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Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry
Published on: December 7, 2019
Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids
D Vanhauteghem1, K Demeyere1, N Callaert1
1Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
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).
Insights
Flow cytometry (FCM) offers a rapid and accurate method for detecting fungal viability in metalworking fluids (MWF). This method predicts the fungicidal capacity of MWF, aiding in controlling fungal contamination in industrial settings.
Area of Science:
- Industrial Microbiology
- Analytical Chemistry
- Mycology
Background:
- Fungal contamination of metalworking fluids (MWF) causes significant issues in automated processing, including machine obstruction by biofilms.
- Some MWF fungi, like Fusarium solani, are pathogenic, necessitating accurate viability assessment.
- Current methods for evaluating fungal viability in MWF are often slow and less accurate.
Purpose of the Study:
- To develop and validate a flow cytometric method for measuring fungal viability in MWF.
- To assess the fungicidal activity of commercial MWF formulations.
- To establish the predictive value of flow cytometry for fungal load in MWF.
Main Methods:
- Development of a flow cytometry (FCM) assay using Fusarium solani conidia.
- Validation by mixing viable and dead conidia in various proportions for FCM analysis.
- Comparison of FCM with microscopic analysis and plating for viability assessment.
- Evaluation of MWF fungicidal activity and correlation with quantitative PCR (qPCR) for fungal biomass.
Main Results:
- FCM accurately distinguished live from dead conidia as early as 5 hours post-exposure to MWF.
- Microscopic methods detected viability later and less accurately than FCM.
- FCM results at 5 and 24 hours correlated well with fungal biomass measured by qPCR after 7 days.
- The FCM method demonstrated high reproducibility and sensitivity in assessing MWF fungicidal properties.
Conclusions:
- Flow cytometry is a rapid, sensitive, and accurate tool for assessing fungal viability in MWF.
- FCM can predict the fungicidal capacity of MWF formulations.
- FCM can be implemented as an industrial tool for estimating viable fungal load in MWF.
