minutes to allow for similar growth times. Thus, total bacteria number could be compared.
Free full text: Click here
