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.

Keywords: Amphibian declines; Batrachochytrium dendrobatidis; Batrachochytrium salamandrivorans; Disease; MTT assay; Pathogen.

Figure 1

A comparison of optical density (OD) measurements at multiple incubation time points and two different concentrations of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in wells containing Batrachochytrium dendrobatidis (Bd). The MTT assay provides a colorimetric measurement of metabolically active cells. A volume of 20 μL of 5 mg/mL MTT added to 100 μL of Bd culture (purple) produced a greater color change than 10 μL (pink). With the addition of 20 μL 5 mg/mL MTT, the assay was maximized after a 2–4-h incubation (Color figure online).

Figure 2

Optical density (OD) measurements in wells of serially diluted Batrachochytrium dendrobatidis (Bd). The wells containing Bd were incubated with 20 μL 5 mg/mL MTT for 2 h. OD readings were directly proportional to Bd densities.

Figure 3

Growth of Batrachochytrium salamandrivorans (Bsal) over time using the MTT assay. Optical density measurements (OD₅₇₀) were collected after incubating Bsal in 20 μL 5 mg/mL MTT (n = 12 randomly selected wells per day). The peak in OD readings coincided with maximum zoospore densities (gray bars) on day 8.

Figure 4

A comparison of optical density (OD) measurements of Batrachochytrium dendrobatidis (Bd) over time with and without incubation with MTT. OD measurements with MTT (solid dark blue, read at an optimum wavelength of 570 nm, n = 8 randomly selected wells per day) were greater than measurements without MTT (dashed light blue, read at an optimum wavelength of 490 nm). Maximum Bd growth was evident on days 10–12 and coincided with maximum zoospore densities on day 10 (gray bars) (Color figure online).