Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids

Abstract

Fungal contamination of metalworking fluids (MWF) is a dual problem in automated processing plants because resulting fungal biofilms obstruct cutting, drilling, and polishing machines. Moreover, some fungal species of MWF comprise pathogens such as Fusarium solani. Therefore, the development of an accurate analytical tool to evaluate conidial viability in MWF is important. We developed a flow cytometric method to measure fungal viability in MWF using F. solani as the model organism. To validate this method, viable and dead conidia were mixed in several proportions and flow was cytometrically analyzed. Subsequently, we assessed the fungicidal activity of two commercial MWF using flow cytometry (FCM) and compared it with microscopic analyses and plating experiments. We evaluated the fungal growth in both MWF after 7 days using quantitative PCR (qPCR) to assess the predictive value of FCM. Our results showed that FCM distinguishes live from dead conidia as early as 5 h after exposure to MWF, whereas the microscopic germination approach detected conidial viability much later and less accurately. At 24 h, microscopic analyses of germinating conidia and live/dead analyses by FCM correlated well, although the former consistently underestimated the proportion of viable conidia. In addition, the reproducibility and sensitivity of the flow cytometric method were high and allowed assessment of the fungicidal properties of two commercial MWF. Importantly, the obtained flow cytometric results on viability of F. solani conidia at both early time points (5 h and 24 h) correlated well with fungal biomass measurements assessed via a qPCR methodology 7 days after the start of the experiment. IMPORTANCE This result shows the predictive power of flow cytometry (FCM) in assessing the fungicidal capacity of MWF formulations. It also implies that FCM can be implemented as a rapid detection tool to estimate the viable fungal load in an industrial processing matrix (MWF).

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

“…mirabilis . Of relevance, such predictive power was recently also described by our group for the antifungal capacity, after incubation of different commercial MWF with Fusarium solani [29].…”

“…There are only a handful of studies describing the use of FCM to evaluate microbial viability in MWF. Recently, our group has developed a novel method that allows the accurate prediction of the fungicidal potential of MWF using FCM viability measurements [29]. This method uses a simple centrifugation protocol to isolate fungal conidia from MWF.…”

“…The LIVE/DEAD BacLight TM dual staining used in the current study has been described for viability analysis of bacterial species in several biological matrices such as seawater, drinking water, soil and food products [45,46,47,48]. Only in a few studies the use of this staining has been reported in MWF for either Mycobacteria [25] or by our group for fungal conidia [29]. Nonetheless, the ability of the SYTO 9/PI staining to differentiate different viability states of bacteria within a bacterial population has been widely described including also reports from our group [30,31,49].…”

“…In the present study, an isolation method of selected Gram-negative bacterial species all relevant in the context of MWF is at first described complementary to our recent report on fungal contamination of MWF [29]. Secondly, this optimized sample preparation was the basis for the subsequent FCM evaluation of real-time bacterial viability using carefully optimised conditions.…”

Brewing yeast viability measured using a novel fluorescent dye and image cytometer

“…Currently, with the emergence of automated cell counters, researchers can analyze a large number of samples faster [15,16] and with less variability stemming from human-error. The development of computers, automation software, optics, fluorescent dyes and precision manufacturing techniques together with modern technologies such as flow cytometry and image cytometry, has helped to overcome many of the drawbacks associated with the hemacytometer [17][18][19][20][21][22][23][24][25].…”