Evaluation of in-vitro growth assays for Batrachochytrium dendrobatidis revealed methylene blue colorimetry is the most accurate

Batrachochytrium dendrobatidis (Bd) is a destructive fungal pathogen, having caused the global decline and extinction of numerous amphibian species. Robust methodologies to accurately measure fungal growth in-vitro are needed for experiments aimed at understanding the physiology, pathogenicity, and virulence of this fungus. Despite their importance, no studies have directly compared Bd growth assays or evaluated their performance across different cell concentrations. This study is the first to compare assays and identify the most suitable methods for quantifying Bd growth in-vitro under different experimental conditions. To determine the optimal protocol for measuring the growth of Bd cells, we compared four methods: optical density, BacTiter-Glo™ microbial cell viability assay, methylene blue colorimetry, and microscopic image analysis. The performance of each assay was determined over a range of growing conditions. These were created by applying two stressors (cadmium and hygromycin) on two concentrations of Bd cells and replicated in two different Bd isolates. Our results conclude methylene blue colorimetry as the most reliable technique for measuring Bd growth in-vitro as it consistently yields accurate and precise results across a spectrum of cell concentrations regardless of the stressor or Bd isolate. Imaging and optical density analyses appear moderately accurate and are non-destructive options. BacTiter-Glo™ assay is suitable to assess growth in low cell concentrations. The optimal choice of growth assays depends on experimental designs and available laboratory facilities.