Non-perturbative Bacterial Identification Directly from Solid Agar Plates Using Raman

TL;DR

This study presents a non-perturbative Raman spectroscopy method for directly identifying bacteria from unopened agar plates with high accuracy, integrating DFT calculations and machine learning for real-time microbiological analysis.

Contribution

The paper introduces a novel Raman-based bacterial identification technique that works directly through agar without opening plates, using DFT and machine learning to achieve high accuracy.

Findings

Achieved over 97.7% classification accuracy.

Surpassed standard open-plate measurements by 10.8% in accuracy.

Demonstrated robustness across different media and substrate conditions.

Abstract

Raman spectroscopy is a promising tool for microbial identification, yet its implementation in microbiology and clinical workflow is still restricted due to the accompanying additional preparation required to focus on microbial signals. Here, we demonstrate Raman-based bacterial identification directly from unopened, inverted agar plates, the same conditions used during incubation. Our approach enabled identification with single gene-level sensitivity using two Escherichia coli variants, differing only in green fluorescent protein (GFP) expression, across diverse media and substrate material conditions, despite the interrogation path traversing 3-4 mm thick background material. We integrated traditional density functional theory (DFT)-based material computation with machine learning analysis, achieving over 97.7% classification accuracy, surpassing the performance of standard measurements from opened plates by 10.8% higher mean accuracy and 0.76% less variance. We further demonstrated Raman mapping-based colony identification via Raman peaks characteristic to GFPmut3 chromophore structure generated by DFT. Our approach is robust to changes in algorithms or substrate materials and promises real-time, non-perturbative monitoring of bacterial growth, biofilm formation, and antimicrobial resistance development.