Protocols and Programs for High-Throughput Growth and Aging Phenotyping in Yeast

Abstract

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting “Colony Forming Units”. To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.

Multiple Origins of the Pathogenic Yeast Candida orthopsilosis by Separate Hybridizations between Two Parental Species

“…A 600 measurements were taken every 15 min over 48 h using a shaking plate reader (Biotek Synergy HT: Multi-Detection Microplate Reader), to generate growth curves. The doubling times of the isolates were calculated from three biological replicates, each with three technical replicates, using the software tool GATHODE [53]. …”

“…Doubling times were calculated using GATHODE [53] from growth curves of three biological replicates with three technical replicates growing in YPD broth at 30°C shaking for 48 h. The graph shows the average and standard deviation. Each isolate is colored with respect to its clade.…”

Dietary restriction and lifespan: Lessons from invertebrate models

“…Similar approaches but with different initial culture media including liquid YPD (Ashrafi et al, 1999) or amino acid restricted solid synthetic media (Madia et al, 2007) have also been used. Higher throughput approaches involving monitoring survival based on outgrowth by optical density in liquid culture using multi-well plate readers have also become common (Jung et al, 2015; Murakami and Kaeberlein, 2009). …”

An optimized competitive-aging method reveals gene-drug interactions underlying the chronological lifespan of Saccharomyces cerevisiae

“…Measuring death rate in monoculture from outgrowth kinetics. CLS estimates based on outgrowth kinetics of single-population aging cultures were adapted from a well established high-throughput method 21,22 . Culture density was monitored by increase in absorbance at OD 600nm after subtracting background signal, defined as…”

“…Importantly, this previous study assumed that mutant strains had no effects in doubling time, which biased relative-survivorship measurements of slow-growing strains. For nine knockout strains, we compare the replicability of our results with those obtained with a useful parallelizable approach based on outgrowth kinetics 21,22 . Finally, we take advantage of our improved method to derive gene-environment interactions by measuring the relative effects on survival of metformin in 76 single-gene deletion strains, revealing some of the genes that mediate longevity by metformin in yeast.…”