Yin-Fong Su

Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces

Aims:  Having and executing a well‐defined and validated sampling protocol is critical following a purposeful release of a biological agent for response and recovery activities, for clinical and epidemiological analysis and for forensic purposes. The objective of this study was to address the need for validated sampling and analysis methods called out by the General Accounting Office and others to systematically compare the collection efficiency of various swabs and wipes for collection of bacterial endospores from five different surfaces, both porous and nonporous. This study was also designed to test the collection and extraction solutions used for endospore recovery from swabs and wipes.

Quantitative reflectance spectra of solid powders as a function of particle size

We have recently developed vetted methods for obtaining quantitative infrared directional-hemispherical reflectance spectra using a commercial integrating sphere. In this paper, the effects of particle size on the spectral properties are analyzed for several samples such as ammonium sulfate, calcium carbonate, and sodium sulfate as well as one organic compound, lactose. We prepared multiple size fractions for each sample and confirmed the mean sizes using optical microscopy. Most species displayed a wide range of spectral behavior depending on the mean particle size. General trends of reflectance versus particle size are observed such as increased albedo for smaller particles: for most wavelengths, the reflectivity drops with increased size, sometimes displaying a factor of 4 or more drop in reflectivity along with a loss of spectral contrast. In the longwave infrared, several species with symmetric anions or cations exhibited reststrahlen features whose amplitude was nearly invariant with particle size, at least for intermediate and large size sample fractions: that is, ≳150  μm. Trends of other types of bands (Christiansen minima, transparency features) are also investigated as well as quantitative analysis of the observed relationship between reflectance versus particle diameter.

Chemometric analysis of multiple species of Bacillus bacterial endospores using infrared spectroscopy: discrimination to the strain level.

Previous work using infrared spectroscopy has shown potential for rapid discrimination between bacteria in either their sporulated or vegetative states, as well as between bacteria and other common interferents. For species within one physiological state, however, distinction is far more challenging, and requires chemometrics. In the current study, we have narrowed the field of study by eliminating the confounding issues of vegetative cells as well as growth media and focused on using IR spectra to distinguish only between different species all in the sporulated state. Using principal component analysis (PCA) and a classification method based upon similarity measurements, we demonstrate a successful identification rate to the species level of 85% for Bacillus spores grown and sporulated in a glucose broth medium.

Intensity-Value Corrections for Integrating Sphere Measurements of Solid Samples Measured Behind Glass

Accurate and calibrated directional-hemispherical reflectance spectra of solids are important for both in situ and remote sensing. Many solids are in the form of powders or granules and to measure their diffuse reflectance spectra in the laboratory, it is often necessary to place the samples behind a transparent medium such as glass for the ultraviolet (UV), visible, or near-infrared spectral regions. Using both experimental methods and a simple optical model, we demonstrate that glass (fused quartz in our case) leads to artifacts in the reflectance values. We report our observations that the measured reflectance values, for both hemispherical and diffuse reflectance, are distorted by the additional reflections arising at the air–quartz and sample–quartz interfaces. The values are dependent on the sample reflectance and are offset in intensity in the hemispherical case, leading to measured values up to ∼6% too high for a 2% reflectance surface, ∼3.8% too high for 10% reflecting surfaces, approximately correct for 40–60% diffuse-reflecting surfaces, and ∼1.5% too low for 99% reflecting Spectralon® surfaces. For the case of diffuse-only reflectance, the measured values are uniformly too low due to the polished glass, with differences of nearly 6% for a 99% reflecting matte surface. The deviations arise from the added reflections from the quartz surfaces, as verified by both theory and experiment, and depend on sphere design. Empirical correction factors were implemented into post-processing software to redress the artifact for hemispherical and diffuse reflectance data across the 300–2300 nm range.

The infrared spectra of Bacillus bacteria part I: vegetative Bacillus versus sporulated cells and the contributions of phospholipids to vegetative infrared spectra.

This paper highlights the distinctions between the infrared (IR) absorption spectra of vegetative versus sporulated Bacillus bacteria. It is observed that there are unique signatures clearly associated with either the sporulated or vegetative state and that vegetative cells (or cell debris) can contribute to the spore spectra. A distinct feature at ∼1739 cm−1 appears to be unique to vegetative cell spectra and can also be used as an indicator of vegetative cells or cell debris in the spore spectra. The data indicate that the band arises from a lipid-soluble species such as an ester or phospholipid carbonyl bond and are consistent with it being either phosphatidyl glycerol (PG) or phosphatidylethanolamine (PE), two major classes of phospholipids found in vegetative cells of Bacillus species. A companion work discusses bands associated with the sporulated state.

The Infrared Spectra of Bacillus Bacteria Part II: Sporulated Bacillus-the Effect of Vegetative Cells and Contributions of Calcium Dipicolinate Trihydrate, CaDP•3H2O

Our previous paper showed that certain infrared (IR) peaks, e.g., the peak at 1739 cm−1, are due to varying (trace) amounts of vegetative cells amongst the Bacillus spores and that these and other vegetative bands are associated with lipid-soluble compounds, likely an ester or phospholipid. This work investigates the infrared spectra of eight different sporulated Bacillus bacteria. For the endospores it is observed that peaks at 1441, 1277, and 1015 cm−1 along with a distinct quartet of peaks at 766, 725, 701, and 659 cm−1 are clearly associated with calcium dipicolinate trihydrate, CaDP·3H2O. It is emphasized that the spore peaks, especially the quartet, arise from the calcium dipicolinate trihydrate and not from dipicolinic acid or other dipicolinate hydrate salts. The CaDP-3H2O infrared peaks and the effects of hydration are studied using quantum chemistry in the PQS software package. The quartet is associated with many modes including contributions from the Ca2+ counterion and hydration waters including Ca–O–H bends, H2O–Ca–O torsions, and O–C–O bends. The 1441 and 1015 cm−1 modes are planar pyridine modes with the 1441 cm−1 mode primarily a ring C–N stretch and the 1015 cm−1 mode primarily a ring C–C stretch.

Adapting Raman Spectra from Laboratory Spectrometers to Portable Detection Libraries

Raman spectral data collected with high-resolution laboratory spectrometers are processed into a format suitable for importing as a user library on a 1064 nm DeltaNu first generation, field-deployable spectrometer prototype. The two laboratory systems used are a 1064 nm Bruker Fourier transform (FT)-Raman spectrometer and a 785 nm Kaiser dispersive spectrometer. The steps taken to adapt for device-dependent spectral resolution, wavenumber shifts between instruments, and relative intensity response are described. Effects due to the differing excitation laser wavelengths were found to be minimal, indicating—at least for the near-infrared (NIR)—that data can be ported between different systems, so long as certain measures are taken with regard to the reference and field spectra.

Demonstrated Wavelength Portability of Raman Reference Data for Explosives and Chemical Detection

As Raman spectroscopy continues to evolve, questions arise as to the portability of Raman data: dispersive versus Fourier transform, wavelength calibration, intensity calibration, and in particular the frequency of the excitation laser. While concerns about fluorescence arise in the visible or ultraviolet, most modern (portable) systems use near-infrared excitation lasers, and many of these are relatively close in wavelength. We have investigated the possibility of porting reference data sets from one NIR wavelength system to another: We have constructed a reference library consisting of 145 spectra, including 20 explosives, as well as sundry other compounds and materials using a 1064 nm spectrometer. These data were used as a reference library to evaluate the same 145 compounds whose experimental spectra were recorded using a second 785 nm spectrometer. In 128 cases of 145 (or 88.3% including 20/20 for the explosives), the compounds were correctly identified with a mean “hit score” of 954 of 1000. Adding in criteria for when to declare a correct match versus when to declare uncertainty, the approach was able to correctly categorize 134 out of 145 spectra, giving a 92.4% accuracy. For the few that were incorrectly identified, either the matched spectra were spectroscopically similar to the target or the 785 nm signal was degraded due to fluorescence. The results indicate that imported data recorded at a different NIR wavelength can be successfully used as reference libraries, but key issues must be addressed: the reference data must be of equal or higher resolution than the resolution of the current sensor, the systems require rigorous wavelength calibration, and wavelength-dependent intensity response should be accounted for in the different systems.

Quantitative Total and Diffuse Reflectance Laboratory Measurements for Remote, Standoff, and Point Sensing

Methods for making total and diffuse directional/hemispherical reflectance measurements in the shortwave to longwave infrared using an integrating sphere are described. The sphere is a commercial, off-the-shelf optical device with its sample port at the bottom, which is essential for examining powdered samples without using a cover glass. The reflectance spectra of recently-developed National Institute of Standards and Technology (NIST, USA) infrared reflectance standards have been measured using the sphere. Reflectance spectra of other materials such as Spectralon and Infragold were also measured. The relative systematic error for the total reflectance measurements is estimated to be on the order of 3%, and random measurement error for multiple samples of each material is on the order of 0.5%.

Infrared reflectance spectra: effects of particle size, provenance and preparation

We have recently developed methods for making more accurate infrared total and diffuse directional - hemispherical reflectance measurements using an integrating sphere. We have found that reflectance spectra of solids, especially powders, are influenced by a number of factors including the sample preparation method, the particle size and morphology, as well as the sample origin. On a quantitative basis we have investigated some of these parameters and the effects they have on reflectance spectra, particularly in the longwave infrared. In the IR the spectral features may be observed as either maxima or minima: In general, upward-going peaks in the reflectance spectrum result from strong surface scattering, i.e. rays that are reflected from the surface without bulk penetration, whereas downward-going peaks are due to either absorption or volume scattering, i.e. rays that have penetrated or refracted into the sample interior and are not reflected. The light signals reflected from solids usually encompass all such effects, but with strong dependencies on particle size and preparation. This paper measures the reflectance spectra in the 1.3 – 16 micron range for various bulk materials that have a combination of strong and weak absorption bands in order to observe the effects on the spectral features: Bulk materials were ground with a mortar and pestle and sieved to separate the samples into various size fractions between 5 and 500 microns. The median particle size is demonstrated to have large effects on the reflectance spectra. For certain minerals we also observe significant spectral change depending on the geologic origin of the sample. All three such effects (particle size, preparation and provenance) result in substantial change in the reflectance spectra for solid materials; successful identification algorithms will require sufficient flexibility to account for these parameters.