Rapid quantitative laboratory test methods are needed to screen potential antifungal agents. Existing laboratory test methods are relatively time consuming, may require specialized test equipment and rely on subjective visual ratings. A quantitative, colorimetric micro-assay has been developed that uses XTT tetrazolium salt to metabolically assess mold spore germination. Using isothiazolinone as a model mold inhibitor, the minimum inhibitory concentration (MIC) for the XTT assay was compared with the 4 week ASTM plate method (D4445) to test fungicides for controlling mold and the 8 week AWPA environmental chamber method (E24) for evaluating wood product surfaces to mold growth. Quantitative values for the 2-day XTT assay estimated the same MIC for isothiazolinone (50 ppm) for all three test fungi. For the ASTM and AWPA methods, there was a sharp cutoff at a MIC of 50 ppm and 100 ppm isothiazolinone, respectively that was sustained for 12 weeks. While XTT and ASTM methods utilized the same test fungi, the AWPA method exposed treated specimens to a broad range of mold spores naturally present in unsterilized soil so a higher MIC might be expected. Because the XTT assay is conducted in a microplate, it saves resources and reagents in addition to time. The XTT microplate assay can be used to demonstrate the relative resistance of different mold species to individual antifungal agents, to compare the relative inhibition of a new antifungal agent to existing antifungal agents, or to screen new wood preservative formulations for their susceptibility to mold growth.
Clausen, Carol A.; Yang, Vina W. 2014. Accelerated Colorimetric Micro-assay for Screening Mold Inhibitors. V.W. 2014. Accelerated colorimetric micro-assay for screening mold inhibitors. Proceedings, 108th Annual Meeting of the American Wood Protection Association, April 29 – May 2, 2012. Nashville, TN. 108: pp. 150-155. 2014.