Abstract
Bacterial growth in batch cultures occurs in four phases (lag, exponential/log, stationary and death phase) that differ distinctly in number of different bacteria, biochemistry and physiology. Knowledge regarding the growth phase and its kinetics is essential for bacterial research, especially in taxonomic identification and monitoring drug interactions. However, the conventional methods by which to assess microbial growth are based only on cell counting or optical density, without any insight into the biochemistry of cells or processes. Both Raman and Fourier transform infrared (FTIR) spectroscopy have shown potential to determine the chemical changes occurring between different bacterial growth phases. Here, we extend the application of spectroscopy and for the first time combine both Raman and FTIR microscopy in a multimodal approach to detect changes in the chemical compositions of bacteria within the same phase (intra-phase). We found a number of spectral markers associated with nucleic acids (IR: 964, 1082, 1215 cm−1; RS: 785, 1483 cm−1), carbohydrates (IR: 1035 cm−1; RS: 1047 cm−1) and proteins (1394 cm−1, amide II) reflecting not only inter-, but also intra-phase changes in bacterial chemistry. Principal component analysis performed simultaneously on FTIR and Raman spectra enabled a clear-cut, time-dependent discrimination between intra-lag phase bacteria probed every 30 min. This demonstrates the unique capability of multimodal vibrational spectroscopy to probe the chemistry of bacterial growth even at the intra-phase level, which is particularly important for the lag phase, where low bacterial numbers limit conventional analytical approaches.
Highlights
Bacterial growth in batch cultures typically occurs in four distinct phases that are clearly visible through the growth curve: (1) lag, (2) exponential, (3) stationary and (4) death phase [1,2,3]
IR in its conventional application enables the study of bacteria on a population level because the diffraction limit of IR light prevents a resolution from being achieved that is adequate for single bacterial cells
This limitation was recently overcome by combining atomic force microscopy with IR spectroscopy (AFM-IR) and probing the photothermal expansion resulting from IR absorption [12]
Summary
Bacterial growth in batch cultures typically occurs in four distinct phases that are clearly visible through the growth curve: (1) lag, (2) exponential (log), (3) stationary and (4) death phase [1,2,3]. Conventional methods of determining the growth phase are based on cell numbers, which are established either through standard plate counting or through optical density [5] These methods, cannot describe any biochemical changes, which would reflect microbial physiology. IR in its conventional application enables the study of bacteria on a population level because the diffraction limit of IR light prevents a resolution from being achieved that is adequate for single bacterial cells. This limitation was recently overcome by combining atomic force microscopy with IR spectroscopy (AFM-IR) and probing the photothermal expansion resulting from IR absorption [12]. Together with an analysis of spectral patterns and integral intensities of selected bands for single and combined modalities, we identified a range of spectra markers, reflective of inter- and intra-phase biochemical changes
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