Chemical fixation methods for Raman spectroscopy-based analysis of bacteria

• Chemically fixing biological samples can influence downstream analysis.
• We examined the effect of fixation on the Raman spectra of six species of bacteria.
• Fixation caused significant, species-specific changes in the composition of Raman spectra.
• Sodium azide (NaN3) conserved spectral features best compared to unfixed cells.

Preservation of biological samples for downstream analysis is important for analytical methods that measure the biochemical composition of a sample. One such method, Raman microspectroscopy, is commonly used as a rapid phenotypic technique to measure biomolecular composition for the purposes of identification and discrimination of species and strains of bacteria, as well as investigating physiological responses to external stressors and the uptake of stable isotope-labelled substrates in single cells. This study examines the influence of a number of common chemical fixation and inactivation methods on the Raman spectrum of six species of bacteria. Modifications to the Raman-phenotype caused by fixation were compared to unfixed control samples using difference spectra and Principal Components Analysis (PCA). Additionally, the effect of fixation on the ability to accurately classify bacterial species using their Raman phenotype was determined. The results showed that common fixatives such as glutaraldehyde and ethanol cause significant changes to the Raman spectra of bacteria, whereas formaldehyde and sodium azide were better at preserving spectral features.