Raman Spectroscopy Compatible Inactivation Method for Pathogenic Endospores

Micro-Raman spectroscopy is a fast and sensitive tool for the detection, classification, and identification of biological organisms. The vibrational spectrum inherently serves as a fingerprint of the biochemical composition of each bacterium and thus makes identification at the species, or even subspecies level possible. Therefore, microorganisms in areas susceptible to bacterial contamination, e.g., clinical environments or food-processing technology can be sensed. Within the scope of point-of-care-testing also a detection of intentionally released biosafety level (BSL) 3 agents, like Bacillus anthracis endospores, or their products, is attainable. However, as a matter of fact, no Raman spectroscopic compatible inactivation method for the notoriously resistant Bacillus endospores has been elaborated so far. Within this work we present an inactivation protocol of endospores that permits on one hand a sufficient microbial inactivation and allows on the other hand a subsequent recording of Raman spectroscopic signatures of single endospores making a species-specific identification by means of highly sophisticated chemometrical methods possible. Several physical and chemical inactivation methods were assessed and eventually a treatment with 20% formaldehyde proved to be superior to the other methods in terms of sporicidal capacity and information conservation in the Raman spectra. The latter fact has been verified by successfully applying self-learning machines (like support vector machines or artificial neural networks) to identify B. anthracis related, inactivated endospores with adequate accuracies within the range of the employed limited model database