High-throughput antimicrobial efficacy tests on a 384PillarPlate

Preservatives, including antimicrobial compounds, are routinely added to home and personal care products such as dishwashing detergents to prevent microbial contamination during repeated consumer use. Traditional antimicrobial efficacy testing (T-AET) relies on colony counting methods, where microbial cocktails are inoculated into preservative-containing products and plated on agar to measure microbial survival. While considered a gold standard, T-AET is time-consuming, labor-intensive, costly, and prone to variability and error due to manual handling. To address these limitations, we developed a high-throughput, miniature, ATP-based, on-chip AET (O-AET) using a 384PillarPlate platform. Pseudomonas aeruginosa, Enterobacter cloacae, and Staphylococcus aureus were encapsulated in 3% agarose and exposed to test compounds and detergent formulations. ATP levels in viable microbes were quantified using the BacTiter-Glo™ luminescence assay to generate dose-response curves, IC₅₀ values, and log reduction measurements. Two industrial antimicrobials, Acticide LG and Acticide MBR2, were evaluated, showing CV values below 8%. Nine detergent formulations from Colgate-Palmolive, including both fresh and aged samples (stored for 8 weeks at 40 °C), were tested. Log reduction data from O-AET showed strong correlation with T-AET. Using a 3-log reduction cutoff, O-AET achieved 98% sensitivity, 44% specificity, and 71% overall predictivity. The lower specificity was partly attributed to limited true negative samples used and nutrient deficiencies. The O-AET enables significant reduction in reagent volume, rapid turnaround, and compatibility with standard plate readers, making it cost-effective and scalable. This ATP bioluminescence-based platform provides a robust, reproducible, and high-throughput alternative to T-AET, with high potential to streamline product development and preservative screening in consumer care applications.