Vital stains were used in combination with fluorimetry for the elaboration of a new method to quantify Streptomyces programmed cell death, one of the key events in Streptomyces differentiation. The experimental approach described opens the possibility of designing online protocols for automatic monitoring of industrial fermentations.Streptomyces is an extremely important bacterium for industry, since approximately two-thirds of all antibiotics are synthesized by members of this genus (4). Furthermore, streptomycetes produce large numbers of eukaryotic cell differentiation inducers and apoptosis inhibitors and inducers (19,24,25). Moreover, some authors consider that bacteria with complex life cycles (streptomycetes, cyanobacteria, etc.) are the evolutionary origin of some of the protein domains involved in programmed cell death (PCD) processes, including eukaryotic apoptosis: APATPases (apoptotic ATPases), kinases, caspases, nucleases, etc. As such, these bacteria would constitute a simple and convenient model by which to study this important phenomenon (1,3,9,12,21,26).The classical Streptomyces developmental model in confluent solid cultures assumed that differentiation processes took place along the transverse axis of the cultures (bottom up): completely viable vegetative mycelia (substrate) grew on the surface and inside agar until they underwent a PCD process, after which they differentiated into a reproductive (aerial) mycelium that grew into the air (reviewed in reference 8). Although most industrial processes for secondary metabolite production are performed in liquid cultures, Streptomyces strains generally do not sporulate under these conditions (6,18,22), and most authors assumed that differentiation did not take place. Recently, a detailed analysis of Streptomyces differentiation in surface and submerged cultures has been performed, describing novel aspects of the differentiation processes of this bacterium (10-17). A previously unidentified compartmentalized mycelium (MI) initiates the developmental cycle and then dies following a highly ordered sequence (PCD) (10,11,14). Subsequently, the remaining viable segments enlarge, yielding a multinucleated mycelium (MII) that grows in successive waves that determine the characteristic complex growth curves of this microorganism. In surface cultures, two types of second mycelium were defined, based on the absence (in early development) or presence (in late development) of the hydrophobic layers characteristic of aerial hyphae (5). The traditionally denominated substrate (vegetative) mycelium corresponds, in fact, to the early second multinucleated mycelium that still lacks the hydrophobic layers coating the aerial mycelium (15). We proposed that the first compartmentalized mycelium fulfills the true vegetative role in Streptomyces development in soil (17). According to this scheme, the second early and late multinucleated mycelia should be considered jointly as part of the reproductive phase, since they are destined to sporulate (17). The second multinuclea...