Applications of Raman Spectroscopy to Virology and Microbial Analysis - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 440

between bacteria but also between bacteriophages [49]. In addition, closely
related Bacillus species [50] as well as various strains of tuberculosis and non-
tuberculosis mycobacteria [51] could be distinguished.
Beside identification, also a molecular characterization of bacteria by
means of Raman spectroscopy was performed. Escoriza et al. reported about
variations in bacterial Raman spectra in different metabolic states [52]. Here,
the intensity of nucleic acid signals showed a maximum at the beginning of
the exponential phase and then slowly decreased with time, the other bands
remaining relatively constant.
Raman spectroscopy can also be used to investigate the changes in chem-
ical composition induced by various chemical and physical inactivation meth-
ods [51]. A clear differentiation between normal and deactivated cells was
possible based on the Raman spectra.
The correlation between chemical composition of microorganisms and their
susceptibility to sakacin P, a bacteriocin produced by some lactic acid bacteria,
was carried out by Oust et al. [53]. It could be shown that at least some of
the variations in the susceptibility to sakacin P in Listeria monocytogenes can
be correlated to alterations in the chemical composition of the bacterial cell
wall.
Berger et al. used Raman spectroscopy for oral bacteria identification of
Streptococcus species [54]. Furthermore, it was possible to determine the rel-
ative concentrations of different Streptococcus species in a polymicrobial mix-
ture [55]. De Gelder et al. used the Raman signal to monitor the amount of
PHB in bacterial cells [56]. In addition, the amount of bacteria in drinking
water was quantified by Escoriza et al. [57].
Foodborne microorganisms were analyzed directly on the food surface
by means of FT-Raman spectroscopy [58]. These results indicate that FT-
Raman has the ability to discriminate between pathogenic and non-pathogenic
bacteria.

19.3.2 Single Cell Identification of Bacteria by Means
of Micro-Raman Spectroscopy

By using high numerical aperture illumination and light gathering optics,
micro-Raman setups allow for Raman studies on a single organism level, thus
making the time-consuming cultivation step to generate enough biomass for
bulk measurements unnecessary. The combination of an optical microscope
and a common Raman setup leads to a lateral spatial resolution of approxi-
mately 1 m. The application of a confocal detection regime, i.e., by integrat-

ing a pinhole at the image point of the focused laser spot, leads to an even
higher spatial resolution. By doing so, the scattering signals emerging from
material outside the measuring volume are removed. However, the application
of a confocal pinhole is attended by a loss of signal, depending on the pinholes
and the objects' size. But if the latter has the dimension of the laser beam fo-
cus only suppression of the background signal will occur. Therefore, the study

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