Detection and Classification of Bacterial Cells After Centrifugation and Filtration of Liquid Specimens Using Laser-Induced Breakdown Spectroscopy.

Five species of bacteria including Escherichia coli, Mycobacterium smegmatis, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Enterobacter cloacae were deposited from suspensions of various titers onto disposable nitrocellulose filter media for analysis by laser-induced breakdown spectroscopy (LIBS). Bacteria were concentrated and isolated in the center of the filter media during centrifugation using a simple and convenient sample preparation step. Summing all the single-shot LIBS spectra acquired from a given bacterial deposition provided perfectly sensitive and specific discrimination from sterile water control specimens in a partial least squares discriminant analysis (PLS-DA). Use of the single-shot spectra provided only a 0.87 and 0.72 sensitivity and specificity, respectively. To increase the statistical validity of chemometric analyses, a library of pseudodata was created by adding Gaussian noise to the measured intensity of every emission line in an averaged spectrum of each bacterium. The normally distributed pseudodata, consisting of 4995 spectra, were used to compare the performance of the PLS-DA with a discriminant function analysis (DFA) and an artificial neural network (ANN). For the highly similar bacterial data, no algorithm showed significantly superior performance, although the PLS-DA performed least accurately with a classification error of 0.21 compared to 0.16 and 0.17 for ANN and DFA, respectively. Single-shot LIBS spectra from all of the bacterial species were classified in a DFA model tested with a tenfold cross-validation. Classification errors ranging from 20% to 31% were measured due to repeatability limitations in the single-shot data.

Silver Microparticle-Enhanced Laser-Induced Breakdown Spectroscopy.

Enhanced emission was observed in the laser-induced breakdown spectroscopy (LIBS) atomic emission spectra of bacterial cells deposited upon a nitrocellulose filtration medium in the presence of one-micron silver microparticles. A deposition chamber was constructed that allowed a uniform coating of the filter with trace amounts of silver microparticles. Masses from 10 to 100 µg were deposited in a circular area of 52.18 mm2. A 30 s deposition time was used for all experiments resulting in a mass deposition of 39 µg ± 17 µg. This mass coverage on the filter provided for a single laser shot silver mass ablation of 3.3 ng per laser shot. LIBS spectra were acquired with single-shot 1064 nm laser pulses from specimens of E. coli, M. smegmatis, and E. cloacae deposited on both microparticle-coated filters and blank filters. An increase in emission intensity for all elements detected in the bacterial LIBS spectrum as well as the carbon emission which derives in part from the nitrocellulose filter medium was observed due to the ablation with silver microparticles relative to the intensity measured from the ablation of bacterial cells deposited on a blank filter. The ratio of emission intensity with microparticles to emission intensity without microparticles was measured to be 3.6 for phosphorus, 4.5 for magnesium, 5.3 for calcium, 4.0 for sodium, and 1.2 for carbon. An enhancement in LIBS emission intensity in the range of 1-10 was observed for all the spectra, with an average enhancement ratio of 4.3.