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Title: Minimization of Between-well Sample Variance of Antifungal Activity Measurements Using a High-Throughput Screening Microplate Bioassay

Author
item PRYOR, SCOTT - CORNELL UNIVERSITY
item Gibson, Donna
item BERGSTROM, GARY - CORNELL UNIVERSITY
item WALKER, LARRY - CORNELL UNIVERSITY

Submitted to: Biotechniques
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/30/2006
Publication Date: 2/15/2007
Citation: Pryor, S.W., Gibson, D.M., Bergstrom, G.C., Walker, L.P. 2007. Minimization of Between-well Sample Variance of Antifungal Activity Measurements Using a High-Throughput Screening Microplate Bioassay. Biotechniques. 42(2):168-172.

Interpretive Summary: Microplate assays have been useful to rapidly screen materials for biological activity, and we and others have used these methods to identify and quantify antifungal and antibacterial activity in screening programs for novel chemicals. Part of the difficulty of this assay system, however, is that the optical readings are subject to variability due to the variation in microbial growth across the well. We developed am improved method in order to more accurately quantify activity, based on taking into account the variation in optical measurements across the well surface. This technique should be useful for improving the accuracy of optical readings for microplate assays.

Technical Abstract: The use of microplate bioassays, or broth microdilution assays, to measure the activity of biological and synthetic compounds against fungal pathogens has increased in recent years; this technique has been identified as the most promising in vitro bioassay for quantifying antifungal activity. Quantification of fungal growth by spectrophotometric methods can be imprecise, however, because mycelial growth by nature is filamentous and therefore heterogeneous in liquid media. Studies using these methods have shown that there is variability in mycelial growth within a single well but such variability has not been quantified. Sample variability among replicate wells has been reported, but variability of absorbance readings within a single well has not been taken into account. Despite the growing popularity of this type of high-throughput assay and possible limitations because of high within-well variance, sample variability and the introduction of sampling error have received little attention. Significance of within-well variation differs between samples, inhibitor concentrations, and assay test species and can adversely affect assay results particularly for high-throughput screening. This study presents an improvement of a high-throughput screening method presented earlier and based upon a previously published method. The assay was used to quantify the antifungal activity of a B. subtilis biocontrol product against plant pathogenic fungi of the genus Fusarium. The new method allows for more precise quantification of antifungal activity by accounting for variation in optical density within a single well of a 96-well plate.