Capture and Cultivation of Microorganisms Using Magnetic, Lipid-Bound Antibodies

University of Minnesota M.S. thesis. May 2015. Major: Earth Sciences. Advisor: Jake Bailey. 1 computer file (PDF); v, 43 pages.In order to uncover and fully understand the genetic potential of the microbial "uncultivable majority,"� novel methods for isolation are needed. Here we report on efforts to develop a method for physical separation and isolation of viable microorganisms. This method uses differences in the molecular composition of membrane lipids between bacteria and archaea to segregate the desired organisms while keeping them alive and viable for reproduction. Magnetic antibodies bound to the molecule squalene, which is found in the cell membranes of certain archaea, but not bacteria, may enable separation of archaea from bacteria in mixed samples. We tested our technique using the squalene-bearing archaeon, Halobacterium NRC-1, and an analogous bacterium, Salinibacter ruber. Cells were subject to partial fixation and digestion to allow for antibodies to access the cell membrane. Viability of cells was demonstrated by growth of treated cells in batch culture. Specificity of the antibody probe to archaeal cells was demonstrated using immunofluorescence microscopy. Evaluation of separation efficiency using qPCR indicated that samples were not enriched in the target strain and that archaeal cells were preferentially lost throughout the procedure. FISH-probed cell counts suggested that cell clumping during centrifugation negatively impacted separation efficiency. Future efforts will aim to increase efficiency by minimizing the existence of cellular aggregates throughout the procedure