Working on a diverse species of bacteria that have hundreds of pathotypes representing hundreds of strains and many closely related family members is a challenge. Appropriate research design is required not only to achieve valid desired outcome but also to minimize the use of resources, including time to outcome and intervention. This chapter outlines basics of Escherichia coli isolation and characterization strategies that can assist in research designing that matches the set objectives. Types of samples to be collected, collection and storage strategies, and processing of samples are described. Different approaches to isolation, confirmation and concentration of various E. coli strains are summarized in this chapter. Characterization and typing of E. coli isolates by biochemical, serological, and molecular methods have been explained so that an appropriate choice is made to suite a specific E. coli strain/pathotype. Some clues on sample and isolate preservation for future use are outlined, and general precautions regarding E. coli handling are also presented to the researcher to avoid improper planning and execution of E. coli-related research. Given different options, the best E. coli research design, however, should try as much as possible to shorten the length of time to outcomes.