AN-RS-047
2025-06
Rapid phenotypic identification of microorganisms with Raman
A simple and nondestructive method for bacterial analysis
Summary
Microorganisms are among the most diverse life forms on Earth. They exhibit unique characteristics and play crucial roles in ecological nutrient and material cycles. Microorganisms are essential to food production, including yogurt and alcoholic beverages, and in the remediation of environmental contaminants. Additionally, genetic modification of microorganisms facilitates production of valuable products like insulin. Given their importance, many countries maintain specialized repositories like the American Type Culture Collection (ATCC) and the Swiss Collection of Microorganisms (SCM) to preserve and accumulate microorganisms.
Traditionally, identifying microorganisms such as bacteria involved sequencing their genetic makeup. This expensive process requires specialized training and equipment. However, Raman spectroscopy is a potential tool for identifying bacteria and detecting metabolites produced by the culture, providing insights into the bioprocesses and function in an ecosystem. Metrohm’s laboratory Raman portfolio contains options for 785 nm and 1064 nm Raman interrogation of bacterial cultures.
Introduction
Raman spectroscopy is used in microbiology for its potential to identify bacteria and monitor metabolites. All living organisms on Earth are composed of carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, and other trace elements. These elements bond together to form DNA, lipids, amino acids, and other biomolecules. The composition of these biomolecules varies between organisms. Some bacteria store metabolites (e.g., polyphosphate and glycogen) depending on environmental conditions. The Raman spectra of bacteria reflect these chemical differences, enabling their identification and elucidating their roles in bioprocesses.
Experiment
Lysogeny broth (LB) agar culture media was prepared by dissolving LB powder and agar powder in deionized water following manufacturer specifications (Sigma-Aldrich). After autoclaving, the mixture was poured into sterilized glass petri dishes and cooled. Once the LB agar solidified, fingers were pressed onto the surface to transfer bacteria to the media. The petri dish was then incubated at room temperature until bacterial colonies were observed.
The petri dish was placed on a BAC150B probe holder and BAC151C Video microscope, and Raman spectra were collected from colonies and the culture media (Figure 1). Instrument setup and acquisition parameters are summarized in Table 1.
| Instrument | Probe holder (BAC150B) |
Video microscope (BAC151C) |
|---|---|---|
| i-Raman Prime 785 | BAC102-785HT | 50x objective |
| i-Raman EX | BAC102-1064HT | 50x objective |
| BWSpec Software | ||
| Acquisition Parameters* | ||
| Laser power (%) | 30–100 | |
| Integration time | 3–60 s | |
| Averages | 3–5 | |
Raman sprectra of bacterial colony
Raman spectra of the bacterial colony (Figure 2) contained peaks representing various amino acids (1001, 1156, and 1654 cm-1) and DNA (723, 669, and 1337 cm−1). These features, commonly observed in bacteria, confirm the success of i-Raman Prime 785 in microbial analysis [1].
Raman excitation at 785 nm provided stronger and sharper peaks than excitation at 1064 nm. This is attributed to the higher scattering power of the 785 nm laser and better resolution of the silicon CCD detector compared to the InGaAs array detector with a lower pixel density. However, 1064 nm excitation may mitigate fluorescence associated with darkly colored substrates, such as chocolate agar or blood agar.
Differentiating bacteria
Bacteria with two distinct morphologies (white and yellow) formed on the LB agar, suggesting that they are different organisms (Figure 3). The Raman spectra of these two bacteria were markedly different, with the yellow bacteria displaying peaks associated with colored pigments commonly found in plants and microorganisms [1].
Principal Component Analysis (PCA) may be suitable for differentiating bacteria with distinct phenotypic features in small bacterial communities, as in this experiment (Figure 4). However, researchers typically develop machine-learning algorithms to detect subtle differences in minor peaks for more detailed characterization.
Field test note
- Using glass petri dishes avoids spectral contributions from plastic.
- Raman spectra of colonies may change after low-temperature storage and extended culturing.
- A video microscope is used with 1064 nm laser excitation to visualize the laser spot
Conclusion
Raman spectroscopy can be used to acquire spectra of bacterial colonies directly from solid culture media. Raman spectra collected with 785 nm excitation provides higher resolution, while excitation at 1064 nm reduces fluorescence from culture media.
Simple bacterial colonies can be differentiated using PCA models, but advanced machine-learning algorithms can be used to characterize more complex microbial communities.
Users can easily export the spectral files from i-Raman instruments for further analysis using BWSpec software or other more advanced machine learning tools.
Reference
- Paret, M. L.; Sharma, S. K.; Green, L. M.; et al. Biochemical Characterization of Gram-Positive and Gram-Negative Plant-Associated Bacteria with Micro-Raman Spectroscopy. Appl Spectrosc 2010, 64 (4), 433–441. DOI:10.1366/000370210791114293
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