Establishment of Appropriate Conditions for Culturing Actinobacteria in Picolitre Droplets: A Microfluidic Approach for Novel Antibiotic Screening
Miguel Angel Tovar Ballen
Master Thesis, TU Dresden, October 2012
Loss of effectiveness in antimicrobial agents is currently a major challenge for medical applications and microbiological research. Desp ite ongoing efforts for the discovery of novel natural compounds with antibiotic activity, classic screening techniques frequently fail to detect new active secondary metabolites. However, landmark studies agree that more than 97 % of the antibiotics produced by Actinobacteria are yet to be found. As an innovative strategy to overcome such difficulties, we are developing a droplet-based microfluidic approach for high-throughput cultivation of Actinobacteria with subsequent whole cell screening for new antimicrobial compounds. After standardizing the conditions for droplet generation in the microfluidic devices, Actinobacteria spores were encapsulated in ~100 pL monodisperse droplets of culture medium generated at frequencies above 100 Hz. In order to achieve suitable off-chip droplet incubation and reinjection, we tested different containers and reduced the surfactant concentration in the carrier oil. Germination and growth was observed within hours after droplet generation, indicating the feasibility of high-throughput cultivation of actinomycetes in droplets. However, metabolic activity inside the droplets generates an osmotic imbalance that results in water flux between droplets and therefore leads to an increase in polydispersity of the droplet population. Additionally, first experiments regarding reporter-cell dosing into reinjected droplets were carried out. By identifying the most relevant sources of variation during droplet manipulation for actinomycete cultures, we significantly contributed to the future implementation of this technology in the discovery of novel antibiotics.