Bacterial Suspensions Deposited on Microbiological Filter Material for Rapid Laser-Induced Breakdown Spectroscopy Identification

Abstract

Four species of bacteria, E. coli, S. epidermidis, M. smegmatis, and P. aeruginosa, were harvested from agar nutrient medium growth plates and suspended in water to create liquid specimens for the testing of a new mounting protocol. Aliquots of 30 µL were deposited on standard nitrocellulose filter paper with a mean 0.45 µm pore size to create highly flat and uniform bacterial pads. The introduction of a laser-based lens-to-sample distance measuring device and a pair of matched off-axis parabolic reflectors for light collection improved both spectral reproducibility and the signal-to-noise ratio of optical emission spectra acquired from the bacterial pads by laser-induced breakdown spectroscopy. A discriminant function analysis and a partial least squares-discriminant analysis both showed improved sensitivity and specificity compared to previous mounting techniques. The behavior of the spectra as a function of suspension concentration and filter coverage was investigated, as was the effect on chemometric cell classification of sterilization via autoclaving.

A review of the use of laser-induced breakdown spectroscopy for bacterial classification, quantification, and identification

“…The linear dependence of the total LIBS emission intensity with concentration was confirmed in 2018 by Liao et al who demonstrated this for both E. coli and S. aureus concentrations spanning four decades, from 10 4 CFU/mL up to 10 8 CFU/ml with R 2 values greater than 0.97 [51]. Malenfant et al also reported a linear dependence of the total LIBS emission intensity on the cell concentration when a large number of cells were deposited on a nitrocellulose filter testing medium [70]. In this study, approximately 10 5 cells were ablated per laser shot.…”

“…1513 spectra were classified using both DFA and PLS-DA in an externally validated classification. Both algorithms possessed sensitivities and specificities greater than 97% in a four-genus library composed of Escherichia, Staphylococcus, Pseudomonas, and Mycobacterium [70].…”

“…In the work of Putnam et al [67] the X-variable datasets possessed either 24 or 80 independent variables, constructed from ratios of emission line intensities as described above. In Malenfant et al [70] the X-variable datasets were 164 independent variables constructed from ratios of the emission line intensities.…”

“…This medium provided an essentially background-free LIBS spectrum, allowing a very sensitive biochemical discrimination down to the strain-level. More recently, we have utilized a highly convenient microbiological filter medium, which provides a highly stable, reproducible, but non-background-free (especially from carbon) mounting substrate [70]. Such filters, fabricated from nitrocellulose paper, nylon, or other material, have been used extensively by other groups due to their ubiquity and availability [44,47,49,75].…”

Diagnostic Molecular Microbiology

“…As yet, there is no FDA-cleared LIBS system for rapid identification of pathogen microorganisms. 51,52 Raman spectroscopy…”