Making permanent stocks of dictyostelium.

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrients are depleted from the immediate environment of the cells. Dictyostelium can be grown axenically or in the presence of bacteria, either on agar plates or in suspension. Because Dictyostelium growth rates are relatively slow compared to those of bacteria or yeast, laboratories commonly maintain stocks of growing cultures in order to start experiments rapidly. However, it is important to remember that the genome of Dictyostelium, like that of any living organism, is subject to genetic modification. It is well documented that cell lines that are kept in culture for an extended period of time exhibit undesirable changes that yield unreliable experimental results (Hughes et al. 2007). Dictyostelium strains from different laboratories are known to contain various large genome duplications, presumably due to clone selection. Thus, good handling of the cells is essential. To obtain consistent results, new cultures must be started every 2-4 wk, and cultures should never be allowed to grow beyond 4 × 10(6) cells/mL. If overgrowth occurs, a new culture should be started. This protocol describes two methods for preparing long-term stocks of Dictyostelium, either as frozen cells or as spores.