The purpose of this study was to develop sensitive and accurate methods of quantitating bacterial glycocalyx. Coagulase-negative staphylococci were cultured in trypticase soy broth. Quantitation of slime production was evaluated using various methods of fixation and staining. The amount of dye associated with bacterial slime was assessed spectrophotometrically following solubilization of the dye-biofilm complex by 0.2 M NaOH at 85 degrees C for 1 h. Carnoy's solution was optimal for fixation of the slime, and toluidine blue staining was most reproducible. Fifteen strains of Staphylococcus epidermidis showed a gradation in biofilm production ranging from high, medium, to low that was strain stable. Irrespective of the technique used, high, medium, and low producers of bacterial slime remained in the same category and always showed significantly different optical density readings (p less than 0.05). In our experiments, solubilization of a toluidine blue-bacterial biofilm complex was a direct, simple, and efficient method for reproducibily quantitating glycocalyx. This method provides a useful means of rapid quantitation of biofilm production to assess its role in the infection process and in the response to antibiotic therapy.