Experimental validation of stability and applicability of Start Growth Time method for high-throughput bacterial...

Ecotoxicity assessments based on bacteria as model organisms are widely used for routine toxicity screening because it has the advantages of time-saving, high sensitivity, cost-effectiveness, and less ethical responsibility. Determination of ecotoxicity effect via bacterial growth can avoid the restriction of model bacteria selection and unique equipment requirements, but traditional viable cell count methods are relatively labor- and time-intensive. The Start Growth Time method (SGT) is a high-throughput and time-conserving method to determine the amount of viable bacterial cells. However, its usability and stability for ecotoxicity assessment are rarely studied. This study confirmed its applicability in terms of bacterial types (gram-positive and gram-negative), growth phases (middle exponential and early stationary phases), and simultaneous existence of dead cells (adjustment by flow cytometry). Our results verified that the stability of establishing SGT correlation is independent of the bacterial type and dead-cell portion. Moreover, we only observed the effect of growth phases on the slope value of established SGT correlation in Shewanella oneidensis, which suggests that preparing inoculum for the SGT method should be consistent in keeping its stability. Our results also elucidate that the SGT values and the live cell percentages meet the non-linear exponential correlation with high correlation coefficients from 0.97 to 0.99 for all the examined bacteria. The non-linear exponential correlation facilitates the application of the SGT method in the ecotoxicity assessment. Finally, applying the exponential SGT correlation to evaluate the ecotoxicity effect of copper ions on E. coli was experimentally validated. The SGT-based method would require about 6 to 7 h to finish the assessment and obtain an estimated EC₅₀ at 2.27 ± 0.04 mM. This study demonstrates that the exponential SGT correlation can be a high-throughput, time-conversing, and wide-applicable method for bacterial ecotoxicity assessment.