Reproducibility assessment of the crystal violet biofilm assay in Clostridioidesdifficile

Crystal violet staining is widely used assay to quantitate bacterial biofilms. However, reproducibility remains a persistent challenge, particularly for anaerobic pathogens such as Clostridioides difficile. The purpose of this study was to assess the reproducibility of the crystal violet biofilm assay in C. difficile, systematically assessing the impact of microplate surface chemistry, plate format, and isolate-level heterogeneity on assay reproducibility.

Reference strains (CD630 and R20291) and fifteen clinical isolates were grown anaerobically in tissue-culture treated and untreated 24-, 48-, and 96-well microplates. Biofilm biomass was quantified spectrophotometrically after 48h using crystal violet staining. All experiments were performed in three independent biological replicates. Reproducibility was assessed using within-plate and across-experiment coefficients of variation (CV%).

Tissue-culture treated 96-well plates consistently reduced variability relative to untreated plates in within-plate and across-experiment comparisons. Increasing well size in tissue-culture treated plates markedly improved reproducibility, with 24-well plates yielding the lowest CV% values and 96-well plates showing the greatest variability. Linear mixed-effects modeling confirmed these trends in effect direction and showed substantial experiment-to-experiment noise in small-volume plate formats. Mixed-effects variance partitioning across the fifteen isolate panel demonstrated that biological heterogeneity between isolates accounted for ∼20% of total within-plate CV% variance, whereas experiment-level variance was negligible, indicating high day-to-day assay stability under optimized conditions.

Crystal violet assay reproducibility for C. difficile biofilm quantitation is governed by technical parameters and isolate-specific biofilm physiology. Tissue-culture treated, 24-well plates can limit technical variability between experiments.