Nonequilibrium phase transitions from survival to extinction have recently been observed in computational models of evolutionary dynamics. Dynamical signatures predictive of population collapse have been observed in yeast populations under stress. We experimentally investigate the population response of the budding yeast Saccharomyces cerevisiae to biological stressors (temperature and salt concentration) in order to investigate the system’s behavior in the vicinity of population collapse. While both conditions lead to population decline, the dynamical characteristics of the population response differ significantly depending on the stressor. Under temperature stress, the population undergoes a sharp change with significant fluctuations within a critical temperature range, indicative of a continuous absorbing phase transition. In the case of salt stress, the response is more gradual. A similar range of response is observed with the application of various antibiotics to Escherichia coli, with a variety of patterns of decreased growth in response to antibiotic stress both within and across antibiotic classes and mechanisms of action. These findings have implications for the identification of critical tipping points for populations under environmental stress.

To investigate the dynamics of yeast population growth under stress, two environmental stressors were utilized, temperature and elevated NaCl concentration. In both cases, S. cerevisiae (strain yWO3) was initially grown at 30 °C in standard medium (YEPD) containing 10 g/L yeast extract, 20 g/L peptone and 2% dextrose. These initial 50 mL cultures were inoculated with cells to an OD (optical density) of 0.0001. From this initial concentration, the cells typically grew to an OD of 2.5 in 24 hours. All OD measurements were taken at 600 nm.

To study the response to temperature stress, the initial culture, prepared as described above, was inoculated into 50 mL of YEPD in 250 mL flasks to a resulting OD of 0.05. The samples were then placed in orbital shaking water baths that had already reached the temperature of interest.  Sample ODs were then measured every 24 hours.  If the sample had grown, the sample was diluted back to an OD of 0.05 in a volume of 50 mL YEPD in a new 250 mL flask. If the sample had not grown significantly (less than 0.01 OD growth), or if the OD of the sample was lower than 0.05, the sample was placed back into the water bath for another 24 hours.  Measurements were taken over eight 24-hour periods. Growth was measured by calculating the number of times the population doubled between measurement cycles, or number of doublings.

For the measurement of yeast growth under high salt stress, separate YEPD + NaCl media were prepared for each NaCl concentration.  The ratio of yeast extract, peptone, and dextrose per liter was unchanged, but salt was added at appropriate g/L concentrations. An initial culture in YEPD was inoculated into 50 mL of YEPD + NaCl in 125 mL flasks to a resulting OD of 0.05. The samples were then placed in an orbital shaking water bath at 30 ºC. As with the temperature studies described above, sample ODs were measured after 24 hours.  If growth had occurred, the sample was diluted back to an OD of 0.05 in a volume of 50 mL YEPD + NaCl in a new 125 mL flask. If the sample did not have significant growth, or if the OD was less than 0.05, the sample was returned to the incubator until the next measurement period. Measurements were taken over eight 24-hour periods, and doublings were calculated.

Escherichia coli (MG1655) were grown in M9 minimal glucose media overnight, shaking at 37 °C. This bacterial culture was used to inoculate 96-well plates containing a dilution series of 10 different antibiotics, at a starting OD (600 nm) of 0.005 (i.e., 2 µl of culture in 198 µl media). We selected a panel of 10 antibiotics across different antibiotic classes with a variety of mechanisms of action. Each antibiotic was added at a starting concentration of 100 µg/ml and subsequently serially diluted by ¾ to give 40 different concentrations, ranging from 100 µg/ml to 0.001 µg/ml. There were 6 replicates for each antibiotic. 96-well plates were incubated, shaking at 37 °C, for 24 hours. The OD (600nm) for each well was measured using a Cytation 3 multimode plate reader (BioTek). Doubling times for each well were calculated as a function of initial and final OD as they were for the yeast data.