Chemotaxonomic identification of single bacteria by micro-Raman spectroscopy: application to clean-room-relevant biological contaminations

Abstract

Microorganisms, such as bacteria, which might be present as contamination inside an industrial food or pharmaceutical clean room process need to be identified on short time scales in order to minimize possible health hazards as well as production downtimes causing financial deficits. Here we describe the first results of single-particle micro-Raman measurements in combination with a classification method, the so-called support vector machine technique, allowing for a fast, reliable, and nondestructive online identification method for single bacteria.

FIG. 1.

(A) Possible planes for separating the two classes. (B) The optimal separation plane has the largest margin and is defined only by the adjacent training samples. Support vectors are marked by circles. (C) Classification of simulated spectra. The SVM automatically detects relevant and irrelevant peaks. The third peaks of class +1 differ in size, and so compared to the third peak in class −1, those peaks contain no discriminative information and are irrelevant for the SVM classification.

FIG. 2.

Micro-Raman spectra of nine different strains (bulk). The numbers in the figure are the strain numbers.

FIG. 3.

Raman mapping experiment of a single bacterium (B. sphaericus DSM 28). (A) Micrograph of a single bacterium. The white frame indicates the mapping area (0.3 by 0.3 μm²) for taking the Raman images shown in panel C. (B) Micro-Raman spectra from selected positions within the marked scan area. The marked bands are used to calculate the Raman images plotted in panel C. (C) Raman maps for three different wavenumber regions labeled in panel B: a, 2,851 to 2,964 cm⁻¹; b, 1,604 to 1,671 cm⁻¹; and c, 1,410 to 1,455 cm⁻¹. For details, see the text.

FIG. 4.

(A) Schematic diagram of a spore. (B) Chemical structure of CaDPA, which is a marker substance and can be found in all spores. (C) Raman spectra of a vegetative cell and a spore of B. sphaericus DSM 28.

FIG. 5.

Raman mapping experiment on single spores and vegetative cells (B. sphaericus DSM 28). (A) Micrograph of two spores surrounded by vegetative cells. The white frame indicates the mapping area. (B) Micro-Raman spectra from selected positions within the marked scan area. The spectrum at the bottom corresponds to a background spectrum, while the other spectra are taken from a vegetative cell or at two different positions within the spore, respectively. The marked bands are used to calculate the Raman images plotted in panel D. (D) Raman maps for the two different wavenumber regions labeled in panel B (a, 2,871 to 2,991 cm⁻¹; b, 993 to 1,034 cm⁻¹) for three different depths positions indicated by the three horizontal lines within the schematic sketch of a spore shown in panel C. Position 3, −1.0 μm; position 2, −0.5 μm; position 1, 0 μm. For details, see the text.

FIG. 6.

(A) Raman spectra of single B. subtilis cells recorded for different growth times as indicated. (B) Raman spectra of single colored M. luteus bacteria for various growth times as indicated.

FIG. 7.

(A) Micro-Raman spectra of M. luteus DSM 348 recorded after irradiating the single M. luteus cell at 0, 60, and 360 s with the 532-nm laser, which is resonant with an electronic absorption of the chromophore sacinaxanthin of this microorganism. (B) Micrograph of various single M. luteus bacteria. The cell with which the Raman spectra in panel A was obtained is marked by a circle. (C) Dependency of the three bands labeled a, b, and c in panel A on the irradiation time. Only the band which corresponds to the C=C stretch vibration of the protection pigment sacinaxanthin of M. luteus shows a bleaching effect, while the other two vibrations, namely, the C-H vibration (b) and the amide-I band (c), are almost not affected by irradiation with 532 nm. For details, see the text.

FIG. 8.

Micro-Raman spectra of single living bacteria of nine different strains. The numbers in the figure are the strain numbers. *, fused silica.