Molecular Tools for the Characterization of Bacterial Strains in Modern Microbiology
Students will be exposed to fundamental molecular microbiology used for the characterization of bacterial isolates and communities, complement phenotype-based taxonomy with basic molecular methods and reinforce a research oriented approach to the characterization of unknown isolates and microbial communities, recognize advantages and limitations of the experimental methods used and how these affect the interpretation of results. Most of the exercises currently offered in the course are limited to basic staining procedures, conventional light microscopy and culture-based techniques. Bacterial characterization is based solely on traditional biochemical tests with limited exposure to modern, molecular tools, inconsistent with the increasing prevalence of DNA-based methods in modern microbiology. Furthermore, recent recommendations from the FDA are to implement DNA-based identification of microbes of relevance in the sterile drug manufacturing setting and to increase the frequency of DNA amplification methods in clinical diagnosis of infectious diseases. In this module the students will do DNA extraction from a Gram+ and a Gram- culture using a bead beating-based protocol using zirconia beads and a standard chemical lysis method. The lysates will then be used for the amplification of 16S rRNA genes. The recovered material can be used for performing PCR genomic fingerprinting of a collection of isolates in order to discriminate among strains of the same species. Isolates will be provided from real research projects. The students will be introduced to the concepts of molecular characterization of bacteria and polymerase chain reaction (PCR). The small subunit (16S) rRNA genes will be amplified from DNA extracted in Session 1, hence conducting genomic fingerprinting (BOX-PCR) allowing the characterization of bacterial cultures beyond the species level. The students will then clone 16S rRNA genes amplified in Session 2 into plasmid vectors followed by the transformation of E. coli cells using commercially available kits. A questionnaire on research content, skills, tools and techniques will be offered on the first day of lab and after completing the lab module.