Development of a rapid and highly sensitive biochemical method for the measurement of fungal spore viability. An alternative to the CFU method

A biochemical method, based on dehydrogenase activity (DRA) measurement, ha s been developed as an alternative to colony forming unit (CFU) enumeration , for assessing the viability of fungal spores. In viable cells, a tetrazol ium salt (MTT) is reduced to a coloured formazan (MTTf) by cellular dehydro genase enzymes. From the colorimetric assay developed by Mosmann for mammal ian cells, the procedure has been adapted and optimised using P. digitatum spores as a model. Propan-2-ol has been selected as the best solvent to ext ract the MTTf` from the. spores. The sensitivity of the method has been con siderably increased by determining the optimal conditions of incubation for the MTT reduction by spores: temperature at 50 degreesC and pH 8. Using th e assay, a correlation has been established between DHA and the number of v iable spores as measured by the CFU method (10(5) CFU = 1.14 DHA -26; r(2) = 0.94). This standardised procedure allows the viability of P. digitatum s pores to be estimated with accuracy and reliability. Moreover, the extensio n of this assay to other strains has been demonstrated (Aspergillus niger a nd Metarhizium flavoviride). (C) 2001 Elsevier Science Inc. All rights rese rved.