Evaluation of the additive effects of volatile fatty acids and moderate heat treatment for enhancing the inactivation of vegetative cells and spores of Clostridium perfringens by flow cytometry.

OBJECTIVES: Clostridium perfringens is a well-known spore-forming bacterium that can resist the environment. A mixture of volatile fatty acids or thermal treatments can interact with these bacteria. The aim of this study was to evaluate the effects of different volatile fatty acid concentrations and moderate heat treatment on Clostridium perfringens sporulation. METHODS: A pure culture of Clostridium perfringens type A in Duncan Strong medium was treated with a mixture of volatile fatty acids at several concentrations. A thermal treatment was also tested. To evaluate the effects, a double staining method was employed, and treatments on Clostridium perfringens were analysed by flow cytometry. RESULTS: Moderate heat treatment destroyed vegetative forms but had no effect on sporulating forms. Volatile fatty acids combined with moderate heat treatment inhibited Clostridium perfringens sporulation. CONCLUSIONS: The use of flow cytometry as an original method for evaluating the treatment of Clostridium perfringens is of interest because of its simplicity, short time to obtain results, and the level of information provided on the microbial population (impact on metabolism). A combination of mild treatments (moderate heat treatment + volatile fatty acids) to decrease the Clostridium perfringens concentration when these bacteria sporulate is a very promising finding for inhibiting Clostridium perfringens propagation.

A computational two-photon fluorescence approach for revealing label-free the 3D image of viruses and bacteria.

Current solutions for bacteria and viruses identification are based on time-consuming technics with complex preparation procedures. In the present work, we revealed label-free the presence of free viral particles and bacteria with a computational two-photon fluorescence (C-TPF) strategy. Six bacteria were tested: Escherichia coli, Staphylococcus epidermidis, Proteus vulgaris, Pseudomonas fluorescens, Bacillus subtilis, and Clostridium perfringens. The two families of viral particles were the herpes virus with the cytomegalovirus (CMV, 300 nm of diameter) and the coronavirus with the SARS-CoV-2 (100 nm of diameter). The instrumental and computational pipeline FAMOUS optimized the produced 3D images. The origin of the fluorescence emission was discussed for bacteria regarding to their two-photon excitation spectra and attributed to the metabolic indicators (FAD and NADH). The optical and computational strategy constitute a new approach for imaging label-free viral particles and bacteria and paves the way to a new understanding of viral or bacterial ways of infection.