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The effect of surface properties and application method on the retention of Pseudomonas aeruginosa on uncoated and titanium-coated stainless steel
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International Biodeterioration & Biodegradation 60 (2007) 74–80 www.elsevier.com/locate/ibiod
The effect of surface properties and application method on the retention of Pseudomonas aeruginosa on uncoated and titanium-coated stainless steel Kathryn A. Whitehead, Joanna Verran Division of Biology, Department of Biology, Chemistry and Health Sciences, Manchester Metropolitan University, Chester St, Manchester M1 5GD, UK Received 28 September 2006; received in revised form 21 November 2006; accepted 23 November 2006 Available online 9 January 2007
Abstract Four commercially available stainless steel surfaces 316 2B, 304 2B, 304 coarse abraded and 304 fine polished with defined surface topographies (Ra 0.3–0.5 mm, Rz 2.5–3.8 mm) and comparable hydrophobicities (85.574.51) were coated with 0.8 mm thick layers of titanium using magnetron sputtering. Coating the surfaces did not alter the surface Ra, Rz or surface hydrophobicity values. Substrata were immersed in a Pseudomonas aeruginosa cell suspension (retention assay), or were spray coated with the same suspension (spray assay). When cells were applied via retention assays, the pattern of surface topography affected the pattern of microbial retention on both uncoated and coated surfaces. After spraying application on 2B surfaces the same trends were noted. However, cells clusters were observed on coarse abraded and fine polished uncoated substrata. When substrata were coated with titanium, cells were more evenly spread across the surface and numbers were higher. This work illustrates that method of application and surface chemistry affects the overall distribution of cells, whereas surface topography influences the pattern of cell retention. This has implications in terms of material selection and cost for industries with an environment where corrosion of the stainless steel is likely to be minimal. r 2006 Elsevier Ltd. All rights reserved. Keywords: Stainless steel; Titanium; Pseudomonas; Microorganisms; Surface topography
1. Introduction Hygienic food contact surfaces may vary in their topography and chemistry, with both factors potentially affecting hygienic status. Stainless steel is the most commonly used food contact material because its properties make it robust and easy to clean (Holah and Thorpe, 1990; Boulange-Petermann, 1996; Boulange-Petermann et al., 1997; Boyd et al., 2001). Finishes applied to stainless steel will have a direct bearing on surface appearance, and the environmental performance of the material. Grade 304 stainless steel (SS304) is found throughout the food production chain, from manufacture to storage, in largescale catering, and domestic kitchens (Driessen et al., 1984; Corresponding author. Tel.: +44 0161 247 1157; fax: +44 0161 247 6365. E-mail address: [email protected] (K.A. Whitehead).
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Lewis and Gilmour, 1987; Boulange-Petermann, 1996). Grade 316 stainless steel (SS316) contains molybdenum which increases resistance to surface pitting in aggressive environments, therefore it is more widely used in the environment (Little et al., 1991). A 2B finish is the most widely used surface finish, since it ensures good corrosion resistance, smoothness and flatness. Since the surface of a 2B finish stainless steel is composed of grains and grain boundaries, grain boundary segregation can affect the composition of the surface of that material, making it heterogeneous (Adams, 1983), thus providing ‘‘microniches’’ of chemistry and topography that may favour microbial retention. Other surface finishes are polished versions of the 2B stainless steels, which are usually used for cosmetic function and improved cleanability. Titanium exhibits well-known properties such as very high resistance to corrosion, low specific weight, very low toxicity, and high biocompatibility (World Health Organisation, 1982).
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Along with the current lowering of the price of titanium such properties suggest that titanium could be considered as a material intended to come into contact with foodstuffs (Feliciani et al., 1998). The Ra value (the arithmetic average height parameter) is generally used as a parameter for comparison of surface roughness (Anonymous, 1988; Boulange-Petermann, 1996; Verran and Maryan, 1997; Flint et al., 2000; Hilbert et al., 2003; Whitehead et al., 2005), but it has been argued that parameters such as 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) (Korber et al., 1997; Mettler and Carpentier, 1998a; Verran and Boyd, 2001) surface features (Mettler and Carpentier, 1998b; Whitehead et al., 2006) and three-dimensional surface images (Whitehead et al., 2005) should also be used. An Ra value of less than 0.8 mm has been ascribed to a hygienic stainless steel surface (Standard ISO/DIS 14159, 1998; Flint et al., 1997), the implication being that surfaces with higher values would be less easy to clean (Verran et al., 2001). In order to assess the propensity of microbial cells to attach to and be retained on test surfaces whilst evaluating cleanability, some thought must be given to the methods employed. In retention assays, surfaces are immersed in cell suspension, removed, rinsed and attached cells are enumerated (Verran et al., 1991; Whitehead et al., 2005). The removal of test pieces from the liquid and the subsequent rinsing step will cause shear which may remove highly attached cells. In effect cell retention is assessed rather than cell attachment, thus mimicking the conditions that might be found during cleaning regimes in the food industry, or where surfaces have been left under liquid. Enumeration of cells remaining, enable comparison of attachment to and retention on surfaces. In order to provide a more standard starting point for comparison of factors affecting removal of cells from a surface, a known concentration of cells may be sprayed onto a surface and dried; cells therefore remain on the surface (Verran et al., 2001) before a cleaning system is applied. This application method might be compared to contact of aerosol, rain, or spray with a surface. Depending on the surface orientation—vertical or horizontal, attached cells within the spray droplets may remain and dry in situ or roll downwards. The aim of this work was to compare the effect of a titanium coating on microbial retention on commercially available stainless steel finishes. The effect of application method of cells to the surfaces was also compared.
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Samples were cleaned by washing in 50% nitric acid, sterile distiled water, then soaking for 2 min in acetone, methanol and ethanol and then were finally rinsed in sterile distiled water and dried by blowing nitrogen gas over the surfaces (Whitehead et al., 2005). This enabled good adhesion of the titanium film onto the stainless steel surface (Whitehead et al., 2004). A number of the cleaned SS316 and SS304 surfaces were coated with a layer of titanium using ion beam assisted magnetron sputtering (Whitehead et al., 2004). Roughness parameters and images were obtained using an AFM (Quesant Instruments, CA, USA) operated in contact mode using silicon nitride tips with a force constant of 0.12 N m1. Surfaces were examined and visualised by scanning electron microscopy (SEM). Dynamic contact angle analysis was carried out using DCA 322-1 (Cahn Instruments, USA). Contact angle measurements of clean, dry substrata were taken in HiPerSolv HPLC grade H2O (BDH, Basingstoke, Hampshire, England).
2.2. Microbiology Stocks of Pseudomonas aeruginosa (NCIMB 10421) were prepared by adding 0.5 ml aliquots of 18 h culture to 1.0 ml glycerol (BDH, Basingstoke, Hampshire, England) and storing at 20 1C, being plated onto nutrient agar monthly. Microorganisms were incubated for 24 h at 37 1C, and maintained at 4 1C on nutrient agar. In preparation for assays a single colony of P. aeruginosa was inoculated into 100 ml nutrient broth and incubated for 18 h with shaking, at 37 1C so cells were in stationary phase. Cells were harvested by centrifugation (3600g for 12 min) and washed 3 times in sterile distiled water. The resultant cell suspension was adjusted to an OD 1.0 at 540 nm corresponding to 6.3570.74 108 cfu ml1.
2.3. Retention assays Retention assays were carried out as previously described (Whitehead et al., 2005). Three replicate test pieces were placed horizontally in a glass Petri dish, to which 25 ml of cell suspension were added, and incubated for 1 h without agitation. Test pieces were removed, rinsed once for 5 s with sterile distiled water and air dried in a microbiological class 2 hood. Retained cells were stained for 2 min using 0.03% acridine orange in 2% glacial acetic acid (Sigma, Poole, Dorset, England), rinsed, and air dried. Substrata plus adherent microorganisms were visualised using epifluorescence microscopy (Nikon Eclipse E600, Nikon, Kingston, Surrey, England). The numbers of cells cm2 was determined.
2.4. Spraying assay The spraying assay was adapted from that described by Verran et al. (2001). Three replicate test pieces were placed onto a stainless steel tray using adhesive gum (Impega, Malaysia). The tray and attached test pieces were placed vertically into a microbiological class 2 hood. 20 ml of bacterial culture was placed into the spray reservoir of a Badger Airbrush (USA), propelled by a Letraset 600 ml liquid gas canister (Esselte Letraset Ltd., Ashford, Kent, UK). The airbrush was set to the finest spray setting, and at a distance of 10 cm the airbrush was passed once from left to right at a speed of 50 mm s1 delivering 0.2 ml of solution over the substrata. This speed was determined using previously timed experiments. Immediately following spraying the tray containing substrata was laid horizontally, and allowed to dry. Cells were stained with 0.03% acridine orange in 2% glacial acetic acid, visualised and enumerated as for the retention assays.
2. Materials and methods 2.1. Substrata SS316 with a 2B finish and SS304 with three different topographies; 2B finish, coarse abraded, and fine polished were used. Stainless steel samples 1 mm thick, in sheets 300 600 mm, were guillotined into 1 1 cm squares.
3. Results 3.1. Substrata SEM images of the uncoated test substrata (Fig. 1) were used to visualise the surface features of the substrata. Using
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Fig. 1. SEM images illustrating surface topography and different features: (a) 316 2B finish stainless steel, (b) 304 2B finish stainless steel, (c) 304 coarse abraded stainless steel, and (d) 304 fine polished stainless steel. Scale ¼ 5 mm.
Fig. 2. 20 20 mm 3D atomic force microscope images illustrating the surface topography and features of uncoated stainless steel: (a) SS316 2B finish (b) SS304 2B finish (c) SS304 coarse abraded and (d) SS304 fine polished finish and (e–h) titanium-coated stainless steel with (e and f) a 2B finish (g) coarse abraded and (h) fine polished finish. (z heights ¼ a) 2.98 mm, (b–d) 2.5 mm, (e–f) 4 mm and (g) 1.24 mm. The z heights are not the same for all images to allow the visualisation of the surface features.
AFM imaging (Fig. 2) and 2-D surface profiles (not shown) it was found that the SS316 and SS304 2B finished surfaces had grains 1–3 mm wide and small and larger grain boundaries 0.2–1 mm deep. Surface features on the coarse abraded SS304 were found to be up to 6 mm wide and 0.5–2 mm in depth, whilst the fine polished stainless steel had surface features of 1–2 mm wide and 0.25 mm deep. The Ra values (Table 1 and Fig. 3a) for the uncoated stainless steels ranged from 0.31 to 0.48 mm and for the titaniumcoated stainless steels from 0.24 to 0.52 mm. Rz values (Fig. 3b) ranged from 2.5 to 2.6 mm (uncoated) and 2.5 to 3.8 mm (titanium coated), respectively. Thus there was little difference between the uncoated and coated samples in terms of roughness descriptors. The surface wettabilities of the stainless steel and titanium-coated stainless steel were found to be in the range of 8574.51 (data not shown), with no noticeable difference between samples. 3.2. Retention assays Microorganisms were retained in grain boundaries on the 2B finish SS316 and SS304 (Fig. 4a and b) rather than
Table 1 Values for Ra and Rz values of uncoated and titanium coated stainless steels Substratum
Ra (mm)
Rz (mm)
316 2B finish stainless steel 304 2B finish stainless steel 304 coarse abraded stainless steel 304 fine polished stainless steel Titanium coated 316 2B finish stainless steel Titanium coated 304 2B finish stainless steel Titanium coated 304 coarse abraded stainless steel Titanium coated 304 fine polished stainless steel
0.32 0.31 0.42 0.48 0.24 0.33 0.52 0.50
2.7 2.7 2.5 2.6 2.5 3.5 3.8 3.5
on the flatter areas of the surface. On the coarse abraded SS304 surfaces, the microorganisms were retained in the large and small surface grooves (Fig. 4c). On the fine polished surfaces, the microorganisms were retained in the surface grooves (Fig. 4d), but the pattern of retention was not as pronounced as on the coarse abraded surface. On the titanium-coated stainless steels, microorganisms were again retained at the grain boundaries on the 316 and 304 2B finish coated samples (Fig. 4e and f). However, on
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a
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b
Fig. 3. (a) Ra and (b) Rz values for uncoated and titanium-coated stainless steels. 3162Bun ¼ 316 2B finish uncoated; 3162BTi ¼ 316 2B finish titanium coated; 3042Bun ¼ 304 2B finish uncoated; 3042Bti ¼ 304 2B finish titanium coated; CAbUn ¼ 304 coarse abraded uncoated; CAbTi ¼ 304 coarse abraded titanium coated; FPolUn ¼ 304 fine polished uncoated; FPolTi ¼ Fine polished titanium coated (n ¼ 3).
Fig. 4. Epifluorescence images of P. aeruginosa following retention assays on (a–d) uncoated and (e–h) titanium-coated stainless steel showing that the pattern of cell retention was determined by the pattern of surface topography. The titanium caused better spreading of cells. (a and e) 316 2B finish; (b and f) 304 2B finish; (c and g) 304 course abraded; and (d and h) 304 fine polished.
the titanium-coated 304 coarse abraded (Fig. 4g) and fine polished (Fig. 4h) stainless steel samples, retained microorganisms were more evenly distributed across the surfaces, although the substratum features were still apparent. Cells were usually present individually rather than in aggregates. There was no relationship found between the Ra (or Rz) values of the substratum and the numbers of microorganisms retained. Following retention assays the number of cells retained on the titanium-coated surfaces was found to be in the same order ( 106 cm2) as for the stainless steels, although generally in higher numbers. There was a significant difference ðpo0:001Þ in the numbers of cells retained on the equivalent uncoated and coated stainless steel surfaces.
brushed and polished stainless steel (Fig. 5c and d); cells were clumped together. As previously on the 316 and 304 2B finish titanium-coated samples, bacteria were attached at grain boundaries (Fig. 5e and f), and on the coarse abraded and fine polished titanium stainless steel surfaces cells were spread out evenly across the surface (Fig. 6g and h). Following spray assays (Fig. 6) the number of cells retained on the titanium-coated surfaces was found to be in the order ( 106 cm2) as for the stainless steels, although cell numbers were only higher on the 2B finish titaniumcoated surfaces. There was no relationship between the Ra (or Rz) values and cell numbers retained. 4. Discussion
3.3. Spray assays As for the retention assays on the uncoated stainless steel surfaces cells were retained in grain boundaries (Fig. 5a and b). A hydrophobic drying effect was apparent on the
The SS316 and SS304 were chosen because of their slight differences in chemistry, and since SS316 is usually used in more aggressive environments. The 2B finish was chosen because of its extensive use in the food industry and
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Fig. 5. Epifluorescence images of P. aeruginosa following spray assays on (a–d) uncoated and (e–h) titanium-coated stainless steel. On the 304 uncoated coarse abraded and fine polished surfaces there is a droplet drying effect evident. Titanium increased cell spreading. (a and e) 316 2B finish; (b and f) 304 2B finish; (c and g) 304 course abraded; and (d and h) 304 fine polished.
700
1.80E+07
-2 cm2 Cells cm
1.60E+07
Ra
600
1.40E+07
500
1.20E+07
400
1.00E+07 8.00E+06
300
6.00E+06
200
4.00E+06
Ra (nm)
Cells cm-2
2.00E+07
100
2.00E+06 Ti Ab C
n
Ti ol FP
n
U ol FP
U Ab C
BT 42
n
30
Bu 62
BU
n 31
42
BT
30
62 31
i
0 i
0.00E+00
Fig. 6. Effect of Ra on the retention of cell numbers (cm2) following spray assays, illustrating that at this level of surface roughness neither Ra nor surface chemistry has an effect on the numbers of cells retained. 3162Bun ¼ 316 2B finish uncoated; 3162BTi ¼ 316 2B finish titanium coated; 3042Bun ¼ 304 2B finish uncoated; 3042Bti ¼ 304 2B finish titanium coated; CAbUn ¼ 304 coarse abraded uncoated; CAbTi ¼ 304 coarse abraded titanium coated; FPolUn ¼ 304 fine polished uncoated; FPolTi ¼ Fine polished titanium coated (n ¼ 60).
domestic kitchens (Driessen et al., 1984; Lewis and Gilmour, 1987; Boulange-Petermann, 1996). The polished surfaces were used because of their use in applications where cosmetic appearance is of importance. The titaniumcoated surfaces were used to determine the effect of a homogeneous chemistry (with surfaces of similar topography and wettability) on cell retention. This work was focused on the effect of surface topography, and method of application of cells to a surface. The Ra values of the uncoated and coated substrata used were below 0.8 mm, the value considered to represent a hygienic food surface (Flint et al., 1997). The topography of the titanium-coated stainless steel did not differ greatly from that of the uncoated stainless steel; hydrophobicities were also comparable. The Ra results for
the 2B finished surfaces are in agreement with those of the manufacturer: data for the coarse abraded and fine polished stainless steels were higher than those of the manufacturer (Avesta Polarit, 2000), probably because the AFM measured across a small area where vertical differences have more effect for brushed rather than grain finishes. Coarse abraded stainless had higher Ra and Rz values than the other stainless steel finishes. There was also a higher variation in the Ra and Rz values for the fine polished finishes. The chemical composition of the stainless steel was determined using energy dispersive X-ray spectroscopy (data not shown), a qualitative method. Both stainless steels contained the usual elements, iron, chromium and nickel, but the SS316 also contained molybdenum as
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expected. However, other chemical elements were detected such as silicon, sulphur, calcium, manganese and carbon on the stainless steel surface. On stainless steel surfaces, contaminants can become trapped in grain boundaries, surface defects and grooves. The small concentrations of elements may be important in terms of microbial retention, since microbes can become attached to the trace chemical elements on the surface of the stainless steel. For this work, it was hypothesised that the presence of trace elements on surfaces might be important, since previous work had shown preferential microbial retention at grain boundaries (Fang et al., 2002). The different chemical composition microniche in comparison to the bulk surface properties may cause microorganisms to behave differently. This feature of heterogeneous surface chemistry may become relevant where surfaces are used in nano-scale applications. However the use of the titanium-coated surface has illustrated that on both the uncoated and coated stainless steels, following retention and spray assays, cell retention followed the pattern of the grain boundaries. Therefore it can be concluded that the pattern of retention seen is an effect of surface topography and not of chemistry, or application method. The retention assay facilitates specific interactions between cell and substratum, and the washing step removes irreversibly attached cells. Thus differences in retained cell numbers should be related to the properties of the cells and surface. Following retention assays on the uncoated and coated stainless steel, in general cells were retained in features as expected (Timperley et al., 1992; Quirynen and Bollen, 1995; Verran and Boyd, 2001). On the uncoated fine polished stainless steel surfaces, the microorganisms were retained in the smaller surface grooves, but the pattern of retention was not as pronounced as on the coarse abraded surface, possibly because there were higher numbers of organisms lodged across the wider surface features. This is feasible since surface features on the coarse abraded SS304 were found to be up to 6 mm wide and 0.5–2 mm in depth, which would accommodate the rod shaped cells, in any orientation. The fine polished SS304 had surface features of 1–2 mm wide and 0.25 mm deep, which may result in a lowered cell binding energy with the surface (Edwards and Rutenberg, 2001; Whitehead et al., 2005; Whitehead and Verran, 2006). However, there were lower numbers of cells counted on the coarse abraded than the fine polished surface; this may be explained if the cells had stacked above one another on the deeper features posing difficulty in visually differentiating and counting cells, resulting in a lower cell count. Following the retention assays there were higher numbers of microorganisms retained on the titanium surfaces than on the stainless steel surfaces, which in this instance suggests the altered surface chemistry is responsible for the increase in cell retention, since there is no difference in the surface topography or wettability of the uncoated and coated substrata. This may be due to the manner in which the cell surface interacts/influenced by substratum chemistry. In
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agreement with our data, Verran et al. (2001) found that at Ra values below 0.8 mm the surface topography did not affect the surface cleanability, but the pattern of retention was heavily affected by surface topography (as was the strength of attachment (Boyd et al., 2002; Whitehead et al., 2006)). Boulange-Petermann et al. (1997) also showed no clear relationship between the number of adhering cells and the average roughness of stainless steel in the range of 0.015–1.04 mm. Other authors have reported similar findings in this Ra range (Barnes et al., 1999; Tide et al., 1999). Thus hygienic finishes behave comparably in terms of microbial retention and cleanability. The spray assay applies a fixed number of cells to a surface, does not include a rinse step, and the cells are dried onto the surface. Therefore, there should be comparable numbers of cells on the surface, providing a useful baseline for comparison of cell removal under a given cleaning procedure. The spray assays generally generated higher numbers of attached cells than retention because the cells applied were not removed/rinsed off the surface. As with the retention assays, bacteria were retained at the grain boundaries of the uncoated and coated SS316 and SS304 2B finish surfaces. However on the uncoated SS304 coarse abraded and fine polished surfaces, cells clustered in droplets, within which they were retained within the brushed features. On the coated surfaces the cells were evenly distributed across all the surfaces. Therefore on the brushed, uncoated stainless steel surfaces the spray method allowed the surface wettability to affect the distribution of the cells during drying, whereas the retention assays did not. Presumably in the retention assays the shear force applied on the removal of substrata from the cell suspension, or following rinsing effectively pulled cells from the surface or into surface features. On all surfaces where grain boundaries were present, these features provided a niche for cell retention. This is in agreement with work by Fang et al. (2002) who reported that bacteria tend to preferentially colonise the grain boundaries of steel in comparison with smoother areas. This may be due to the residual fluid from the spraying or shear pulls the cells into the grain boundaries on drying, an effect absent in the linear features. Although the 2B finish is used in the food industry, a polished or brushed treatment is always applied where it may be presumed that the linear features and/or smoother surfaces are easier to clean. 5. Conclusion Findings form this study illustrate many of the problems associated with the study of microbial retention on surfaces. Although surface topography effects are relatively well defined the surface chemistry and method of cell application to a surface may also play a part in determining the distribution of cells on test substrata and may affect subsequent removal/detachment/cleanability findings. However, despite the method used for cell application this work found that there was no benefit in using grade SS316
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over grade SS304 in terms of cleanability and supports the use of SS304 in industries with an environment where corrosion of the stainless steel is likely to be minimal (dry areas food industry) in place of the more expensive SS316. Acknowledgement The authors wish to give special thanks to John Colligon and Reza Valizadeh for their advice and expertise on magnetron sputtering systems. References Adams, R.O., 1983. A review of the stainless steel surface. Journal of Vacuum Science and Technology A 1, 12–18. Anonymous, 1988. BS1134-1: Assessment of Surface Texture—Part 1: Methods and Instrumentation, British Standards Institute, Milton Keynes, UK. Avesta Polarit, 2000. Stainless steel: steel grades, properties and global standards. Online at: /www.industry.siemens.com/metals.mining/en/S. Barnes, L-M., Lo, M.F., Adams, M.R., Chamberlain, A.L.H., 1999. Effect of milk proteins on adhesion of bacteria to stainless steel surfaces. Applied and Environmental Microbiology 65, 4543–4548. Boulange-Petermann, L., 1996. Processes of bioadhesion on stainless steel surfaces and cleanability: a review with special reference to the food industry. Biofouling 10, 275–300. Boulange-Petermann, L., Rault, J., Bellon-Fontaine, M.-N., 1997. Adhesion of Streptococcus thermophilus to stainless steel with different surface topography and roughness. Biofouling 11, 201–216. Boyd, R.D., Rowe, D., Cole, D., Verran, J., Hall, K.E., Underhill, C., Hibbert, S., West, R., 2001. The cleanability of stainless steel as determined by X-ray photoelectron spectroscopy. Applied Surface Science 172, 135–143. Boyd, R.D., Verran, J., Jones, M.V., Bhakoo, M., 2002. Use of the atomic force microscope to determine the effect of substratum surface topography on bacterial adhesion. Langmuir 18, 2343–2346. Driessen, F.M., De Vries, J., Kingma, F., 1984. Adhesion and growth of thermoresistant Streptococci on stainless steel during heat treatment of milk. Journal of Food Protection 47, 848–852. Edwards, K.J., Rutenberg, A.D., 2001. Microbial response to surface microtopography: the role of metabolism in localised mineral dissolution. Chemical Geology 180, 19–32. Fang, H.H.P., Xu, L-C., Chan, K-Y., 2002. Effects of toxic metals and chemicals on biofilm and biocorrosion. Water Research 36, 4709–4716. Feliciani, R., Migliorelli, D., Maggio, A., Gramiccioni, L., 1998. Titanium: a promising new material for food contact. A study of titanium resistance to some aggressive food stimulants. Food Additives and Contaminants 15, 237–242. Flint, S.H., Bremer, P.J., Brooks, J.D., 1997. Biofilms in the dairy manufacturing plant—description, current concerns and method of control. Biofouling 11, 81–97. Flint, S.H., Brooks, J.D., Bremer, P.J., 2000. Properties of the stainless steel substrate influencing the adhesion of thermo resistant Streptococci. Journal of Food Engineering 43, 235–242. Hilbert, L.R., Bagge-Ravn, D., Kold, J., Gram, L., 2003. Influence of surface roughness of stainless steel on microbial adhesion and corrosion resistance. International Biodeterioration & Biodegradation 52, 175–185.
Holah, J.T., Thorpe, R.H., 1990. Cleanability in relation to bacterial retention on unused and abraded domestic sink materials. Journal of Applied Bacteriology 69, 599–608. Korber, D.R., Choi, A., Woolfaardt, G.M., Ingham, S.C., Caldwell, D.E., 1997. Substratum topography influences susceptibility of Salmonella enteritidis biofilms to sodium phosphate. Applied and Environmental Microbiology 63, 3352–3358. Lewis, S.J., Gilmour, A., 1987. Microflora associated with the internal surfaces of rubber and stainless steel milk transfer pipelines. Journal of Applied Bacteriology 62, 327–333. Little, B., Wagner, P., Mansfield, F., 1991. Microbiologically induced corrosion of metals and alloys. International Material Reviews 36, 253–272. Mettler, E., Carpentier, B., 1998a. Hygienic quality of floors in relation to surface texture. Transactions Institute Chemical Engineers 77C, 90–96. Mettler, E., Carpentier, B., 1998b. Variations over time of microbial load and physicochemical properties of floor materials after cleaning in food industry premises. Journal of Food Protection 61, 57–65. Quirynen, M., Bollen, C.M.L., 1995. The influence of surface roughness and surface free energy on the supra and subgingival plaque formation in man. A review of the literature. Journal Clinical Periodontology 22, 1–14. Standard ISO/DIS 14159, 1998. Safety of machinery—hygiene requirement for the design of machinery. Tide, C., Harkin, S.R., Geesey, G.G., Bremer, P.J., Scholz, W., 1999. The influence of welding procedures on bacterial colonisation of stainless steel weldments. Journal of Food Engineering 42, 85–96. Timperley, D.A., Thorpe, R.H., Holah, J.T., 1992. Implications of engineering design in food industry hygiene. In: Melo, L.F., et al. (Eds.), Biofilms-Science and Technology. Kluwer Academic Publishers, Netherlands, pp. 379–393. Verran, J., Boyd, R.D., 2001. The relationship between substratum surface roughness and microbiological and organic soiling: a review. Biofouling 17, 59–71. Verran, J., Maryan, C.J., 1997. Retention of Candida albicans on acrylic resin and silicone of different surface topography. Journal of Prosthetic Dentistry 77, 535–539. Verran, J., Lees, G., Shakespeare, A.P., 1991. The effect of surface roughness on the adhesion of Candida albicans to acrylic. Biofouling 3, 183–192. Verran, J., Rowe, D.L., Boyd, R.D., 2001. The effect of nanometer dimension topographical features on the hygienic surface of stainless steel. Journal of Food Protection 64, 1183–1187. Whitehead, K.A., Verran, J., 2006. The effect of surface topography on the retention of microorganisms: a review. Transactions of the IchemE, in press. Whitehead, K.A., Colligon, J., Verran, J., 2004. The production of surfaces of defined topography and chemistry for microbial retention studies using ion beam sputtering technology. International Biodeterioration & Biodegradation 54, 143–151. Whitehead, K.A., Colligon, J., Verran, J., 2005. Retention of microbial cells in substratum surface features of micrometer and sub-micrometer dimensions. Colloids and Surfaces B 41, 129–138. Whitehead, K.A., Rodgers, D., Colligon, J., Wright, C., Verran, J., 2006. Use of the atomic force microscope to determine the effect of substratum surface topography on the ease of bacterial removal. Colloids and Surfaces B 51, 44–53. World Health Organisation, 1982. Environmental Health Criteria 24, Titanium, WHO, Geneva.
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The effect of surface properties and application method on the retention of Pseudomonas aeruginosa on uncoated and titanium-coated stainless steel Kathryn A. Whitehead, Joanna Verran Division of Biology, Department of Biology, Chemistry and Health Sciences, Manchester Metropolitan University, Chester St, Manchester M1 5GD, UK Received 28 September 2006; received in revised form 21 November 2006; accepted 23 November 2006 Available online 9 January 2007
Abstract Four commercially available stainless steel surfaces 316 2B, 304 2B, 304 coarse abraded and 304 fine polished with defined surface topographies (Ra 0.3–0.5 mm, Rz 2.5–3.8 mm) and comparable hydrophobicities (85.574.51) were coated with 0.8 mm thick layers of titanium using magnetron sputtering. Coating the surfaces did not alter the surface Ra, Rz or surface hydrophobicity values. Substrata were immersed in a Pseudomonas aeruginosa cell suspension (retention assay), or were spray coated with the same suspension (spray assay). When cells were applied via retention assays, the pattern of surface topography affected the pattern of microbial retention on both uncoated and coated surfaces. After spraying application on 2B surfaces the same trends were noted. However, cells clusters were observed on coarse abraded and fine polished uncoated substrata. When substrata were coated with titanium, cells were more evenly spread across the surface and numbers were higher. This work illustrates that method of application and surface chemistry affects the overall distribution of cells, whereas surface topography influences the pattern of cell retention. This has implications in terms of material selection and cost for industries with an environment where corrosion of the stainless steel is likely to be minimal. r 2006 Elsevier Ltd. All rights reserved. Keywords: Stainless steel; Titanium; Pseudomonas; Microorganisms; Surface topography
1. Introduction Hygienic food contact surfaces may vary in their topography and chemistry, with both factors potentially affecting hygienic status. Stainless steel is the most commonly used food contact material because its properties make it robust and easy to clean (Holah and Thorpe, 1990; Boulange-Petermann, 1996; Boulange-Petermann et al., 1997; Boyd et al., 2001). Finishes applied to stainless steel will have a direct bearing on surface appearance, and the environmental performance of the material. Grade 304 stainless steel (SS304) is found throughout the food production chain, from manufacture to storage, in largescale catering, and domestic kitchens (Driessen et al., 1984; Corresponding author. Tel.: +44 0161 247 1157; fax: +44 0161 247 6365. E-mail address: [email protected] (K.A. Whitehead).
0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2006.11.009
Lewis and Gilmour, 1987; Boulange-Petermann, 1996). Grade 316 stainless steel (SS316) contains molybdenum which increases resistance to surface pitting in aggressive environments, therefore it is more widely used in the environment (Little et al., 1991). A 2B finish is the most widely used surface finish, since it ensures good corrosion resistance, smoothness and flatness. Since the surface of a 2B finish stainless steel is composed of grains and grain boundaries, grain boundary segregation can affect the composition of the surface of that material, making it heterogeneous (Adams, 1983), thus providing ‘‘microniches’’ of chemistry and topography that may favour microbial retention. Other surface finishes are polished versions of the 2B stainless steels, which are usually used for cosmetic function and improved cleanability. Titanium exhibits well-known properties such as very high resistance to corrosion, low specific weight, very low toxicity, and high biocompatibility (World Health Organisation, 1982).
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Along with the current lowering of the price of titanium such properties suggest that titanium could be considered as a material intended to come into contact with foodstuffs (Feliciani et al., 1998). The Ra value (the arithmetic average height parameter) is generally used as a parameter for comparison of surface roughness (Anonymous, 1988; Boulange-Petermann, 1996; Verran and Maryan, 1997; Flint et al., 2000; Hilbert et al., 2003; Whitehead et al., 2005), but it has been argued that parameters such as 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) (Korber et al., 1997; Mettler and Carpentier, 1998a; Verran and Boyd, 2001) surface features (Mettler and Carpentier, 1998b; Whitehead et al., 2006) and three-dimensional surface images (Whitehead et al., 2005) should also be used. An Ra value of less than 0.8 mm has been ascribed to a hygienic stainless steel surface (Standard ISO/DIS 14159, 1998; Flint et al., 1997), the implication being that surfaces with higher values would be less easy to clean (Verran et al., 2001). In order to assess the propensity of microbial cells to attach to and be retained on test surfaces whilst evaluating cleanability, some thought must be given to the methods employed. In retention assays, surfaces are immersed in cell suspension, removed, rinsed and attached cells are enumerated (Verran et al., 1991; Whitehead et al., 2005). The removal of test pieces from the liquid and the subsequent rinsing step will cause shear which may remove highly attached cells. In effect cell retention is assessed rather than cell attachment, thus mimicking the conditions that might be found during cleaning regimes in the food industry, or where surfaces have been left under liquid. Enumeration of cells remaining, enable comparison of attachment to and retention on surfaces. In order to provide a more standard starting point for comparison of factors affecting removal of cells from a surface, a known concentration of cells may be sprayed onto a surface and dried; cells therefore remain on the surface (Verran et al., 2001) before a cleaning system is applied. This application method might be compared to contact of aerosol, rain, or spray with a surface. Depending on the surface orientation—vertical or horizontal, attached cells within the spray droplets may remain and dry in situ or roll downwards. The aim of this work was to compare the effect of a titanium coating on microbial retention on commercially available stainless steel finishes. The effect of application method of cells to the surfaces was also compared.
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Samples were cleaned by washing in 50% nitric acid, sterile distiled water, then soaking for 2 min in acetone, methanol and ethanol and then were finally rinsed in sterile distiled water and dried by blowing nitrogen gas over the surfaces (Whitehead et al., 2005). This enabled good adhesion of the titanium film onto the stainless steel surface (Whitehead et al., 2004). A number of the cleaned SS316 and SS304 surfaces were coated with a layer of titanium using ion beam assisted magnetron sputtering (Whitehead et al., 2004). Roughness parameters and images were obtained using an AFM (Quesant Instruments, CA, USA) operated in contact mode using silicon nitride tips with a force constant of 0.12 N m1. Surfaces were examined and visualised by scanning electron microscopy (SEM). Dynamic contact angle analysis was carried out using DCA 322-1 (Cahn Instruments, USA). Contact angle measurements of clean, dry substrata were taken in HiPerSolv HPLC grade H2O (BDH, Basingstoke, Hampshire, England).
2.2. Microbiology Stocks of Pseudomonas aeruginosa (NCIMB 10421) were prepared by adding 0.5 ml aliquots of 18 h culture to 1.0 ml glycerol (BDH, Basingstoke, Hampshire, England) and storing at 20 1C, being plated onto nutrient agar monthly. Microorganisms were incubated for 24 h at 37 1C, and maintained at 4 1C on nutrient agar. In preparation for assays a single colony of P. aeruginosa was inoculated into 100 ml nutrient broth and incubated for 18 h with shaking, at 37 1C so cells were in stationary phase. Cells were harvested by centrifugation (3600g for 12 min) and washed 3 times in sterile distiled water. The resultant cell suspension was adjusted to an OD 1.0 at 540 nm corresponding to 6.3570.74 108 cfu ml1.
2.3. Retention assays Retention assays were carried out as previously described (Whitehead et al., 2005). Three replicate test pieces were placed horizontally in a glass Petri dish, to which 25 ml of cell suspension were added, and incubated for 1 h without agitation. Test pieces were removed, rinsed once for 5 s with sterile distiled water and air dried in a microbiological class 2 hood. Retained cells were stained for 2 min using 0.03% acridine orange in 2% glacial acetic acid (Sigma, Poole, Dorset, England), rinsed, and air dried. Substrata plus adherent microorganisms were visualised using epifluorescence microscopy (Nikon Eclipse E600, Nikon, Kingston, Surrey, England). The numbers of cells cm2 was determined.
2.4. Spraying assay The spraying assay was adapted from that described by Verran et al. (2001). Three replicate test pieces were placed onto a stainless steel tray using adhesive gum (Impega, Malaysia). The tray and attached test pieces were placed vertically into a microbiological class 2 hood. 20 ml of bacterial culture was placed into the spray reservoir of a Badger Airbrush (USA), propelled by a Letraset 600 ml liquid gas canister (Esselte Letraset Ltd., Ashford, Kent, UK). The airbrush was set to the finest spray setting, and at a distance of 10 cm the airbrush was passed once from left to right at a speed of 50 mm s1 delivering 0.2 ml of solution over the substrata. This speed was determined using previously timed experiments. Immediately following spraying the tray containing substrata was laid horizontally, and allowed to dry. Cells were stained with 0.03% acridine orange in 2% glacial acetic acid, visualised and enumerated as for the retention assays.
2. Materials and methods 2.1. Substrata SS316 with a 2B finish and SS304 with three different topographies; 2B finish, coarse abraded, and fine polished were used. Stainless steel samples 1 mm thick, in sheets 300 600 mm, were guillotined into 1 1 cm squares.
3. Results 3.1. Substrata SEM images of the uncoated test substrata (Fig. 1) were used to visualise the surface features of the substrata. Using
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Fig. 1. SEM images illustrating surface topography and different features: (a) 316 2B finish stainless steel, (b) 304 2B finish stainless steel, (c) 304 coarse abraded stainless steel, and (d) 304 fine polished stainless steel. Scale ¼ 5 mm.
Fig. 2. 20 20 mm 3D atomic force microscope images illustrating the surface topography and features of uncoated stainless steel: (a) SS316 2B finish (b) SS304 2B finish (c) SS304 coarse abraded and (d) SS304 fine polished finish and (e–h) titanium-coated stainless steel with (e and f) a 2B finish (g) coarse abraded and (h) fine polished finish. (z heights ¼ a) 2.98 mm, (b–d) 2.5 mm, (e–f) 4 mm and (g) 1.24 mm. The z heights are not the same for all images to allow the visualisation of the surface features.
AFM imaging (Fig. 2) and 2-D surface profiles (not shown) it was found that the SS316 and SS304 2B finished surfaces had grains 1–3 mm wide and small and larger grain boundaries 0.2–1 mm deep. Surface features on the coarse abraded SS304 were found to be up to 6 mm wide and 0.5–2 mm in depth, whilst the fine polished stainless steel had surface features of 1–2 mm wide and 0.25 mm deep. The Ra values (Table 1 and Fig. 3a) for the uncoated stainless steels ranged from 0.31 to 0.48 mm and for the titaniumcoated stainless steels from 0.24 to 0.52 mm. Rz values (Fig. 3b) ranged from 2.5 to 2.6 mm (uncoated) and 2.5 to 3.8 mm (titanium coated), respectively. Thus there was little difference between the uncoated and coated samples in terms of roughness descriptors. The surface wettabilities of the stainless steel and titanium-coated stainless steel were found to be in the range of 8574.51 (data not shown), with no noticeable difference between samples. 3.2. Retention assays Microorganisms were retained in grain boundaries on the 2B finish SS316 and SS304 (Fig. 4a and b) rather than
Table 1 Values for Ra and Rz values of uncoated and titanium coated stainless steels Substratum
Ra (mm)
Rz (mm)
316 2B finish stainless steel 304 2B finish stainless steel 304 coarse abraded stainless steel 304 fine polished stainless steel Titanium coated 316 2B finish stainless steel Titanium coated 304 2B finish stainless steel Titanium coated 304 coarse abraded stainless steel Titanium coated 304 fine polished stainless steel
0.32 0.31 0.42 0.48 0.24 0.33 0.52 0.50
2.7 2.7 2.5 2.6 2.5 3.5 3.8 3.5
on the flatter areas of the surface. On the coarse abraded SS304 surfaces, the microorganisms were retained in the large and small surface grooves (Fig. 4c). On the fine polished surfaces, the microorganisms were retained in the surface grooves (Fig. 4d), but the pattern of retention was not as pronounced as on the coarse abraded surface. On the titanium-coated stainless steels, microorganisms were again retained at the grain boundaries on the 316 and 304 2B finish coated samples (Fig. 4e and f). However, on
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a
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b
Fig. 3. (a) Ra and (b) Rz values for uncoated and titanium-coated stainless steels. 3162Bun ¼ 316 2B finish uncoated; 3162BTi ¼ 316 2B finish titanium coated; 3042Bun ¼ 304 2B finish uncoated; 3042Bti ¼ 304 2B finish titanium coated; CAbUn ¼ 304 coarse abraded uncoated; CAbTi ¼ 304 coarse abraded titanium coated; FPolUn ¼ 304 fine polished uncoated; FPolTi ¼ Fine polished titanium coated (n ¼ 3).
Fig. 4. Epifluorescence images of P. aeruginosa following retention assays on (a–d) uncoated and (e–h) titanium-coated stainless steel showing that the pattern of cell retention was determined by the pattern of surface topography. The titanium caused better spreading of cells. (a and e) 316 2B finish; (b and f) 304 2B finish; (c and g) 304 course abraded; and (d and h) 304 fine polished.
the titanium-coated 304 coarse abraded (Fig. 4g) and fine polished (Fig. 4h) stainless steel samples, retained microorganisms were more evenly distributed across the surfaces, although the substratum features were still apparent. Cells were usually present individually rather than in aggregates. There was no relationship found between the Ra (or Rz) values of the substratum and the numbers of microorganisms retained. Following retention assays the number of cells retained on the titanium-coated surfaces was found to be in the same order ( 106 cm2) as for the stainless steels, although generally in higher numbers. There was a significant difference ðpo0:001Þ in the numbers of cells retained on the equivalent uncoated and coated stainless steel surfaces.
brushed and polished stainless steel (Fig. 5c and d); cells were clumped together. As previously on the 316 and 304 2B finish titanium-coated samples, bacteria were attached at grain boundaries (Fig. 5e and f), and on the coarse abraded and fine polished titanium stainless steel surfaces cells were spread out evenly across the surface (Fig. 6g and h). Following spray assays (Fig. 6) the number of cells retained on the titanium-coated surfaces was found to be in the order ( 106 cm2) as for the stainless steels, although cell numbers were only higher on the 2B finish titaniumcoated surfaces. There was no relationship between the Ra (or Rz) values and cell numbers retained. 4. Discussion
3.3. Spray assays As for the retention assays on the uncoated stainless steel surfaces cells were retained in grain boundaries (Fig. 5a and b). A hydrophobic drying effect was apparent on the
The SS316 and SS304 were chosen because of their slight differences in chemistry, and since SS316 is usually used in more aggressive environments. The 2B finish was chosen because of its extensive use in the food industry and
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Fig. 5. Epifluorescence images of P. aeruginosa following spray assays on (a–d) uncoated and (e–h) titanium-coated stainless steel. On the 304 uncoated coarse abraded and fine polished surfaces there is a droplet drying effect evident. Titanium increased cell spreading. (a and e) 316 2B finish; (b and f) 304 2B finish; (c and g) 304 course abraded; and (d and h) 304 fine polished.
700
1.80E+07
-2 cm2 Cells cm
1.60E+07
Ra
600
1.40E+07
500
1.20E+07
400
1.00E+07 8.00E+06
300
6.00E+06
200
4.00E+06
Ra (nm)
Cells cm-2
2.00E+07
100
2.00E+06 Ti Ab C
n
Ti ol FP
n
U ol FP
U Ab C
BT 42
n
30
Bu 62
BU
n 31
42
BT
30
62 31
i
0 i
0.00E+00
Fig. 6. Effect of Ra on the retention of cell numbers (cm2) following spray assays, illustrating that at this level of surface roughness neither Ra nor surface chemistry has an effect on the numbers of cells retained. 3162Bun ¼ 316 2B finish uncoated; 3162BTi ¼ 316 2B finish titanium coated; 3042Bun ¼ 304 2B finish uncoated; 3042Bti ¼ 304 2B finish titanium coated; CAbUn ¼ 304 coarse abraded uncoated; CAbTi ¼ 304 coarse abraded titanium coated; FPolUn ¼ 304 fine polished uncoated; FPolTi ¼ Fine polished titanium coated (n ¼ 60).
domestic kitchens (Driessen et al., 1984; Lewis and Gilmour, 1987; Boulange-Petermann, 1996). The polished surfaces were used because of their use in applications where cosmetic appearance is of importance. The titaniumcoated surfaces were used to determine the effect of a homogeneous chemistry (with surfaces of similar topography and wettability) on cell retention. This work was focused on the effect of surface topography, and method of application of cells to a surface. The Ra values of the uncoated and coated substrata used were below 0.8 mm, the value considered to represent a hygienic food surface (Flint et al., 1997). The topography of the titanium-coated stainless steel did not differ greatly from that of the uncoated stainless steel; hydrophobicities were also comparable. The Ra results for
the 2B finished surfaces are in agreement with those of the manufacturer: data for the coarse abraded and fine polished stainless steels were higher than those of the manufacturer (Avesta Polarit, 2000), probably because the AFM measured across a small area where vertical differences have more effect for brushed rather than grain finishes. Coarse abraded stainless had higher Ra and Rz values than the other stainless steel finishes. There was also a higher variation in the Ra and Rz values for the fine polished finishes. The chemical composition of the stainless steel was determined using energy dispersive X-ray spectroscopy (data not shown), a qualitative method. Both stainless steels contained the usual elements, iron, chromium and nickel, but the SS316 also contained molybdenum as
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expected. However, other chemical elements were detected such as silicon, sulphur, calcium, manganese and carbon on the stainless steel surface. On stainless steel surfaces, contaminants can become trapped in grain boundaries, surface defects and grooves. The small concentrations of elements may be important in terms of microbial retention, since microbes can become attached to the trace chemical elements on the surface of the stainless steel. For this work, it was hypothesised that the presence of trace elements on surfaces might be important, since previous work had shown preferential microbial retention at grain boundaries (Fang et al., 2002). The different chemical composition microniche in comparison to the bulk surface properties may cause microorganisms to behave differently. This feature of heterogeneous surface chemistry may become relevant where surfaces are used in nano-scale applications. However the use of the titanium-coated surface has illustrated that on both the uncoated and coated stainless steels, following retention and spray assays, cell retention followed the pattern of the grain boundaries. Therefore it can be concluded that the pattern of retention seen is an effect of surface topography and not of chemistry, or application method. The retention assay facilitates specific interactions between cell and substratum, and the washing step removes irreversibly attached cells. Thus differences in retained cell numbers should be related to the properties of the cells and surface. Following retention assays on the uncoated and coated stainless steel, in general cells were retained in features as expected (Timperley et al., 1992; Quirynen and Bollen, 1995; Verran and Boyd, 2001). On the uncoated fine polished stainless steel surfaces, the microorganisms were retained in the smaller surface grooves, but the pattern of retention was not as pronounced as on the coarse abraded surface, possibly because there were higher numbers of organisms lodged across the wider surface features. This is feasible since surface features on the coarse abraded SS304 were found to be up to 6 mm wide and 0.5–2 mm in depth, which would accommodate the rod shaped cells, in any orientation. The fine polished SS304 had surface features of 1–2 mm wide and 0.25 mm deep, which may result in a lowered cell binding energy with the surface (Edwards and Rutenberg, 2001; Whitehead et al., 2005; Whitehead and Verran, 2006). However, there were lower numbers of cells counted on the coarse abraded than the fine polished surface; this may be explained if the cells had stacked above one another on the deeper features posing difficulty in visually differentiating and counting cells, resulting in a lower cell count. Following the retention assays there were higher numbers of microorganisms retained on the titanium surfaces than on the stainless steel surfaces, which in this instance suggests the altered surface chemistry is responsible for the increase in cell retention, since there is no difference in the surface topography or wettability of the uncoated and coated substrata. This may be due to the manner in which the cell surface interacts/influenced by substratum chemistry. In
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agreement with our data, Verran et al. (2001) found that at Ra values below 0.8 mm the surface topography did not affect the surface cleanability, but the pattern of retention was heavily affected by surface topography (as was the strength of attachment (Boyd et al., 2002; Whitehead et al., 2006)). Boulange-Petermann et al. (1997) also showed no clear relationship between the number of adhering cells and the average roughness of stainless steel in the range of 0.015–1.04 mm. Other authors have reported similar findings in this Ra range (Barnes et al., 1999; Tide et al., 1999). Thus hygienic finishes behave comparably in terms of microbial retention and cleanability. The spray assay applies a fixed number of cells to a surface, does not include a rinse step, and the cells are dried onto the surface. Therefore, there should be comparable numbers of cells on the surface, providing a useful baseline for comparison of cell removal under a given cleaning procedure. The spray assays generally generated higher numbers of attached cells than retention because the cells applied were not removed/rinsed off the surface. As with the retention assays, bacteria were retained at the grain boundaries of the uncoated and coated SS316 and SS304 2B finish surfaces. However on the uncoated SS304 coarse abraded and fine polished surfaces, cells clustered in droplets, within which they were retained within the brushed features. On the coated surfaces the cells were evenly distributed across all the surfaces. Therefore on the brushed, uncoated stainless steel surfaces the spray method allowed the surface wettability to affect the distribution of the cells during drying, whereas the retention assays did not. Presumably in the retention assays the shear force applied on the removal of substrata from the cell suspension, or following rinsing effectively pulled cells from the surface or into surface features. On all surfaces where grain boundaries were present, these features provided a niche for cell retention. This is in agreement with work by Fang et al. (2002) who reported that bacteria tend to preferentially colonise the grain boundaries of steel in comparison with smoother areas. This may be due to the residual fluid from the spraying or shear pulls the cells into the grain boundaries on drying, an effect absent in the linear features. Although the 2B finish is used in the food industry, a polished or brushed treatment is always applied where it may be presumed that the linear features and/or smoother surfaces are easier to clean. 5. Conclusion Findings form this study illustrate many of the problems associated with the study of microbial retention on surfaces. Although surface topography effects are relatively well defined the surface chemistry and method of cell application to a surface may also play a part in determining the distribution of cells on test substrata and may affect subsequent removal/detachment/cleanability findings. However, despite the method used for cell application this work found that there was no benefit in using grade SS316
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over grade SS304 in terms of cleanability and supports the use of SS304 in industries with an environment where corrosion of the stainless steel is likely to be minimal (dry areas food industry) in place of the more expensive SS316. Acknowledgement The authors wish to give special thanks to John Colligon and Reza Valizadeh for their advice and expertise on magnetron sputtering systems. References Adams, R.O., 1983. A review of the stainless steel surface. Journal of Vacuum Science and Technology A 1, 12–18. Anonymous, 1988. BS1134-1: Assessment of Surface Texture—Part 1: Methods and Instrumentation, British Standards Institute, Milton Keynes, UK. Avesta Polarit, 2000. Stainless steel: steel grades, properties and global standards. Online at: /www.industry.siemens.com/metals.mining/en/S. Barnes, L-M., Lo, M.F., Adams, M.R., Chamberlain, A.L.H., 1999. Effect of milk proteins on adhesion of bacteria to stainless steel surfaces. Applied and Environmental Microbiology 65, 4543–4548. Boulange-Petermann, L., 1996. Processes of bioadhesion on stainless steel surfaces and cleanability: a review with special reference to the food industry. Biofouling 10, 275–300. Boulange-Petermann, L., Rault, J., Bellon-Fontaine, M.-N., 1997. Adhesion of Streptococcus thermophilus to stainless steel with different surface topography and roughness. Biofouling 11, 201–216. Boyd, R.D., Rowe, D., Cole, D., Verran, J., Hall, K.E., Underhill, C., Hibbert, S., West, R., 2001. The cleanability of stainless steel as determined by X-ray photoelectron spectroscopy. Applied Surface Science 172, 135–143. Boyd, R.D., Verran, J., Jones, M.V., Bhakoo, M., 2002. Use of the atomic force microscope to determine the effect of substratum surface topography on bacterial adhesion. Langmuir 18, 2343–2346. Driessen, F.M., De Vries, J., Kingma, F., 1984. Adhesion and growth of thermoresistant Streptococci on stainless steel during heat treatment of milk. Journal of Food Protection 47, 848–852. Edwards, K.J., Rutenberg, A.D., 2001. Microbial response to surface microtopography: the role of metabolism in localised mineral dissolution. Chemical Geology 180, 19–32. Fang, H.H.P., Xu, L-C., Chan, K-Y., 2002. Effects of toxic metals and chemicals on biofilm and biocorrosion. Water Research 36, 4709–4716. Feliciani, R., Migliorelli, D., Maggio, A., Gramiccioni, L., 1998. Titanium: a promising new material for food contact. A study of titanium resistance to some aggressive food stimulants. Food Additives and Contaminants 15, 237–242. Flint, S.H., Bremer, P.J., Brooks, J.D., 1997. Biofilms in the dairy manufacturing plant—description, current concerns and method of control. Biofouling 11, 81–97. Flint, S.H., Brooks, J.D., Bremer, P.J., 2000. Properties of the stainless steel substrate influencing the adhesion of thermo resistant Streptococci. Journal of Food Engineering 43, 235–242. Hilbert, L.R., Bagge-Ravn, D., Kold, J., Gram, L., 2003. Influence of surface roughness of stainless steel on microbial adhesion and corrosion resistance. International Biodeterioration & Biodegradation 52, 175–185.
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