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

The use of laser-induced breakdown spectroscopy to determine the elemental composition of bacterial cells has been described in the peer-reviewed literature since 2003. Fifteen years on, significant accomplishments have been reported that have served to clarify and underscore the areas of bacteriological investigation that LIBS is well-suited for as well as the challenges that yet remain to be faced. This review will attempt to summarize the state of the field by surveying the available body of knowledge. The early days of these experiments, roughly from 2003 to 2007, in which many of the most fundamental experiments were initially conducted will be described. The more in-depth investigations that followed in the subsequent decade will then be detailed. Many important aspects of performing LIBS on bacterial cells were reported on and are summarized here including: the use of chemometric algorithms for statistical classification of unknown spectra; the influence of the mounting substrate on classification; the effect of the testing gas atmosphere and the choice of bacterial cell growth nutrient medium on the measured LIBS spectrum; the efficacy of a LIBS-based test as a genus-level or strain–level discrimination test; the ability of LIBS to determine the cell titer or concentration of cells in the initial sample; the effects that possible contaminations or interferents within the sample would have on the LIBS spectrum; the influence that environmental stresses the cells may be exposed to during growth and the state of reproductive health of the cells could have on the LIBS spectrum; the use of standoff or remote apparatus to minimize the risk to the operators during bacteriological identification of unknown specimens; and the combination of other optical modalities with LIBS to enhance the sensitivity or specificity of identification. Lastly, tables are provided which summarize both every species of bacteria ever tested with LIBS as well as the major lessons learned by the community through 15 years of careful investigation

Detection of trace Al in model biological tissue with laser-induced breakdown spectroscopy

Laser-induced breakdown spectroscopy (LIBS), which is an excellent tool for trace elemental analysis, was studied as a method of detecting sub-part-per-106 (ppm) concentrations of aluminum in surrogates of human tissue. Tissue was modeled using a 2% agarose gelatin doped with an Al2O3 nanoparticle suspension. A calibration curve created with standard reference samples of known Al concentrations was used to determine the limit of detection, which was less than 1 ppm. Rates of false negative and false positive detection results for a much more realistic sampling methodology were also studied, suggesting that LIBS could be a candidate for the real-time in vivo detection of metal contamination in human soft tissue

Pathogenic Escherichia coli strain discrimination using laser-induced breakdown spectroscopy

A pathogenic strain of bacteria, Escherichia coli O157:H7 (enterohemorrhagic E. coli or EHEC), has been analyzed by laser-induced breakdown spectroscopy (LIBS) with nanosecond pulses and compared to three nonpathogenic E. coli strains: a laboratory strain of K-12 (AB), a derivative of the same strain termed HF4714, and an environmental strain, E. coli C (Nino C). A discriminant function analysis (DFA) was performed on the LIBS spectra obtained from live colonies of all four strains. Utilizing the emission intensity of 19 atomic and ionic transitions from trace inorganic elements, the DFA revealed significant differences between EHEC and the Nino C strain, suggesting the possibility of identifying and discriminating the pathogenic strain from commonly occurring environmental strains. EHEC strongly resembled the two K-12 strains, in particular, HF4714, making discrimination between these strains difficult. DFA was also used to analyze spectra from two of the nonpathogenic strains cultured in different media: on a trypticase soy (TS) agar plate and in a liquid TS broth. Strains cultured in different media were identified and effectively discriminated, being more similar than different strains cultured in identical media. All bacteria spectra were completely distinct from spectra obtained from the nutrient medium or ablation substrate alone. The ability to differentiate strains prepared and tested in different environments indicates that matrix effects and background contaminations do not necessarily preclude the use of LIBS to identify bacteria found in a variety of environments or grown under different conditions

Escherichia coli identification and strain discrimination using nanosecond laser-induced breakdown spectroscopy

Three strains of Escherichia coli, one strain of environmental mold, and one strain of Candida albicans yeast have been analyzed by laser-induced breakdown spectroscopy using nanosecond laser pulses. All microorganisms were analyzed while still alive and with no sample preparation. Nineteen atomic and ionic emission lines have been identified in the spectrum, which is dominated by calcium, magnesium, and sodium. A discriminant function analysis has been used to discriminate between the biotypes and E. coli strains. This analysis showed efficient discrimination between laser-induced breakdown spectroscopy spectra from different strains of a single bacteria species

Laser collimation of an atomic gallium beam

The linear-perpendicular-linear polarization gradient technique was used for investigating laser collimation of a gallium atomic beam in one dimension. The full angular divergence of the atomic beam was reduced to 0.3 mrad by operating on a particular electron transition at 294.45 nm. The transverse velocity of the atoms was reduced to 11 cm/s, which was about half of the Doppler cooling limit. The one-dimensional kinetic energy of atoms was reduced to 6 neV. The transition state exhibited optical pumping of the atoms by the cooling laser

Towards the clinical application of laser-induced breakdown spectroscopy for rapid pathogen diagnosis: The effect of mixed cultures and sample dilution on bacterial identification

Laser-induced breakdown spectroscopy has been utilized to classify and identify bacterial specimens on the basis of their atomic composition. We have characterized the effect that the presence of a second bacterial species in the ablated specimen had on the identification of the majority species. Specimens with a reduced number of bacterial cells (approximately 2500) were identified with 100% accuracy when compared to undiluted specimens. In addition, a linear dependence of the total spectral power as a function of cell number was determined. Lastly, a high selectivity was obtained for a LIBS-based analysis of nine separate bacterial strains from four genera

The effect of bacterial environmental and metabolic stresses on a laser-induced breakdown spectroscopy (LIBS) based identification of Escherichia coli and Streptococcus viridans

In this paper we investigate the effect that adverse environmental and metabolic stresses have on the laser-induced breakdown spectroscopy (LIBS) identification of bacterial specimens. Single-pulse LIBS spectra were acquired from a non-pathogenic strain of Escherichia coli cultured in two different nutrient media: a trypticase soy agar and a MacConkey agar with a 0.01% concentration of deoxycholate. A chemometric discriminant function analysis showed that the LIBS spectra acquired from bacteria grown in these two media were indistinguishable and easily discriminated from spectra acquired from two other non-pathogenic E. coli strains. LIBS spectra were obtained from specimens of a nonpathogenic E. coli strain and an avirulent derivative of the pathogen Streptococcus viridans in three different metabolic situations: live bacteria reproducing in the log-phase, bacteria inactivated on an abiotic surface by exposure to bactericidal ultraviolet irradiation, and bacteria killed via autoclaving. All bacteria were correctly identified regardless of their metabolic state. This successful identification suggests the possibility of testing specimens that have been rendered safe for handling prior to LIBS identification. This would greatly enhance personnel safety and lower the cost of a LIBS-based diagnostic test. LIBS spectra were obtained from pathogenic and non-pathogenic bacteria that were deprived of nutrition for a period of time ranging from one day to nine days by deposition on an abiotic surface at room temperature. All specimens were successfully classified by species regardless of the duration of nutrient deprivation

North American Symposium on Laser-Induced Breakdown Spectroscopy: Introduction to the feature issue

This feature issue highlights the topics presented at the 2009 North American Symposium on Laser-Induced Breakdown Spectroscopy, in New Orleans, Louisiana, held 13-15 July 2009. © 2010 Optical Society of America

A membrane basis for bacterial identification and discrimination using laser-induced breakdown spectroscopy

Nanosecond single-pulse laser-induced breakdown spectroscopy (LIBS) has been used to discriminate between two different genera of Gram-negative bacteria and between several strains of the Escherichia coli bacterium based on the relative concentration of trace inorganic elements in the bacteria. Of particular importance in all such studies to date has been the role of divalent cations, specifically Ca 2+ and Mg 2+, which are present in the membranes of Gram-negative bacteria and act to aggregate the highly polar lipopolysaccharide molecules. We have demonstrated that the source of emission from Ca and Mg atoms observed in LIBS plasmas from bacteria is at least partially located at the outer membrane by intentionally altering membrane biochemistry and correlating these changes with the observed changes in the LIBS spectra. The definitive assignment of some fraction of the LIBS emission to the outer membrane composition establishes a potential serological, or surface-antigen, basis for the laser-based identification. E. coli and Pseudomonas aeruginosa were cultured in three nutrient media: trypticase soy agar as a control, a MacConkey agar with a 0.01% concentration of bile salts including sodium deoxycholate, and a trypticase soy agar with a 0.4% deoxycholate concentration. The higher concentration of deoxycholate is known to disrupt bacterial outer membrane integrity and was expected to induce changes in the observed LIBS spectra. Altered LIBS emission was observed for bacteria cultured in this 0.4% medium and laser ablated in an all-argon environment. These spectra evidenced a reduced calcium emission and in the case of one species, a reduced magnesium emission. Culturing on the lower (0.01%) concentration of bile salts altered the LIBS spectra for both the P. aeruginosa and two strains of E. coli in a highly reproducible way, although not nearly as significantly as culturing in the higher concentration of deoxycholate did. This was possibly due to the accumulation of divalent cations around the bacteria by the formation of an extracellular polysaccharide capsule. Lastly, a discriminant function analysis demonstrated that in spite of alterations in the LIBS spectrum induced by growth in the three different media, the analysis could correctly identify all samples better than 90% of the time. This encouraging result illustrates the potential utility of LIBS as a rapid bacteriological identification technology. © 2009 American Institute of Physics

A Simple and Efficient Centrifugation Filtration Method for Bacterial Concentration and Isolation Prior to Testing Liquid Specimens with Laser-Induced Breakdown Spectroscopy

An inexpensive filtration device was designed and constructed to rapidly concentrate bacteria in a liquid suspension on the surface of a disposable filter medium while at the same time separating the bacterial cells from larger contaminants in the suspension on the basis of their size. The device consists of a two-stage insert that is held rigidly in a standard tube during bacterial suspension centrifugation. The filters can be easily removed from the insert for subsequent testing with laser-induced breakdown spectroscopy in a process that takes only three minutes. Filter media of 0.45 micron pore size was found to capture approximately 90-95% of the cells in suspension. A limit of detection of 90,000 cells per laser shot was calculated by constructing a calibration curve from multiple suspensions of varying concentration. Deposition of the bacterial lawn across the surface of the 9.5 mm diameter filters was found to be uniform to within +/- 20% of the mean as determined by the total measured optical breakdown emission. Use of 5 micron pore size filter media in the first stage of the insert was found to remove close to 100% of a 12 micron grain size tungsten contamination from the suspension while removing 10% of the bacterial cells. This mounting protocol provides a very convenient method for sample preparation that makes use of common techniques, apparatus, and procedures that would be familiar to clinicians or microbiological pathology laboratory personnel