Quantifying Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans Viability

Abstract

The disease chytridiomycosis is responsible for global amphibian declines. Chytridiomycosis is caused by Batrachochytrium dendrobatidis ( Bd ) and B. salamandrivorans ( Bsal ), fungal pathogens with stationary and transmissible life stages. Establishing methods that quantify growth and survival of both life stages can facilitate research on the pathophysiology and disease ecology of these pathogens. We tested the efficacy of the MTT assay, a colorimetric test of cell viability, and found it to be a reliable method for quantifying the viability of Bd and Bsal stationary life stages. This method can provide insights into these pathogens’ growth and reproduction to improve our understanding of chytridiomycosis.

Thermal Performance Curves of Multiple Isolates of Batrachochytrium dendrobatidis, a Lethal Pathogen of Amphibians

“…We included five negative control wells with 50 µL of 50 × 10 4 zoospores/mL heat-killed zoospores and 50 µL of TGhL media for each isolate (48). We filled the perimeter wells of the plate with 150 µL TGhL media to provide a buffer against culture evaporation (45).…”

“…It is generally thought that Bd has a thermal tolerance range of 2-28 • C (42), with a T opt of 17-25 • C (8, 43), and CT min, and CT max of 2-5 and 25-28 • C, respectively (42,44). Mounting evidence suggests that Bd isolates differ in their thermal optima (8,42,44), but experimental approaches have not yet explored this idea by comparing isolates collected across a latitudinal gradient (45). We predicted that the TPCs of Bd isolates collected along a latitudinal gradient would differ due to thermal constraints in each region.…”

Daily fluctuating temperatures decrease growth and reproduction rate of a lethal amphibian fungal pathogen in culture

“…We used an MTT assay, a colorimetric test of cell metabolic activity, to quantify differences in culture viability over time among temperature treatments [33]. The MTT assay differentiates between living and dead cells and captures the decay phase of culture growth, providing additional growth metrics that optical density measurements cannot detect [33]. Temperature altered culture metabolic activity, with differences in maximum metabolic activity among all temperature treatments ( Fig.…”

“…We used the ratio of zoospores to culture OD per well as a metric for culture fecundity (n = 6 treatment −1 day −1 ). We used an MTT assay to measure zoosporangia metabolic activity and viability (n = 8 treatment −1 day −1 ; [33]). The MTT assay is a colorimetric test of cell viability in which the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is reduced to purple MTT-formazan crystals in metabolically active cells [34,42].…”

A rapid and inexpensive viability assay for zoospores and zoosporangia of Batrachochytrium dendrobatidis

“…Zoosporangial growth can be estimated by measuring optical density (OD) over time (Rollins-Smith et al, 2002); however, this method cannot distinguish between live and dead cells. Recently, an optimized MTT assay has been developed which enables more accurate measurements of zoosporangial growth and viability (Lindauer et al, 2019). MTT stains viable cells purple due to the reduction of the reagent by cell metabolic activity, and can be quantified using spectrophotometry (Mosmann, 1983).…”