Various embodiments disclosed herein provide for reagents and methods for rapidly isolating viable microbial cells, including S. pneumoniae, from positive blood culture samples. The resulting microbial pellet can be used for both identification and growth-based methods such as antimicrobial susceptibility testing. The buffers described herein may contain a base solution, non-ionic detergents, thiols, and optionally, ammonium chloride. The disclosed methods provide a process for rapidly isolating and concentrating viable microorganism (s) from PBC samples using only one sample preparation tube and centrifugation while removing cellular debris from the mammalian blood cells that may interfere with identification methods.