Methodological modifications, particularly the use of different fixatives, may account for discrepancies between studies of the relation between virulence and biofilm production in vitro by isolates of coagulase negative staphylococci. The efficacy of formalin and Bouin's reagent for fixing coagulase negative staphylococcal biofilms in a microtitre tray assay was compared. The optical density of stained adherent growth by three strains was reduced by an average of 20% following fixation with 10% formaldehyde compared with Bouin's reagent. This difference seemed to be mainly because of increased background staining and blackening of the biofilm when Bouin's reagent was used. Formalin fixation was also effective at identifying early and late biofilm production in adherence growth kinetic experiments with 10 coagulase negative staphylococcal clinical isolates.