How do you select for your cells over contaminants? I'm not familiar with the techniques you're referring to. It looks like I'm out of my depth here, so I can't really say whether going directly from a glycerol stock would be okay with your system.
When I first made my stocks I streaked my mutants on plates w/ gentamicin, then inoculated into broth which I froze down. I don't use antibiotics in the broth when I am sub culturing for an experiment. Contamination never really has been an issue, besides for the wild type, there is no resistance marker anyway.