Biofilm was grown on stainless steel surfaces (AISI 304 and AISI 316) for 2, 5 and 10 days in slime broth inoculated with Pediococcus pentosaceus and Pseudomonas fragi. The steel surfaces on which biofilm had developed were investigated using conventional plating, quantitative glycocalyx determination and epifluorescence microscopy with image analysis. The results showed that after 10 days of growth the cells of P. fragi were difficult to cultivate from the surface and the growth was detected better by microscopy and image analysis. The slime produced by P. pentosaceus could be demonstrated by epifluorescence image analysis and quantitative glycocalyx measurement. The methods for detecting bacterial growth on surfaces are discussed with reference to conventional plating, image analysis and biofilm formation.