Research Article: Flow cytometry, a powerful novel tool to rapidly assess bacterial viability in metal working fluids: Proof-of-principle

Date Published: February 1, 2019

Publisher: Public Library of Science

Author(s): Donna Vanhauteghem, Kris Audenaert, Kristel Demeyere, Fred Hoogendoorn, Geert P. J. Janssens, Evelyne Meyer, Song Liu.


Metalworking fluids (MWF) are water- or oil-based liquids to cool and lubricate tools, work pieces and machines, inhibit corrosion and remove swarf. One of the major problems in the MWF industry is bacterial growth as bacterial enzymes can cause MWF degradation. In addition, bacteria can form biofilms which hamper the functioning of machines. Last but not least, some bacterial by-products are toxic (e.g. endotoxins) and present potential health risks for metalworking machine operators via the formation of aerosols. Therefore, a novel fast yet accurate analytical method to evaluate and predict the antibacterial capacity of MWF would be an important asset. As such a tool is currently lacking, the present study aimed to develop a protocol based on flow cytometry (FCM) to assess the antibacterial potential of newly developed MWF independent of bacterial growth. Results of this novel method were compared to a biochallenge test currently used in MWF industry and also to traditional plate counts. Our results represent a proof-of-principle that FCM can reliably predict the antibacterial capacity of MWF already within one day of incubation with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis, being substantially faster than the current growth-based methods.

Partial Text

The metalworking industry utilizes recirculating metalworking fluids (MWF) to cool, remove metal fines, lubricate and prevent corrosion during metal grinding and cutting procedures [1]. Metalworking fluids are complex mixtures of oils, biocides, dissolved metals, antifoaming agents and many other organic and inorganic components [2,3]. These are formulated to improve longevity of equipment, but their formulation also makes them highly prone to physical, chemical and especially microbial contamination [4,5]. Uncontrolled microbial growth significantly impacts both the MWF performance and the health of the machine operators [3,6,7]. Indeed, the resulting microbial degradation of MWF can cause corrosion of machines, tools and work pieces, as well as loss of lubricity and fluid stability, and decrease of the fluid pH due to organic acid production. Such contamination also often results in biofilm formation (visible as slime which can plug filters) and in unacceptable odors, but also carries important health risks for operators [8,9]. On the other hand, knowledge on the MWF deterioration capacity of microorganisms may also provide an important solution towards a safe and economical disposal of operationally exhausted MWF [10,11,12].

Results show that the susceptibility to the MWF compounds is predominantly bacterial species-dependent. When the results of an incubation time up to 24h are compared, K. pneumoniae and P. mirabilis are the most resistant to the antimicrobial effects of the MWF matrices. When incubated for 1 week, P. aeruginosa and P. mirabilis appeared to be more resistant to the antimicrobial effects of the MWF. When comparing the different MWF, at 5h of incubation P869 has a greater antibacterial effect, however, after 24h of incubation exposure to P270 results in the highest percentage of dead bacteria. In general, the antibacterial effect of the MWF appears stronger at pH 9.5 compared to pH 9.0, however the observed trends of decreased bacterial viability are comparable at both pH values and there was no statistically significant effect of pH.

This paper provides novel data on the predictive power of FCM in assessing the antibacterial capacity of MWF formulations. The development of an accurate analytical tool to evaluate bacterial viability in MWF is an asset important step to tackle this problem. In the present study we developed a FCM method to measure bacterial viability, based on membrane integrity, in MWF using Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis as model organisms. We compared our novel flow cytometric method with a biochallenge test currently used in MWF industry and also with traditional plate counts. This study is a proof-of-principle which is timely as it meets the current increasing needs of industrial stakeholders and the academic world to extend traditional microbiological test methods with novel high-throughput consensus methods which can later be adopted by industrial stakeholders. The combination of bacterial isolation, fluorescent staining and FCM analysis holds large potential to enhance and greatly accelerate the quantitative and qualitative evaluation of bacterial viability in MWF.




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