![]() 10 Currently, the most widespread technique to determine spore viability is culture growth on agar plates with rich media, 11,12 which is a very robust but time-consuming process needing 48 hours of incubation for clinically relevant species like C. 9 Conversely, a spore may be able to germinate but be non-viable and unable to proceed to cell division. 8 Also, monitoring metabolic activity is of importance in decontamination and spore viability determination as spores that appear “dead” may, in fact, be viable and merely have delayed or disrupted germination and therefore fully recover in the presence of nutrients. 6 Fluorescent dyes such as DAPI can be used to monitor the viability of cells and spores, however, they can be toxic to cells in the process. Cell metabolomics can be used to exactly identify particular metabolites, 6,7 but this method is very time-, and resource intensive and requires making mutant strains of the studied bacterial species. To understand the mechanisms behind spore resilience, and evaluate spore response to chemicals, robust methods for monitoring spore metabolic activity are needed. Because of their inherent resilience, pathogenic spore-forming bacterial strains cause problems in food production, healthcare, and homeland security. 1,2 Under favourable conditions, however, spores germinate (turn back into metabolically active vegetative cells) and start dividing. Introduction Bacterial spores are a metabolically dormant form of bacteria that are very resilient to natural deterioration and decontamination methods such as heat, cold, electromagnetic radiation, and chemical treatment. In conclusion, our work proposes tracking the evolution of the C–D Raman peak in spores incubated with D 2O-infused broth as an effective and time-, and cost-efficient method to monitor the outgrowth of a spore population, simultaneously allowing us to track for how long the bacteria have grown and divided. This shows the potential for real-time monitoring of metabolic activity from a bacterial spore to a dividing cell. Lastly, the germination and cell growth rate of spores were not affected by adding 30% heavy water to the broth. Further, we found that the peak appearance coincides with the observed first cell division indicating little metabolic activity during germination. We find that a significant C–D peak appears after 2 h of incubation at 37 ☌. During germination and cell division, water is metabolized and deuterium from the broth is incorporated into proteins and lipids, resulting in the appearance of a Raman peak related to C–D bonds at 2190 cm −1. cereus spores undergoing germination and cell division in D 2O-infused broth. Specifically, we monitor the Raman spectrum of enterotoxic B. This work investigates isotope labeling and Raman microscopy as a low-cost rapid alternative. However, current methods for tracking metabolic activity are time-consuming and resource intensive. Therefore, methods to monitor spore metabolic activity and verify sterilization are of great interest. Endospore-forming bacteria are associated with food spoilage, food poisoning, and infection in hospitals.
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