This protocol describes how to build up a freezer stock of a given bacterial strain. Following this protocol will result in individual, glycercol-suspended aliquots of genetically similar bacteria originating from a clone of cells forming an isolated colony, derived from a single precursor cell. Using an isolated colony to build up a freezer stock of a bacterial strain ensures the individual aliquots are genetically similar and not contaminated with another bacterial strain. For full instructions, users must follow the steps in 'How to isolate a single bacterial colony' by the same author before commensing with the current protocol.