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COMPARISON OF SCANNING ELECTRON AND ATOMIC FORCE MICROSCOPY OF SURFACE FINISHES ON STAINLESS STEEL THAT REDUCE BACTERIAL ATTACHMENT
Citation:
Arnold, J. W. AND G W. Bailey. COMPARISON OF SCANNING ELECTRON AND ATOMIC FORCE MICROSCOPY OF SURFACE FINISHES ON STAINLESS STEEL THAT REDUCE BACTERIAL ATTACHMENT. Presented at Scanning 2000 Meeting, San Antonio, TX, May 9-12, 2000.
Impact/Purpose:
Elucidate and model the underlying processes (physical, chemical, enzymatic, biological, and geochemical) that describe the species-specific transformation and transport of organic contaminants and nutrients in environmental and biological systems. Develop and integrate chemical behavior parameterization models (e.g., SPARC), chemical-process models, and ecosystem-characterization models into reactive-transport models.
Description:
Bacteria adhere to food products and processing surfaces that can cross-contaminate other products and work surfaces (Arnold, 1998). Using materials for food processing surfaces that are resistant to bacterial contamination could enhance food safety. Stainless steel, although susceptible to bacterial attachment, is the most frequently used material for construction of equipment used for food processing. Treatments of stainless steel were evaluated for
effectiveness in improving the resistance of the surface finish to bacterial attachment. Two methods of microscopy were partnered to correlate form and function of surface finishes.
Relative differences in the topography and morphology of surface finishes were measured by atomic force microscopy (AFM) and compared with changes in bacterial attachment and early biofilm. formation as shown by scanning electron microscopy (SEM). Natural bacterial populations collected frqrn the food processing environment were assessed for their affinity to attach to surfaces. Aliquots (I - ml) of meat rinses were incubated in trypticase soy broth 18 hr, 3 7'C. Then the culture was diluted in broth to Absorbance (41 Onm) of .3 as measured by spectrophotometry. Stainless steel disks (I -cm diameter) were added, and the cultures were grown to an Absorbance of .6, 37'C. The disks were removed from the bacterial suspensions, rinsed in 0. 1 M sodium cacodylate buffer, and fixed for 2 h in 2% glutaraldehyde, 2% paraformaldehyde in buffer. After rinsing in buffer, the samples were dehydrated in 50
to 100% ethanol and critical point dried. The disks were mounted on aluminum stubs, sputter- coated with gold-palladium, and examined in a JEOL 6400V SEM at an accelerating voltage of
5 W. Triplicate counts of bacterial cells on micrographs were taken from each of two trials covering 10 random fields of view. Beginning biofilm was measured as clumps of cells that showed extensive intercellular fibrils at high magnification SEM (Arnold and Shinikets, 1988a).
After the finishing treatments the surfaces could be distinguished visually. For example, the untreated surface of the 304 stainless steel was smooth and light gray, the sandblasted surface was a darker gray and uniformly pitted, and the electropolished surface was mirror-like and very smooth and shiny. The differences in the surface finishes were confirmed by SEM. Bacteria readily attach to the untreated surface (Fig. a). As bacteria accumulated on the untreated stainless steel, they exhibited typical phenotypic properties of biofilm formation. The sandblasting pitted the surface, and with SEM the pit-marks seen by visual observation appeared as "craters" to which bacteria attached even more frequently (Fig. b). The steel-ball burnished surface was much smoother, but was not the least resistant to bacteria (Figure c). Stainless steel that had been electropolished showed significantly fewer bacterial cells and beginning biofilm formations than the other treated surfaces. The electropolished surface was difficult to image with SEM because the surface was so smooth and featureless (Fig. d). Surface morphological characteristics are determinants for the attachment of bacteria (Arnold and Shimkets, 1988b). Measurements of the surface topography by AFM confirmed and extended the SEM data. The disks for each finishing treatment analyzed by AFM were stamped from the same sheets used for SEM. Disks were removed from the bacterial suspensions described above and examined directly. Changes in AFM surface measurements had the same relative differences as the data from the bacterial counts for the SEM studies, with the electropolished finish showing the most reduced roughness parameters. These data demonstrate that AFM can predict the potential for bacteria to attach and form biofilms on surfaces, without the time-consuming preparations and tedious counting required for SEM.
Appropriate finishing treatments on stainless steel surfaces can improve the resistance to bacterial contamination and thereby enhance food safety during processing. Of course, final selection of surface finishes would be influenced by ftinction and economy. This research has shown that AFM is a rapid method to predict the potential resistance of a surface to bacterial contamination. Further analysis with AFM could enable a manufacturer to develop specifications for production with maximum and minimum tolerances for components at particular locations. In future work, the AFM might be used to determine the importance of bacterial morphology and chemistry for the pathogens associated with surface biofilms. To understand these processes will enable us to develop interventions that enhance plant sanitation and pathogen control.