Summary

Microorganisms and their by-products are present in all workplaces. They are found in airborne suspensions, liquids or surface deposits and can have a significant impact on companies and worker health. Detecting them through classical approaches involving counting on agar media is time-consuming and quite expensive. The purpose of this study is to explore an alternative and innovative approach using fluorescence signals emitted at different wavelengths to detect and differentiate microorganisms in the workplace.

A mobile fluorescence detection system allowing excitations at wavelengths of 266 nm and 355 nm was built by the National Optics Institute (INO). The wavelengths were selected based on a literature review and technical considerations related to the availability of lasers. The first wavelength (266 nm) yields information on microorganism composition and is more suited to microbial classification. The second wavelength (355 nm) can be used to detect biological material and the metabolic state of microorganisms. However, neither wavelength is ideal for analyzing the Raman signatures of microorganisms.

Fluorescence signatures were obtained for the following microorganisms: Staphylococcus epidermidis, Bacillus subtilis, Bacillus megaterium, Escherichia coli, Enterobacter cloacae, Cladosporium cladosporoides, Penicillium digitatum and Ulocladium chartarum. The research focused on 1) pure and mixed suspensions of these microorganisms to imitate those found in the workplace in biofountains and cutting fluids; 2) surface deposits like the ones that can be found in ventilation ducts; 3) localized deposits imitating dried droplets on stainless steel surfaces in food industries, hospitals and elsewhere.

The position of the fluorescence maxima with excitation wavelengths of 266 nm appears to differ among species and can be used to differentiate between them. They are at lower wavelengths in moulds than in bacteria.

The fluorescence derivative analysis developed in this study is an innovative approach not reported in the literature. It shows major characteristics of the fluorescence signal that are hard to see with the naked eye. This signal processing showed fundamental differences between derivatives of the fluorescence signal of Gram – and Gram + bacteria, allowing direct and rapid classification of bacteria based on their fluorescence signature.

Fluorescence signals can be used to instantaneously qualify and quantify the magnitude of dust deposits and their bacterial content as found, for instance, in ventilation ducts. However, the system must be adapted to confirm this finding for deposits formed by drying droplets. The fluorescence produced by dipicolinic acid establishes the presence of spores (bacterial and mycological) in dust deposits. This is a promising substance for the detection of bacterial spores and should be considered in studying the fluorescence of microorganisms.