be used to address, as the populations' evolution has presented new phenomena to study, and as technology and methodological techniques have advanced. The use of E. coli as the experimental organism has allowed many generations and large populations to be studied in a relatively short period of time. Moreover, due to the long use of E. coli as a principle model organism in molecular biology, a wide array of tools, protocols, and procedures were available for studying changes at the genetic, phenotypic, and physiological levels. The bacteria can also be frozen and preserved while remaining viable. This has permitted the creation of what Lenski describes as a "frozen fossil record" of samples of evolving populations that can be revived at any time. This frozen fossil record allows populations to be restarted in cases of contamination or other disruption in the experiment, and permits the isolation and comparison of living exemplars of ancestral and evolved clones. Lenski chose an E. coli strain that reproduces only asexually, lacks any plasmids that could permit bacterial conjugation, and has no viable prophage. As a consequence, evolution in the experiment occurs only by the core evolutionary processes of mutation, genetic drift, and natural selection. This strict asexuality also means that genetic markers persist in lineages and clades by common descent, but cannot otherwise spread in the populations. Lenski chose to carry out the experiment with the bacteria grown in a glucose-limited medium called DM25, which is based on a minimal medium developed by Bernard Davis for use in isolating auxotrophic mutants of E. coli using penicillin as a selective agent. To make DM25, the minimal medium is supplemented with a low concentration (25 mg/L) of glucose. Lenski chose this concentration to simplify analysis of the populations' evolution by reducing clonal interference, in which multiple versions of alleles are competing in an evolving population, while also reducing the possibility of the evolution of ecological interactions. This concentration of glucose used supports a maximum population of 500 million cells of the ancestor in a 10 mL culture, though the maximum now varies among the evolved populations. DM25 also contains a large amount of citrate (about 11 times the concentration of glucose), which was originally included by Davis because it improved the killing efficiency of penicillin during his experiments, though it is now known to aid in E. colis acquisition of iron from the medium. Methods The 12