Retention of microbial cells in substratum surface features of micrometer and sub-micrometer dimensions

Abstract

Surfaces were produced with defined topographical features and surface chemistry. Silicon wafers, and wafers with attached nucleopore filters and quantifoils were coated with titanium using ion beam sputtering technology. Irregularly spaced, but regularly featured surface pits, sizes 0.2 and 0.5 μm, and regularly spaced pits with regular features (1 and 2 μm) diameter were produced. The smallest surface feature that could be successfully produced using this system was of diameter 0.2 μm. Ra, the average absolute deviation of the roughness irregularities from the mean line over one sampling length, Rz, the difference in height between the average of the five highest peaks, and the five lowest valleys along the assessment length of the profile and surface area values increased with surface feature size, with Ra values of 0.04–0.217 μm. There was no significant difference between the contact angles observed for smooth titanium surfaces with 0.2 and 0.5 μm features. However, a significant difference in contact angle was observed between the 1 and 2 μm featured surfaces (p < 0.005).

Substrata were used in microbial retention assays, using a range of unrelated, differently sized microorganisms. Staphylococcus aureus (cells 0.5–1 μm diameter) were retained in the highest numbers. S. aureus was well retained in the 0.5 μm sized pits and began to accumulate within larger surface features. Rod shaped Pseudomonas aeruginosa (1 μm × 3 μm) were preferentially retained, often end on, within the 1 μm surface features. Some daughter cells of Candida albicans blastospores were retained in 2 μm pits. For S. aureus and P. aeruginosa, the greatest numbers of cells were retained in the largest (2 μm) surface features. The number of C. albicans was similar across all the surfaces. The use of defined surfaces in microbial retention assays may lead to a better understanding of the interaction occurring between cells and surface features.

Introduction

Biofilm formation is initiated by the attachment of a conditioning film, and subsequently microorganisms, to substrata. An approach to reduce initial surface contamination, which may lead to subsequent reduction in biofilm formation, is to modify the substratum surface. One factor contributing to the fouling of surfaces is topography [1]. The Ra is a value used to describe the roughness of a surface, and is the average departure of the surface profile from a mean centre line [2]. A value of 0.8 μm has been ascribed to a hygienic stainless steel surface [2], [3], [4], the implication being that surfaces with values above that point would be less easy to clean [5]. Wear of hygienic food contact surfaces through abrasion may affect the surface roughness and introduce different topographical features again in a random manner [5]. Hence, much of this work has been carried out on surfaces that have roughness features randomised across a surface. To understand the fundamental mechanisms influencing bacterial retention it would be desirable to produce surfaces whose features are of appropriate dimensions, but present in a regular and defined pattern. Although a number of studies have been carried out on the effect of surface topography on microbial retention, findings are often conflicting. Some have suggested that there is no relationship between surface roughness (in terms of Ra) and the ability of bacteria to attach [6], [7], [8]. However, others have suggested that the greater the degree of surface roughness, the greater the retention of microorganisms [9], [10], [11], [12]. These results are explained by the ranges of Ra values of the substrata used in these studies. Surface features whose dimensions greatly exceed those of the microorganisms will have little effect on retention [14]. Features of dimensions smaller than cells also have little effect [10], [11], [15]. Presumably, therefore, a minimum end point Ra could be defined. Enhanced bacterial retention on different surface features could be due to an increase in bacterial attachment sites (for a given surface area), leading to stronger bacterial attachment and enhanced protection from cleaning shear forces [13]. The production of surfaces of defined topography and chemistry (SDTC) and their effect on retention should help to clarify these issues.

Surfaces with regularly shaped surface features in the micron range can be produced using compression moulding [16], electron beam lithography [17] and photolithography [18]. Modification of surface chemistry by ion implantation can produce surfaces with a low fouling potential. Implantation of SiF₄⁺ into a stainless steel surface has been shown to reduce fouling and scale formation [19], [20]. However, production of surfaces with defined surface topography and chemistry is technically more difficult. Using a template technique along with physical vapour deposition, surfaces with defined topography and chemistry were produced in our laboratories [21]. Surfaces were coated with titanium since it is known to exhibit properties such as high resistance to corrosion, low specific weight, low toxicity, and high biocompatibility [22]. These properties suggest that titanium could be conveniently used as material intended to come into contact with foodstuffs [23], and it is already widely used in medicine [24], [25], [26]. In the natural environment titanium is coated by a protective oxidative layer.

The effect of substratum topography and chemistry on microbial retention will depend on the type, shape and properties of the microorganism.

The aim of this study was to compare the retention of a range of unrelated microorganisms of different sizes (Staphylococcus aureus; cocci 1 μm in diameter; Pseudomonas aeruginosa; rods 1 μm width × 3 μm length; Candida albicans yeast, oval 4 μm width × 5 μm length) on fabricated surfaces of defined topography (features 0.2, 0.5, 1 and 2 μm in diameter) and chemistry (titanium).