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Applied and Environmental Microbiology, April 2001, p. 1461-1469, Vol. 67, No. 4
Laboratory for Intensive Care Research and
Optical Spectroscopy, Erasmus University Rotterdam, and Department of
General Surgery 10M1 and Institute of
Medical Microbiology and Infectious Diseases,6
University Hospital Rotterdam "Dijkzigt," 3015 GD Rotterdam,
The Netherlands; Robert Koch Institute, Biophysical Structure
Analyses, 13353 Berlin, Germany2;
UdR INFM Milano-Bicocca, Dipartimento di Biotecnologie e
Bioscienze, 20126 Milan, Italy3; and
Unité MéDIAN, CNRS FRE 2141, IFR 53, UFR de
Pharmacie, Université de Reims Champagne-Ardenne, 51096 Reims
Cedex,4 and Service d'Hygiène
Hospitalière, Center Hospitalier de Versailles, 78157 Le
Chesnay Cedex,5 France
Received 28 September 2000/Accepted 26 January 2001
Fourier transform infrared and Raman microspectroscopy are
currently being developed as new methods for the rapid identification of clinically relevant microorganisms. These methods involve measuring spectra from microcolonies which have been cultured for as little as
6 h, followed by the nonsubjective identification of
microorganisms through the use of multivariate statistical analyses. To
examine the biological heterogeneity of microorganism growth which is reflected in the spectra, measurements were acquired from various positions within (micro)colonies cultured for 6, 12, and 24 h. The
studies reveal that there is little spectral variance in 6-h microcolonies. In contrast, the 12- and 24-h cultures exhibited a
significant amount of heterogeneity. Hierarchical cluster analysis of
the spectra from the various positions and depths reveals the presence
of different layers in the colonies. Further analysis indicates that
spectra acquired from the surface of the colonies exhibit higher levels
of glycogen than do the deeper layers of the colony. Additionally, the
spectra from the deeper layers present with higher RNA levels than the
surface layers. Therefore, the 6-h colonies with their limited
heterogeneity are more suitable for inclusion in a spectral database to
be used for classification purposes. These results also demonstrate
that vibrational spectroscopic techniques can be useful tools for
studying the nature of colony development and biofilm formation.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1461-1469.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Investigating Microbial (Micro)colony Heterogeneity
by Vibrational Spectroscopy
*
Corresponding author. Mailing address: Dept. General
Surgery 10M, University Hospital Rotterdam "Dijkzigt," Dr.
Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Phone:
31-10-4635890. Fax: 31-10-4635307. E-mail:
PUPPELS{at}HLKD.AZR.NL.
Applied and Environmental Microbiology, April 2001, p. 1461-1469, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1461-1469.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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