Differentiating the growth phases of single bacteria using Raman spectroscopy

Marcoux, Pierre R.; Schultz, E; Perenon, R.; Simon, A.; Espagnon, Isabelle; Allier, Cédric; Dinten, Jean‐Marc

doi:10.1117/12.2041446

Cited by 13 publications

(18 citation statements)

References 20 publications

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“…These results maintain a good correlation with the results of the ATR analysis. The differences in Raman spectra between bacteria in different growth phases were previously reported [ 24 , 25 ]. In particular, a decrease in Raman bands related to nucleic acids (1484, 783 cm −1 ) in the log phase compared to lag phase was observed [ 25 ].…”

Section: Resultsmentioning

confidence: 58%

“…In particular, a decrease in Raman bands related to nucleic acids (1484, 783 cm −1 ) in the log phase compared to lag phase was observed [ 25 ]. This were attributed to changes in relative nucleic acids and protein content between various growth phases [ 24 ].…”

Section: Resultsmentioning

confidence: 99%

“…Both IR and Raman were also applied to study various aspects of growth [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. The strong influence of growth conditions and the composition of medium on the biochemistry of bacteria was demonstrated at a population level by FTIR [ 20 ] and at the single-cell level using Raman microspectroscopy [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

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Vibrational Spectroscopy as a Sensitive Probe for the Chemistry of Intra-Phase Bacterial Growth

Kochan

Lai

Richardson

et al. 2020

Sensors

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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.

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Section: Resultsmentioning

confidence: 58%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Vibrational Spectroscopy as a Sensitive Probe for the Chemistry of Intra-Phase Bacterial Growth

Kochan

Lai

Richardson

et al. 2020

Sensors

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“…In order to perform an external validation, the influence of important factors like growing time, or nutrition, or matrix (sterile or real) effects need to be taken into account in the dataset. For instance, we already observed that our system can discriminate between different growth phases of E. coli and B. subtilis bacteria based on Raman analysis 24 . Next work will be then to build a reliable dataset comprising these different parameters, and to investigate how scattering patterns collected using the lensfree module can help the identification of bacteria strains.…”

Section: Resultsmentioning

confidence: 97%

A novel method for single bacteria identification by Raman spectroscopy

et al. 2014

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In this paper we present results on single bacteria rapid identification obtained with a low-cost and compact Raman spectrometer. At present, we demonstrate that a 1 minute procedure, including the localization of single bacterium, is sufficient to acquire comprehensive Raman spectrum in the range of 600 to 3300 cm -1 . Localization and detection of single bacteria is performed by means of lensfree imaging over a large field of view of 24 mm 2 . An excitation source of 532 nm and 30 mW illuminates single bacteria to collect Raman signal into a Tornado Spectral Systems prototype spectrometer (HTVS technology). The acquisition time to record a single bacterium spectrum is as low as 10 s owing to the high light throughput of this spectrometer. The spectra processing features different steps for cosmic spikes removal, background subtraction, and gain normalization to correct the residual inducted fluorescence and substrate fluctuations. This allows obtaining a fine chemical fingerprint analysis. We have recorded a total of 1200 spectra over 7 bacterial species (E. coli, Bacillus species, S. epidermis, M. luteus, S. marcescens). The analysis of this database results in a high classification score of almost 90 %. Hence we can conclude that our setup enables automatic recognition of bacteria species among 7 different species. The speed and the sensitivity (<30 minutes for localization and spectra collection of 30 single bacteria) of our Raman spectrometer pave the way for high-throughput and non-destructive real-time bacteria identification assays. This compact and low-cost technology can benefit biomedical, clinical diagnostic and environmental applications.

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“…It would also allow, if necessary, to guide the choice for further analyses. Among the technologies capable of rapidly analyzing whole colonies on a Petri dish in a nondestructive way, label-free optical methods, such as Raman spectroscopy 7 , elastic light scattering 8,9 or Fourier-transformed infrared spectroscopy (FTIR) 10 are of particular interest. Specifically, infrared spectroscopy is one of the oldest label-free optical identification technologies 11 .…”

Section: Introductionmentioning

confidence: 99%

Mid-infrared multispectral lensless imaging for wide-field and label-free microbial identification

Galudec

Dupoy

Rebuffel

et al. 2020

Biomedical Spectroscopy, Microscopy, and Imaging

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Microbial identification is a critical process aiming at identifying the species contained in a biological sample, with applications in healthcare, industry or even national security. Traditionally, this process relies either on MALDI-TOF mass spectroscopy, on biochemical tests and on the observation of the morphology of colonies after growth on a Petri dish. Here is presented an innovative method for label-free optical identification of pathogens, based on the multispectral infrared imaging of colonies. This lensless imaging technique enables a high-throughput analysis and wide-field analysis of agar plates. It could yield very high correct identification rates as it relies on an optical fingerprint gathering both spectroscopic and morphologic features.The setup consists of a Quantum Cascade Lasers light source and an imager, a square 2.72 by 2.72 mm uncooled bolometer array. Microorganisms to be analyzed are streaked on a porous growth support compatible with infrared imaging, laid on top of an agar plate for incubation. When imaging is performed, growth support is put in close contact with the imaging sensor and illuminated at different wavelengths. After acquisition, an image descriptor based on spectral and morphological features is extracted for each microbial colony. Supervised classification is finally performed with a Support Vector Machine algorithm and tested with tenfold cross-validation. A first database collecting 1012 multispectral images of colonies belonging to five different species has already been acquired with this system, resulting in a correct identification rate of 92%. For these experiments, multispectral images are acquired at nine different wavelengths, between 5.6 and 8 µm.Considering the optimization possibilities of the image descriptors currently used and the ongoing development of the uncooled bolometers technology, these very first results are promising and could be dramatically improved with further experiments. Thereby, mid-infrared multispectral lensless imaging has the potential to become a fast and precise Petri dish analysis technology.

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Differentiating the growth phases of single bacteria using Raman spectroscopy

Cited by 13 publications

References 20 publications

Vibrational Spectroscopy as a Sensitive Probe for the Chemistry of Intra-Phase Bacterial Growth

Vibrational Spectroscopy as a Sensitive Probe for the Chemistry of Intra-Phase Bacterial Growth

A novel method for single bacteria identification by Raman spectroscopy

Mid-infrared multispectral lensless imaging for wide-field and label-free microbial identification

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