Biology Reference
In-Depth Information
Spectroscopy collects fingerprints of microorganisms across a range
of wavelengths. For microbial samples, these spectra are most commonly
DNA/RNA, proteins, and membrane and cell wall amine- and fatty acid-
containing components. While this can provide extremely detailed infor-
mation, there is also a challenge involved in the identification of organisms
from these data. There is the need to build up a spectral library, containing
a number of representative spectra against which unknown samples can be
compared. There is also the requirement to characterize the effects of the
sample matrix on signals and to ensure that the pathogen library is represen-
tative of a range of conditions, e.g. live, newly shed, environmentally aged,
and nonviable pathogens, for which the establishment of a database would
be highly useful.
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Other potential problems with spectroscopic techniques are interfer-
ence from the substrate upon which the sample is immobilized and, in some
setups, the necessity of long detection times to achieve good signal-to-
noise ratios. However, the advantages of these methods are that they are low
energy, noninvasive and nondestructive with the potential to provide highly
detailed information at the single organism level. A detailed review and
comparison of the two main methods covered here—infrared and Raman
spectroscopy—was conducted in 2009 by Harz et al.
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5.2.1. Infrared spectroscopy
IR spectroscopy is a technique by which polychromatic light is applied to
the sample, which absorbs a photon of this light whenever the frequency
(energy) of the light is equal to the energy required for a particular bond to
vibrate. All molecules undergo different forms of vibration, e.g. stretching
and bending, at temperatures above absolute zero. IR spectroscopy works
for bonds for which the molecular dipole moment changes during the
vibration. For more information, we recommend that the reader consult
standard chemistry textbooks. Different modes of IR spectroscopy are illus-
trated in
Fig. 5.8
.
An attenuated total reflectance (ATR) setup was selected in work
aiming at developing automated, continuous monitoring for viruses.
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The ATR crystal is made of geranium (Ge) which is a semiconductor
and therefore also is operated to enable charge-based virus collection.
The collection efficiency depended upon the sample size, e.g. the dis-
tance to be traveled to reach the surface. The technique only measures
close to the surface thus preventing interference from other compo-
nents in the solution, although the sample collection is thus critical.
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