Supercharged MPNs? Automated Determination of High-Throughput Most Probable Number (htMPN) Using Chip-Based 3D...

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Standard bacterial viability assays like surface plating and most probable number (MPN) have limitations in accuracy and speed.
• Quantifying sublethally injured bacterial cells remains a challenge for traditional methods.

Purpose of the Study:

• To introduce a novel high-throughput most probable number (htMPN) method for accelerated and improved bacterial cell enumeration.
• To enable reliable quantification of viable and sublethally injured bacterial cells using digital PCR technology.

Main Methods:

• Utilized a chip-based digital PCR instrument to track the growth of up to 20,000 individual bacterial cells per chip.
• Employed DNA-intercalating fluorescent dyes for detecting cell density after incubation at optimal temperatures.
• Validated the method using various bacterial species including Escherichia coli and Salmonella enterica.

Main Results:

• The htMPN method demonstrated equivalent cell counts to surface plating for several bacterial species, excluding Bacillus subtilis spores.
• Achieved enumeration of viable Escherichia coli within 7 hours, significantly accelerating detection times.
• Successfully detected heat-injured Salmonella enterica cells that were missed by surface plating, highlighting improved sensitivity.

Conclusions:

• The htMPN method offers a reliable, automated, and accelerated approach for viable bacterial cell enumeration.
• This technique enhances single-cell microbiology analysis and improves the recovery rate of sublethally injured cells.
• htMPN has broad applications in environmental, clinical, and food safety testing to prevent underestimation of viable bacterial counts.