Understanding the effects of culture conditions in bacterial growth: A biochemical perspective using Raman microscopy

Rapid, sensitive and label-free methods to probe bacterial growth irrespective of the culture conditions can shed light on the mechanisms by which bacteria adapt to different environmental stimuli. Raman spectroscopy can rapidly and continuously monitor the growth of bacteria under varied conditions. In this study, the growth of Escherichia coli in Luria broth (nutrient rich conditions) and minimal media with either glucose or glycerol as carbon source (nutrient limiting conditions) is profiled using Raman spectroscopy. Moreover, the study also gives insights into the altered bacterial biochemistry upon exposure to low- (25°C) and high-temperature (45°C) stress. Raman spectral measurement was performed on bulk bacteria cultured under laboratory conditions. A detailed analysis of the spectra as a function of bacterial growth reveals changes in Raman band intensities/area of biomolecules such as DNA, proteins and lipids. We also report five novel ratiometric markers (I₈₃₀ /I₈₁₀ , I₁₁₂₆ /I₁₁₀₀ , I₁₃₄₀ /I₁₄₄₀ , I₁₂₀₇ /I₁₂₄₀ and I₁₅₈₀ /I₁₄₄₀ ) that can identify the phase of growth, independent of the culture condition. Unsupervised multivariate methods like Principal Component Analysis also corroborate the aforementioned markers of growth. Altogether, our findings highlight the potential of Raman spectroscopy in yielding universal biochemical signatures that may be indicative of stress and aging in a growth milieu.