Optical methods can offer good sensitivity for detecting small amounts of chemicals and biologicals, and as these
methods mature, are some of the few techniques that can offer true standoff detection. For detection of biological
species, determining the viability is clearly important: Certain species of gram-positive bacteria are capable of forming
endospores, specialized structures that arise when living conditions become unfavorable or little growth medium is
available. Spores are also resistant to many chemicals as well as changes in heat or pH; such spores can remain dormant
from months to years until more favorable conditions arise, resulting in germination back to the vegetative state. This
persistence characteristic of bacterial spores allows for contamination of a surface (e.g. food or medical equipment) even
after the surface has been nominally cleaned. Bacterial spores have also been used as biological weapons, as in the case
of B. anthracis. Thus, having rapid analytical methods to determine a spore's viability after attempts to clean a given
environment is crucial. The increasing availability of portable spectrometers may provide a key to such rapid onsite
analysis. The present study was designed to determine whether infrared spectroscopy may be used to differentiate
between viable vs. dead B. subtilis and B. atrophaeus spores. Preliminary results show that the reproducible differences
in the IR signatures can be used to identify the viable vs. the autoclaved (dead) spores.
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 the vegetative state, and that vegetative cells (and associated 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 the band is caused by a phospholipid carbonyl bond and are consistent with, but do not prove it to be, either phosphatidyl ethanolamine (PE) or phosphatidyl glycerol (PG), the two major classes of phospholipids found in vegetative cells of Bacillus species. The endospore spectra show characteristic peaks
at 1441, 1277, and 1015 cm-1 along with a distinct quartet of peaks at 766, 725, 701, and 659 cm-1. These are clearly
associated with calcium dipicolinate trihydrate,
CaDP•3H2O. We emphasize 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 vibrational peaks and the effects of hydration were studied using quantum chemistry in the PQS software package. The quartet is associated with many motions 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 mode primarily a ring C-N stretch and the 1015 mode primarily a ring C-C stretch.
Pacific Northwest National Laboratory (PNNL) has active programs investigating the optical absorption strengths of several types of molecules including toxic industrial chemicals (TICs), microbiological threats such as bacteria, as well as explosives such as RDX, PETN and TNT. While most of our work has centered on the mid-infrared domain (600 to 6,500 cm-1), more recent work has also included work in the far-infrared, also called the terahertz (THz) region (500 to ~8 cm-1). Using Fourier transform infrared spectroscopy, we have been able to compare the relative, and in some cases absolute, IR/THz cross sections of a number of species in the solid and liquid phases. The relative band strengths of a number of species of interest are discussed in terms of both experimental and computational results.
The ability to distinguish endospores from each other, from vegetative cells, and from background particles
has been demonstrated by PNNL and several other laboratories using various analytical techniques such as
MALDI and SIMS. Recent studies at PNNL using Fourier transform Infrared (FTIR) spectroscopy
combined with statistical analysis have shown the ability to characterize and discriminate bacterial spores
and vegetative bacteria from each other, as well as from background interferents. In some cases it is even
possible to determine the taxonomical identity of the species using FTIR. This effort has now grown to
include multiple species of bacterial endospores, vegetative cells, and background materials. The present
work reports on advances in being able to use FTIR, or IR in combination with other techniques, for rapid
and reliable discrimination.
The detection and interdiction of biological and chemical warfare agents at point-of-entry military, government, and civilian facilities remains a high priority for security personnel. Commercial personnel and mail screening technologies for these harmful agents are still being developed and improved upon to meet all security client requirements. Millimeter-wave holographic imaging technology developed at the Pacific Northwest National Laboratory is an ideal sensor to interrogate objects concealed behind low dielectric barriers such as paper, cardboard, and clothing. It uses harmless millimeter waves to illuminate the object or person under surveillance. The waves penetrate through the low dielectric barrier and either reflects off or pass through the hidden object, depending on its material dielectric properties. The reflected signals are digitized and sent to high-speed computers to form high-resolution, three-dimensional (3-D) images. Feasibility imaging studies have been conducted to determine whether simulated biological or chemical agents concealed in mail packages or under clothing could be detected using holographic radar imaging techniques. The results of this study will be presented in this paper.
We have recently extended our studies of the infrared signatures of Bacillus bacterial spores from the mid-infrared to the far-infrared (sometimes called the terahertz, THz) spectral domain. The ultimate goal is to use such signatures to distinguish different strains of spores from unknowns as well as from one another. Five different strains of Bacillus were prepared by culturing the spores, washing repeatedly in sterile water and drying them onto windows that are simultaneously transparent in both the mid- and far-infrared. The strains include B. globigii BG-01, B. thuringiensis subsp kurstaki ATCC 35866, B. subtilis ATCC 49760, B. subtilis ATCC 6051, and B. atrophaeus ATCC 49337. Using different combinations of hardware in the Fourier transform infrared (FTIR) spectrometer, essentially continuous spectral coverage was obtained from ~8 to 6,000 cm-1. Preliminary results indicate that any THz signatures are at least 25 times weaker (based on p-p noise) than the strongest mid-IR amide I bands near 1657 cm-1.
We have previously reported a combined mid-infrared spectroscopic/statistical modeling approach for the discrimination and identification, at the strain level, of both sporulated and vegetative bacteria. This paper reports on the expansion of the reference spectral library: transmissive Fourier-transform mid-infrared (trans-FTIR) spectra were obtained for three Escherichia bacterial strains (E. coli RZ1032, E. coli W3110, and E. coli HB101 ATCC 33694), and two Pseudomonas putida bacterial strains (P. putida 0301 and P. putida ATCC 39169). These were combined with the previous spectral data of five Bacillus bacterial strains (B. atrophaeus ATCC 49337, B. globigii Dugway, B. thuringiensis spp. kurstaki ATCC 35866, B. subtilis ATCC 49760, and B. subtilis 6051) to form an extended library. The previously developed four step statistical model for the identification of bacteria (using the expanded library) was subsequently used on blind samples including other bacteria as well as non-biological materials. The results from the trans-FTIR spectroscopy experiments are discussed and compared to results obtained using photoacoustic Fourier-transform infrared spectroscopy (PA-FTIR). The advantages, disadvantages, and preliminary detection limits for each technique are discussed. Both methods yield promising identification of unknown bacteria, including bacterial spores, in a matter of minutes.