Combining imaging techniques for studying biofouled stainless steel surfaces

Stainless steel is regarded as the most hygienic surface material in the food and beverage industry due to the fact that it is inert, stable and easy to clean (1). Stainless steel is usually finished by mechanical grinding, lapping and electrolytic or mechanical polishing. The effect of the finishing with regard to biofouling and cleanability is not fully understood. Surface topography may facilitate more firm attachment by providing more contact points and increased area at the microorganism/material interface. Retention of cells after cleaning may be the consequence of protection from shear forces in the surrounding environment. Surface roughness is traditionally defined with the Ra value. However, the Ra value is a statistical value describing a surface with regular topography and does not directly correlate with cleanability or the fouling potential of stainless steel (2). As food contact surfaces are likely to be contaminated by a mixture of organic debris, cleaning agent residues and microorganisms, a method that could to detect all fouling components would be useful. Unfortunately no single method detects both microbial fouling and organic residues on surfaces (3). Thus a combination of complementary methods should be used for a more realistic view. In this study, direct epifluorescence microscopy (DEM), atomic force microscopy (AFM) and laser profilometry were applied to visualise surface structures and biofilm on stainless steel. Contact angle measurements and microbiological cultivation were used to complement the findings. The stainless steel (AISI 304) surfaces studied were 2B (cold rolled, Ra 0.16 um), 4N (wet polished, Ra 0.17 um), DB (dry brushed, Ra 0.07 um) and BA (bright annealed, Ra 0.03 um). The surfaces were exposed to a mixture of pioneer biofilm microorganisms and examined after biofilm formation and after caustic cleaning. Laser profilometric studies performed in the reflectance mode showed that there were clear irregularities present at the steel surfaces. However, epifluorescence microscopy was needed to confirm that the irregularities were of biological origin. Both laser profilometry, DEM and AFM showed the bacteria attached to the groves of the unidirectional 4N and DB surfaces. Microbiological analyses confirmed that live cells were present also after cleaning. Wetting characteristics of the surfaces covered by biofilm and after cleaning were studied by contact angle measurements. Although the results obtained by the different methods supported each other, no method alone would give sufficient information concerning biofouling and cleaning. Therefore, we conclude that complementary methods should be used to study the effect of surface structures on hygienic features. Acknowledgements This work was founded through Project 478/03 by the National Technology Agency (Tekes), by the Finnish malting and brewing research organisation PBL, by Outokumpu Stainless Oy, by Elomatic Pharmaceutical Engineering Oy, by Noiro Oy, Farmos and by Tankki Oy. References [1] L. Boulang-Peternann. Biofouling 10 (1996), p. 275 [2] J. Verran and R.D. Boyd. Biofbuling 17 (2001), p. 59 [3] J. Verran, R.D. Boyd. K.E. Hall and R. West. Biofouling 18 (2002), p. 167