Rapid phenotypic identification of microorganisms with Raman

Importance of the Topic

Rapid identification of microorganisms is critical for clinical diagnostics environmental monitoring and bioprocess control Traditional genetic approaches are expensive and time consuming Raman spectroscopy offers a fast nondestructive method to analyze microbial phenotypes providing molecular signatures that reflect biomolecular composition and metabolic state

Objectives and Study Overview

This study demonstrates phenotypic identification of bacterial colonies on solid culture media using portable Raman spectrometers equipped with 785 nm and 1064 nm excitation It evaluates spectral acquisition parameters differentiates distinct colony types and investigates the use of statistical models for classification

Methodology

LB agar was prepared and sterile glass petri dishes were inoculated by pressing fingers onto the surface to transfer bacteria Following room temperature incubation colonies were analyzed directly on the agar using a Raman probe holder and video microscope Spectra were collected at laser powers from 30 to 100 integration times between 3 and 60 seconds and 3 to 5 averages Baseline correction was applied prior to analysis

Instrumentation

• i-Raman Prime 785 portable Raman spectrometer with CCD detector and 785 nm laser
• i-Raman EX portable Raman spectrometer with InGaAs detector and 1064 nm laser
• Raman probe holder (BAC150B) for fiber optic probes
• Video microscope (BAC151C) with 50x objective for sample visualization
• BWSpec software for data acquisition and peak analysis

Main Results and Discussion

Spectra of bacterial colonies displayed characteristic peaks for amino acids DNA and storage polymers Raman excitation at 785 nm produced stronger sharper peaks due to higher scattering efficiency and superior CCD resolution while 1064 nm excitation reduced fluorescence from colored substrates Distinct white and yellow colonies exhibited unique spectral features associated with pigments Principal component analysis effectively separated the two colony types in a simple community model

Benefits and Practical Applications of the Method

This nondestructive approach enables direct analysis of colonies on agar without extensive sample preparation It accelerates bacterial identification and monitoring in research environmental and quality control settings The technique can detect metabolic markers and pigment chemistry providing functional insights

Future Trends and Potential Applications

Advances in machine learning and multivariate algorithms will allow discrimination of closely related species in complex samples Integration with portable imaging platforms may enable in situ screening Automated libraries of Raman signatures could support rapid field diagnostics and environmental surveillance

Conclusion

The application note demonstrates that portable Raman spectroscopy provides a reliable phenotypic identification tool for bacterial colonies on solid media Excitation at 785 nm offers high resolution spectral data while 1064 nm reduces fluorescence Statistical models such as PCA facilitate rapid differentiation Future developments in data analytics and instrumentation promise broader deployment in diverse analytical fields

Reference

1 Paret ML Sharma SK Green LM 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