Abstract: Methods of the invention include the isolation of intact, viable microorganism(s) from positive blood culture (“PBC”) samples for use in downstream analyses such as identification and antimicrobial susceptibility testing (“AST”). The methods involve collecting a portion of the PBC sample, adding a choline-containing solution, lysing the blood cells, isolating the viable microorganism, and performing downstream analysis of the isolated, viable microorganism. The methods can be applied to a variety of gram-positive bacteria, gram-negative bacteria, and/or yeast, and particularly to strains of S. pneumoniae.