Effects of Defined Linear Features on Surface Hygiene and Cleanability

Packer, Adele (2012) Effects of Defined Linear Features on Surface Hygiene and Cleanability. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Hygienic food contact surfaces are inert, hard and easy to clean. Aggressive cleaning and disinfection regimes, and general usage and wear, may damage the integrity of the surface, and the resultant defects – pits or scratches – increase the roughness of the surface and potentially affect subsequent cleanability by retaining microorganisms and organic soil. It is generally acknowledged that an increase in surface roughness, often measured using the Ra parameter (the average departure of the surface profile from a centre line) increases the retention of microorganisms on a surface, although feature dimension may also have some influence. The retention of more amorphous organic (food) soil is less affected by the feature dimension, but is likely to be enhanced by any increase in Ra value. The aim of this project was to explore the relationship between surface topography and microbial cell retention on surfaces via the use of surfaces with defined linear features, and with defined chemical properties. Stainless steel is the most commonly used material for hygienic surfaces, but its surface chemistry can be complex. Thus, in order to explore the effect of topography in a controlled manner, test surfaces were coated with titanium, using plasma vapour deposition. A novel impression technique was developed, using acetate softened with acetone pressed against in-use stainless steel surfaces, which when hardened could be removed and examined using atomic force microscopy and scanning electron microscopy. The diameter and profile of typical linear features were measured, enabling model surfaces to be constructed in vitro. Thirty micrometre diameter features were reproduced using nano-indentation, but microorganisms tended to be retained on the edges of the features, rather than within them, because there was accumulation of debris at the edges whose smaller feature size provided increased surface area for microbial retention. Consequently, attention was focused on linear features of microbial dimensions approximating to one and 0.5 micrometer width. These were conveniently obtained by titanium-coating CDs (feature size 1.02 µm, Ra 0.042 µm) and DVDs (feature size 0.59 µm, Ra 0.024 µm) respectively. Escherichia coli did not adhere well to the titanium-coated test surfaces. When stainless steel surfaces were coated with titanium, the same phenomenon was observed: thus it was the surface chemistry rather than topography which reduced microbial retention. In the presence of an organic (meat) soil, retention was again lower on the titanium surface. Thus E.coli was not used in subsequent work, although the potential for titanium coatings to reduce fouling by this species should be explored further. Listeria monocytogenes and Staphylococcus sciuri were used subsequently, representing different shaped microorganisms related to food-borne illness (S.scuiri being related to Staphylococcus aureus). Retention of bacteria on the test surfaces was assessed by incubating cells and surfaces for 1h, gently rinsing, and examining and enumerating retained cells via scanning electron microscopy. Retention was related to cell size and feature size: the spherical staphylococci were preferentially retained on the 1.0 micrometer featured surfaces, being effectively wedged within the features, whilst L.monocytogenes was preferentially retained on the 0.5micrometer featured surfaces, because cell-surface contact was maximised by the increased density of ‘peaks’ on the surface, with the rod-shaped cells lying across and along the linear features. Epifluorescence microscopy was attempted, after staining attached cells with acriding orange, but the relationship between cells and surface features could not be visualised. The strength of attachment rather than the amount of attachment was measured using atomic force microscopy, by application of an increasing lateral force on attached cells, and assessment of the number of scans required to remove cells. Results were similar to those obtained in retention assays, with the S. sciuri retained in highest numbers on the 1 µm features and the least on the 0.5 µm features, emphasising the importance of the relationship between cell size and feature size. Again E.coli could not be used, since it did not adhere: when combined with organic material, the AFM probe could not be used. A more realistic physical removal strategy was applied via repeated physical ‘wipes’ with a mechanised device and water, subsequent to fouling of surfaces with soil, or cells, or a cell-soil mixture. Different fluorescent stains were applied that stained either soil or cells, enabling differential analysis of the area of a microscopic field covered by cells or soil. Whether there was a single fouling event, or sequential fouling-cleaning events, increasing wipes removed increasing amounts of cells and/or soil, and wipes applied along surface features were more effective at removal than wipes applied across the features. Results have revealed that the relationship between cell size and linear feature width and orientation is key to determining whether or not cells are retained on surfaces: the Ra value is of less importance. The direct relationship that is often proposed to exist between Ra value and cell retention is only likely to be true within particular ranges: if the surface features are larger than microbial cells, the cells may not be retained; similarly with features smaller than the diameter of cells. If features are of microbial dimensions, then enhanced retention might be anticipated. Organic food soil is more heterogeneous, thus is retained in features irrespective of feature size, although removal is improved from larger features. Thus rather than merely measuring the Ra, it would appear to be important to assess feature dimension in relation to the size of the microorganism of concern in a given environment. The range of methods used in this study have helped interpretation of a complex interaction between cells, soil, surface and its topography and chemistry. The work described will be useful for exploring these phenomena further, and in the assessment of the effectiveness of putative novel antimicrobial surfaces and/or cleaning regimens used in different environments.