title: Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments.
abstract: Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition.
authors.affiliation: Departments of Bioengineering and Medicine, University of California San Diego, La Jolla, California, USA.
authors.forename: Philipp A
authors.identifier: http://orcid.org/0000-0002-3624-0802
authors.initials: PA
authors.lastname: Jaeger
authors.affiliation: Nanoengineering Program, University of California San Diego, La Jolla, California, USA.
authors.forename: Cameron
authors.initials: C
authors.lastname: McElfresh
authors.affiliation: Bioengineering Program, University of California San Diego, La Jolla, California, USA.
authors.forename: Lily R
authors.initials: LR
authors.lastname: Wong
authors.affiliation: Departments of Bioengineering and Medicine, University of California San Diego, La Jolla, California, USA tideker@ucsd.edu.
authors.forename: Trey
authors.initials: T
authors.lastname: Ideker
mesh_terms: D000362:Agar; D055598:Chemical Phenomena; D003470:Culture Media / Q000737:chemistry*; D005782:Gels; D057166:High-Throughput Screening Assays; D008828:Microbiological Techniques / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000254:growth & development
chemical_list: D003470:Culture Media; D005782:Gels; D000362:Agar