School of Biosciences, Faculty of Science, The University of Melbourne, Parkville, Victoria 3010, Australia.
Dis Aquat Organ. 2023 Sep 14;155:141-146. doi: 10.3354/dao03749.
The emerging fungal pathogen Batrachochytrium dendrobatidis (Bd) threatens hundreds of amphibian species globally. During laboratory-based experiments it is often essential to quantify live Bd cells, but a comparison of the effectiveness of methods for counting and assessing the viability of the infectious zoospore life stage has not been done. A direct comparison of staining methods that assess viability will ensure that the most accurate and efficient method is used. Here, we compared the use of 2 relatively cheap common stains, trypan blue and methylene blue, and assessed their accuracy and precision for estimating the viability of Bd zoospores during both manual counting and colorimetric assays. We stained known proportions of killed Bd zoospores (0, 0.25, 0.50, 0.75, and 1.00) with each stain and estimated the proportion of stained (dead) and unstained (viable) cells in each sample using both manual counting and colorimetric assays. Trypan blue was found to be a much more effective stain than methylene blue for both microscopy and colorimetric assays. Additionally, counting zoospores via microscopy was both a more accurate and precise technique. We recommend using manual counts via microscopy using the trypan blue stain for assessing Bd zoospore viability.
新兴真菌病原体蛙壶菌（Batrachochytrium dendrobatidis，Bd）对全球数百种两栖动物物种构成威胁。在基于实验室的实验中，通常需要定量活的 Bd 细胞，但尚未比较用于计数和评估传染性游动孢子阶段活力的方法的有效性。直接比较评估活力的染色方法将确保使用最准确和高效的方法。在这里，我们比较了两种相对便宜的常用染色方法，台盼蓝和亚甲基蓝，并用它们评估了在手动计数和比色测定过程中估计 Bd 游动孢子活力的准确性和精密度。我们用每种染色剂对已知比例的致死 Bd 游动孢子（0、0.25、0.50、0.75 和 1.00）进行染色，并使用手动计数和比色测定来估计每个样品中染色（死亡）和未染色（存活）细胞的比例。与亚甲基蓝相比，台盼蓝对显微镜和比色测定都更有效。此外，通过显微镜计数游动孢子是一种更准确和更精确的技术。我们建议使用台盼蓝染色通过显微镜手动计数来评估 Bd 游动孢子活力。
