Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments

Abstract

c 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. Single-cell organisms such as bacteria and yeasts have been used extensively to study genes and genome organization, proteins and protein interactions, biological pathways, and cellular structure and to address numerous other fundamental biological questions (1, 2). Many microbes, especially the species Escherichia coli and Saccharomyces cerevisiae, combine numerous beneficial properties that make them ideal model organisms: easy laboratory cultivation, easy genetic manipulation, short generation time, and safe handling. Microbial cultures can be maintained in liquid growth medium or on solid/semisolid gel substrates (3). Liquid colony maintenance allows the microbial cultures to expand rapidly and is used in screens that rely on optical density measurements to extract kinetic growth parameters or use downstream liquid-based assays such as flow cytometry or high-throughput microscopy (4-7). While throughput in liquid is generally bound to a maximum of 96 or 384 samples per plate, solid-substrate colony maintenance has the advantage that individual cell clones can be readily separated and that colony parameters such as color, shape, structure, and size can be extracted easily using digital image acquisition (8-10). Additionally, advances in robotic pinning devices allow for much higher throughput on solid substrate, with close to 25,000 colonies possible on a single microtiter footprintsized gel plate (11). This much higher density has led to solidsubstrate screening being the method of choice for many current high-throughput applications (12)(13)(14).Historically, agar has been the predominant gelling agent in microbial research. Agar is a mixture of polysaccharides f...

Macroscale fluorescence imaging against autofluorescence under ambient light

“…Then, we apply our setup to the imaging of colonies and lawns of fluorescent bacteria that are growing on agar plates. Fluorescence plates are widely used to analyze the level of protein production 29 and the organization of microbial biofilm communities 30 and to screen for compounds that select against antibiotic resistance 31 ; however, they often suffer from a significant autofluorescence that originates from the plate material or the growth media 32 . We demonstrate that our macroscope overcomes the strong autofluorescence of the growth media that are used in microbiology and enables multiplexed fluorescence observation with the same configuration of excitation and emission wavelengths.…”

Systematic Gene-to-Phenotype Arrays: A High-Throughput Technique for Molecular Phenotyping

“…In addition to this ~500-fold increase in speed, the nitrocellulose membrane greatly reduced colony autofluorescence compared to growth on agar (Fig. 1D and S2B), superior even to fluorescence-optimized gels (Jaeger et al, 2015). The improvement in signal is approximately 13-fold (Fig.…”

“…This growth setup pairs with an imaging station (Fig. 1C and S2A) to quantify fluorescent reporter signals for all ~6000 mutant strains in <10 seconds per plate (Jaeger et al, 2015). For comparison, high-throughput microscopy of a similar number of mutants in a GE In Cell Analyzer 2200 requires approximately 1.5 hours.…”

Digital, Rapid, Accurate, and Label-Free Enumeration of Viable Microorganisms Enabled by Custom-Built On-Glass-Slide Culturing Device and Microscopic Scanning

“…Typically, agar is added to aqueous nutrition to form gel matrix, which helps immobilize microbial cells and support them to multiply to colonies. However, the opaque appearance of agar gel determines that it is not a good choice for imaging application [ 22 ]. As an alternative, agarose gel has been extensively employed for imaging due to better optical clarity [ 12 , 15 , 23 , 24 , 25 , 26 , 27 , 28 ].…”