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Flow cytometry is a powerful tool for assessment of the viability of fungal conidia in metalworking fluids

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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.
Keywords
MWF, qPCR, microscopy, conidial germination, FCM, viability, SOLID-PHASE CYTOMETRY, METAL-WORKING FLUIDS, FUSARIUM-GRAMINEARUM, QUANTIFICATION, MICROORGANISMS, ASSAY, IDENTIFICATION, MYCOBACTERIA, BIOPROCESSES, ENUMERATION

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Citation

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Chicago
Vanhauteghem, Donna, Kristel Demeyere, N Callaert, A Boelaert, Geert Haesaert, Kris Audenaert, and Evelyne Meyer. 2017. “Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids.” Applied and Environmental Microbiology 83 (16).
APA
Vanhauteghem, D., Demeyere, K., Callaert, N., Boelaert, A., Haesaert, G., Audenaert, K., & Meyer, E. (2017). Flow cytometry is a powerful tool for assessment of the viability of fungal conidia in metalworking fluids. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 83(16).
Vancouver
1.
Vanhauteghem D, Demeyere K, Callaert N, Boelaert A, Haesaert G, Audenaert K, et al. Flow cytometry is a powerful tool for assessment of the viability of fungal conidia in metalworking fluids. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2017;83(16).
MLA
Vanhauteghem, Donna, Kristel Demeyere, N Callaert, et al. “Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids.” APPLIED AND ENVIRONMENTAL MICROBIOLOGY 83.16 (2017): n. pag. Print.
@article{8550297,
  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.},
  articleno    = {e00938-17},
  author       = {Vanhauteghem, Donna and Demeyere, Kristel and Callaert, N and Boelaert, A and Haesaert, Geert and Audenaert, Kris and Meyer, Evelyne},
  issn         = {0099-2240},
  journal      = {APPLIED AND ENVIRONMENTAL MICROBIOLOGY},
  keyword      = {MWF,qPCR,microscopy,conidial germination,FCM,viability,SOLID-PHASE CYTOMETRY,METAL-WORKING FLUIDS,FUSARIUM-GRAMINEARUM,QUANTIFICATION,MICROORGANISMS,ASSAY,IDENTIFICATION,MYCOBACTERIA,BIOPROCESSES,ENUMERATION},
  language     = {eng},
  number       = {16},
  pages        = {11},
  title        = {Flow cytometry is a powerful tool for assessment of the viability of fungal conidia in metalworking fluids},
  url          = {http://dx.doi.org/10.1128/aem.00938-17},
  volume       = {83},
  year         = {2017},
}

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