Y9 - Parallelized multi-volume cell counting assay for screening media- and time-induced effects on tolerance

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

RF

Rena Fukuda, MEng

graduate student
Cornell University
Ithaca, New York, United States

Primary Investigator(s)

NC

Nate J. Cira

Assistant Professor
Cornell University, United States

Co-Author(s)

NN

Nadia Nikulin

Graduate researcher
Cornell University, United States
TD

Tobias Doerr

Associate Professor
Cornell University, United States

Introduction: : Antibiotic tolerance has become increasingly recognized as a method by which microbes evade antibiotic-induced death, despite lacking traditional genetic resistance mechanisms associated with population survival. Instead, tolerant subpopulations display phenotypic changes – frequently a reduced metabolic rate and decreased antibiotic uptake – that enables survival at elevated antibiotic concentrations. However, high-throughput techniques to perform tolerance assays are lacking, with many microbiology labs relying on labor-intensive plate-count methods. Here, we leverage the Surface Patterned Omniphobic Tiles (SPOTs) platform, which enables parallelized plate counting. This parallelization allows us to screen through media and exposure durations to investigate factors that modulate antibiotic tolerance.

Materials and

Methods: : Preparation of SPOTs plate and loader
SPOTs plates were prepared by coating glass with an omniphobic layer and baking at 300 °C for 4 hours. We then use a UV laser to ablate circular patterns in the coating, leaving bare glass “spots” that match the desired volumes and layout required for the assay.
We also spray and bake a loader – a piece of aluminum hardware than enables precise deposition of liquid across the SPOTs plate. Both the loader and plates were UV sterilized.
Tolerance sample preparation
Klebsiella strains (WT, WT delta 4) were incubated in BHI media overnight, then diluted 1/100 into either LB, BHI, MHB, or wound-mimicking media (WMM), a serum-based media. Each media also contained meropenem. Then, the cultures were incubated for 0 or 12 hours. Cultures were then treated with meropenemase to degrade the antibiotic.
Running the cell counting assay
First, we placed the loader on our first SPOTs plate. Next, samples were added to the loader slots and the loader was dragged across the plate, which deposited precise volumes of liquid onto each ‘spot.’ A second SPOTs plate was prepared with resazurin-containing media (0.1 mg/ml). Plates were joined liquid-to-liquid with a spacer and sealed with tape, then incubated at 37 °C for 12 hours to allow for microbial outgrowth indicative of tolerance.
Analyzing the results
Plates were imaged, and we discriminated growth (pink) or no growth (blue) by thresholding in the HSV color space. Poisson statistics were applied to estimate viable cell concentrations.

Results, Conclusions, and Discussions:: Assay Performance
Using calibration samples, we determined that the assay detects concentrations across five orders of magnitude (101 to 106 cells/mL), which contains the range of clinical relevant levels of Klebsiella tolerance. SPOTs-predicted concentrations align well with those obtained by plate counting techniques. Our resazurin-based readout detected SPOTs correctly (growth vs no-growth) with an AUC consistently above 0.95, and above 0.99 for samples within 102 to 105 cells/mL. This system enabled testing of 16 samples within 15 minutes after sample preparation. These results demonstrate the assay’s accuracy clinical utility, and scalability.
Conclusion and Discussion
We have demonstrated a parallelizable, quantitative assay for assessing bacterial tolerance with clinically relevant sensitivity. In preliminary experiments comparing four media types under a single antibiotic exposure duration, we observed some variation in tolerance post-exposure. However, when normalized to their respective no-exposure controls, the differences were less pronounced.
The assay’s parallelizability allows us to explore tolerance across a broader range of conditions. Ongoing and future experiments will expand to include additional media compositions and varied exposure times, enabling a more comprehensive understanding of the factors that influence Klebsiella tolerance.

Acknowledgements and/or References (Optional): :