Vanhauteghem D, Demeyere K, Callaert N, Boelaert A, Haesaert G, Audenaert K, Meyer E
Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Department of Applied Biosciences, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Appl Environ Microbiol. 2017 Aug 1;83(16). doi: 10.1128/AEM.00938-17. Print 2017 Aug 15.
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 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 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 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. 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).
金属加工液（MWF）的真菌污染在自动化加工厂中是一个双重问题，因为由此产生的真菌生物膜会阻塞切割、钻孔和抛光机器。此外，MWF中的一些真菌种类包含病原体，如 。因此，开发一种准确的分析工具来评估MWF中分生孢子的活力很重要。我们开发了一种流式细胞术方法，以 作为模式生物来测量MWF中的真菌活力。为了验证该方法，将活的和死的分生孢子按几种比例混合，并进行流式细胞术分析。随后，我们使用流式细胞术（FCM）评估了两种商用MWF的杀真菌活性，并将其与显微镜分析和平板实验进行了比较。我们使用定量PCR（qPCR）评估了FCM的预测价值，从而评估了7天后两种MWF中的真菌生长情况。我们的结果表明，FCM早在暴露于MWF后5小时就能区分活的和死的分生孢子，而显微镜下的萌发方法检测分生孢子活力的时间要晚得多，且准确性较低。在24小时时，尽管前者一直低估了活分生孢子的比例，但对萌发分生孢子的显微镜分析与FCM的活/死分析相关性良好。此外，流式细胞术方法的重现性和灵敏度很高，可以评估两种商用MWF的杀真菌特性。重要的是，在实验开始7天后，通过qPCR方法评估的分生孢子活力的流式细胞术结果在两个早期时间点（5小时和24小时）与真菌生物量测量结果相关性良好。这一结果显示了流式细胞术（FCM）在评估MWF配方杀真菌能力方面的预测能力。这也意味着FCM可以作为一种快速检测工具来估计工业加工基质（MWF）中活真菌载量。
