Towards Environmental Microbial Analysis with Deep UV fluorescence and Raman Spectroscopy

Abstract:

The study of microbes from the environment is often facilitated by the fixation of samples prior to analyses in the laboratory. Samples not appropriately preserved can show dramatic changes e.g. unwanted growth, loss of biomass and sample degradation between collection and analysis. To move Deep-UV Raman analyses from model lab organisms to environmental samples the effect of preservation must be evaluated.

Deep UV Raman and Fluorescence (i.e. excitation <250 nm) has been shown capable discriminating various types of microbes as well as giving some information on the growth stage of the culture. The fluorescence signal is typically 3-4 orders of magnitude more intense than the Raman signal and enables rapid location of bacteria on a surface and crudely split them into categories. However it suffers from broad spectral features making discrete classification of bacteria problematic. While a far weaker phenomenon, the chemical specificity of Raman spectroscopy has been shown capable of discriminating between different bacterial species and has even shown spectral variation in same species under differing growth conditions or growth stages and has even been used to measure microbial activity by measuring the incorporation of stable isotope labeled substrates. Typically these analyses are carried out on well-studied, lab-grown model organisms and while relatively easy, these analyses are performed on cells grow under non-environmentally relevant conditions using rich media types not often found in nature.

Here we show the effect on the Raman and fluorescence signal (248 nm Deep-UV excitation) from E. coli and other bacteria, grown in more nutrient limited environments, and fixed/preserved in ethanol, PFA and formalin. These fixatives not only preserve the cells for spectroscopic analysis but are compatible with many common techniques that can be used for further characterization of environmental microbial samples. Ethanol appears to heavily degrade the signals from both Raman and fluorescence while formalin and PFA do not. Our ultimate goal is to create an analytical “pipeline” using the Deep UV fluorescence and Raman techniques on environmental samples in concert with other non-destructive (e.g. Green 548_ex nm Raman) and destructive techniques (e.g. SEM, SIMS and 16S and metagenomic community analyses).