False positives in Biolog EcoPlatesTM and MT2 MicroPlatesTM caused by calcium

•The effects of divalent cations on Biolog MicroPlateTM wells were tested.•Calcium inhibits dissolution of the minimal growth medium, causing false positives.•The calcium effect is non-linear, occurring at concentrations greater than 100 ppm.•We advise testing the calcium concentration of samples prior to inoculation.•We suggest a visual check that color development supports high absorbance values.

Biolog MicroPlatesTM (e.g. EcoPlateTM, MT2 MicroPlateTM, GN MicroPlateTM) are useful tools for characterizing microbial communities, providing community-level physiological profiles to terrestrial and aquatic ecologists. The more recently designed Biolog EcoPlates have been used frequently in aquatic ecology with success. This study, however, reveals one major problem when using EcoPlates to evaluate samples within an estuarine or seawater matrix. At concentrations greater than 100 parts per million, the cation calcium begins to interfere with the microplate chemistry, causing false positive readings. Experiments, in which multiple treatments of natural and artificial seawater were tested, as well as calcium-addition experiments, demonstrate that calcium inhibits complete dissolution of the minimal growth medium in wells. Future studies involving Biolog EcoPlates and MicroPlates should take this effect into account, and the dilution of samples is strongly recommended to diminish the “calcium effect.”