International Biodeterioration & Biodegradation 46 (2000) 99–105www.elsevier.com/locate/ibiod

The use of the atomic force microscope to visualise and measurewear of food contact surfaces

J. Verrana ;∗, D.L. Rowea, D. Coleb, R.D. BoydbaDepartments of Biological Sciences, Manchester Metropolitan University, Chester Street, Manchester M1 5GD UK

bChemistry and Materials, Manchester Metropolitan University, Chester Street, Manchester M1 5GD UK

Accepted 6 June 2000

Abstract

The atomic force microscope (AFM) promises to be a valuable tool for visualising and measuring the wear of food contact surfaces.Samples examined by the AFM require virtually no preparation, and the AFM gives roughness information down to the nanometerscale, enabling description of and discrimination between topographies of surfaces at levels below that normally speci�ed as hygienic(Ra0:8 �m). Stand-alone AFM enables direct and sequential measurement of wear of a surface over time. We have also used theAFM to examine less accessible food contact surfaces indirectly, using dental impression materials. Information gained from wornfood contact surfaces sampled in situ has been used to reproduce typical worn stainless-steel surfaces in vitro, for subsequent realisticand rigorous fouling and cleanability testing, which in turn will provide valuable information on the e�ect of wear on the fouling andcleanability of surfaces on the nanometer scale. c© 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Atomic force microscopy; Surface topography; Surface wear; Food hygiene

1. Introduction

In the food industry, and in the home, a hygienic sur-face needs to be smooth, easy to clean, able to resistwear and retain its hygienic qualities. Stainless steel isthe most common food contact material used in industry,being stable at a variety of temperatures, inert, relativelyresistant to corrosion, and it may be treated mechani-cally or electrolytically to obtain surfaces which are easyto clean (Arnold, 1998; Boulange-Petermann et al., 1997;Lee, 1998; Matilla-Sandholm and Wirtanen, 1992). Ce-ramic tiles are used on oors and on walls (the latterless common in industry), and present a hard vitri�edor glazed surface to the environment (Mettler and Car-pentier, 1998; Notermans et al., 1991; Taylor and Holah,1996). Epoxy resins and polyurethanes are used as groutand oorcoverings. Many plastics are soft, relatively easilyscratched, and for this reason are not preferred for use asfood contact surfaces during processing, although for costand convenience they �nd many uses particularly in thehome as containers, chopping boards, waste bins and other

∗ Corresponding author. Tel.: +44-161-247-1206; fax: +44-161-247-6325.E-mail address: j.verran@mmu.ac.uk (J. Verran).

appliances. More exible (and porous) materials, used asgaskets and conveyors are particularly susceptible to foul-ing and deterioration, and are also more di�cult to clean.These di�erent materials, with varying uses, will all ex-perience wear of di�erent degrees and type. Surface wearwill a�ect the subsequent hygienic and cleanability sta-tus of the surface, as it a�ects the surface topographyand chemistry, thus a measure of the type and degree ofroughness of a surface is of considerable importance.The Ra value, usually stated in micrometer units, is

the most commonly used descriptor of surface roughness(Anon, 1988, 1990, 1995). The value is derived using pro-�lometry, whereby a stylus traverses a surface, perpendic-ular to the lay of the most signi�cant surface topographicalfeatures, producing a tracing of the deviation of the sur-face from a calculated centre line. The Ra is the averagearithmetic departure of the surface pro�le from the cen-tre line. An Ra of 0:8 �m is generally used to describe ahygienic surface (Flint et al., 1997).There are several pro�lometers available, the most com-

mon using a solid stylus with a probe size of 25 �m.Laser pro�lometers have a smaller focal point diameter,approximately 2 �m. Di�erences in probe sizes tend toproduce di�erent Ra values for the same surface (laser-generated Ra values are generally higher than solid stylus

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100 J. Verran et al. / International Biodeterioration & Biodegradation 46 (2000) 99–105

pro�lometers – Taylor et al., 1998a), due to di�erences inability to resolve surface defects. The atomic force micro-scope (AFM) has a sharp tip=probe of some 10 nm diam-eter, attached to a cantilever spring, which is scanned inclose proximity to a sample surface. During scanning, theforces between the tip and the surface cause de ection ofthe cantilever. A laser beam is focused onto the back ofthe cantilever, and by monitoring its movements, an imageof the surface is built up (Allen et al., 1997; Smith, 1999).The AFM has been suggested (Boulange-Petermann, 1996;Boulange-Petermann et al., 1997) as a means for measur-ing roughness on a scale comparable with microorganisms,since the probe is of nanometer dimensions. The AFMmay also be used to examine and discriminate betweensurfaces of roughness levels outside the range of otherpro�lometers (Percival et al., 1998).The aim of this study was to investigate the use of the

AFM for measuring surface roughness and wear on thenanometer scale, in order to produce realistic models forin vitro testing. This was achieved by(i) comparing Ra values for a range of smooth surfaces

obtained from solid and laser stylus pro�lometers and theAFM;(ii) using the AFM to visualise and measure the rough-

ness of stainless-steel surfaces in situ in food processingfactories either directly, or indirectly using dental impres-sion materials;(iii) producing abraded stainless-steel surfaces in vitro

which reproduced the typically worn surface of real foodcontact surfaces, for subsequent fouling and cleanabilitystudies.

2. Materials and methods

2.1. Pro�lometric measurements of smooth testmaterials

Materials exhibiting a wide range of surface char-acteristics (hydrophobicity, hardness, uses in the foodindustry and in the home) were selected. Flat, smoothsurfaces were provided by the supplier, to enable comparisonof surface topography using pro�lometry, AFM (QuesantResolver, Quesant Instruments, CA, USA) and scanningelectron microscopy (Cambridge Stereoscan, Cambridge,UK). They were polytetra uoroethylene and polypropy-lene (Goodfellows, She�eld, UK), polymethylmethacry-late (ICI Acrylics, Darwen), polished and unpolished 316Lstainless steel (Avesta, She�eld, UK), glass (ChanceProper, Worley, UK), and porcelain and vitri�ed tiles(Woolliscroft Tiles, Stoke, UK). All materials were usedas supplied, except the stainless steel, which was polishedusing silicon carbide paper (Buehler, Coventry, UK) ofdecreasing grit size (P240, 400, 600, 1200), and then pol-ished with diamond paste, prior to an ultrasonic wash indetergent and methanol to remove any residual polishing

material. All surfaces were washed in a 50 : 50 mixof isopropylalcohol and cyclohexane (BDH, Poole, UK)to remove any adsorbed impurities before any measure-ments were taken [measurements other than topographicincluded contact angle measurement, X-ray photoelectronspectroscopy (XPS), time of ight secondary ion massspectrometry (ToFSIMS) and electron dispersive analysisof X-rays (EDX), and are not reported here].Pro�lometric measurements were made using solid

(Mitutoyo Surftest, Japan) and laser (Perthen, Mahr, Ger-many) styli. The solid stylus measures 5×0:8 mm dis-tances to produce an average calculation. The laserpro�lometer measures a number of parallel lines, and pro-duces a rudimentary three-dimensional tracing, again with0.8 mm as the sample length; the Ra value is calculatedfrom the central �ve of seven lengths sampled. AFM scanswere made over 40× 40 �m areas. Images were captureddigitally using a computer connected to the microscope,which was used to control the tip position whilst moni-toring the de ection of the cantilever. At least 5 readingswere obtained for each material tested, for each piece ofequipment.

2.2. Examination of worn surfaces in situ

2.2.1. IndirectIn order to examine the real wear of in-use materials,

food contact surfaces in a food processing factory weresampled. Flat, horizontal surfaces representing a range offood contact surfaces were identi�ed. Test surfaces werewiped over with 95% ethanol, or detergent if greasy (sincethe impression material will not polymerise in the pres-ence of greasy material). An approximately square frame,2 cm×2 cm was made using a commercially available ad-hesive non-sticky material (Multi Purpose Tac, Ofrex, UK),and placed on the test surface. The two components of ahigh-resolution impression material (Rowe et al., 1999)(Elite Double, Zhermack, Italy) were pipetted and mixedfor 45 s in a weighing boat (working time 5 min) andthen poured into the frame. After 30 min of setting time,the material was peeled from the surface and the top sur-face was attached to the lid of a Petri dish so that thecontact surface was protected during transport to the lab-oratory. The surface was examined via incident light op-tical microscopy, and also by AFM for visualisation, andRa measurement.

2.2.2. DirectThe Quesant Resolver has a stand-alone attachment

which enables direct examination of test surfaces. Thiswas used to examine horizontal in-use stainless-steel work.

2.3. Production of standard worn surfaces in vitro

From the examination of direct and indirectly obtainedsamples of worn stainless-steel surfaces, a range of Ra

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values were obtained, either at or below the 0:8 �m‘hygiene’ level. The visual and measured information en-abled the production of a range of standard worn stainless-steel surfaces, produced in a reproducible and controlledmanner via the use of silicon carbide paper and quartz gritwith a known and constant load, to give surface topogra-phies mirroring those found in situ. Only the AFM coulddiscriminate between these surfaces. These seven surfaceswere then used for soil and microbial fouling and clean-ability studies (Boyd et al., 2000; Verran et al., 1999;Rowe et al., 1999). Linear and two-dimensional abrasiontechniques were used.

3. Results

3.1. Pro�lometry of smooth test surfaces

Ra values for the smooth test surfaces varied consider-ably (Table 1). For the solid stylus pro�lometer, the highlypolished surfaces were not measurable in terms of Ra val-ues. The laser pro�lometer was able to provide a measure-ment for these surfaces, but it was the same value in eachcase. The AFM was able to di�erentiate between all sur-faces in terms of Ra values on the nanometer scale. TheRa values provided by the three methods are not numer-ically comparable, although the ranking is the same. TheRa values increase with decreasing probe size, re ectingthe greater resolving ability of the laser and AFM probes.Both scanning electron microscopy (SEM) and AFM wereused to visualise the surface. The ceramic surfaces posedsome problems for the AFM, being too rough for the depthof �eld. However, for the smooth surfaces, the AFM pro-vided images which gave more detail of surface featuresthan the SEM (Figs. 1 and 2), and also required lesssample preparation.

3.2. Worn surfaces in situ

The impression material provided a safe and conve-nient means of sampling surfaces with minimal disruption.There is some loss of vertical resolution, but lateral

features are well retained (Rowe et al., 1999). The AFMwas the most simple method for examination of the im-pressions (cryoSEM or environmental SEM had to be usedinstead of SEM because of sample distortion), and also forroughness measurement, since the other styli distorted theimpression material. The AFM provided Ra values below0:8 �m, and visualised surfaces – particularly useful if theRa values were the same, yet the surfaces were not. Figs. 3and 4 show optical micrographs and AFM images of newand used brushed stainless steel. It is clear that use doescause wear of the surface (even though there are compa-rable Ra values), which might facilitate fouling, and makecleaning more di�cult.Direct examination of surfaces using the AFM produces

clearer pictures (Fig. 5), but the sensitivity of the AFM tovibration, and the accessibility of some test surfaces maypreclude its use in many food factories.

3.3. Production of abraded surfaces in vitro

Abrasion techniques of stainless-steel samples were usedto produce a range of Ra values (Table 2) which re ectedthe magnitude of those of surfaces sampled in food pro-cessing factories, and also di�erent topographies. Thesesurfaces were then used in routine soil and microbial foul-ing studies to determine the e�ect of small-scale roughnesson retention and cleanability. Preliminary studies indicatethat for microorganisms, the surface topography did nota�ect the number of cell retained, although it clearly af-fected the pattern of attachment (Figs. 6 and 7), and maywell in uence surface cleanability, to be tested in futurestudies.

4. Discussion

It is generally acknowledged that an increase in substra-tum surface roughness increases the retention of micro-organisms on that surface, but there are several issuesconcerning the degree and type of roughness which haveyet to be resolved. An increase in roughness Ra (from0.02 to 1:24 �m) had a signi�cant e�ect on cell reten-tion (Verran et al., 1991; Taylor et al., 1998a) but the

Table 1Ra values of smooth test surfaces as determined using solid stylus and laser pro�lometry, and atomic force microscopy

Material Surface pro�lometer Laser pro�lometer AFM(�m) (�m) (nm)

PP 0:24± 0:11 0:81± 0:06 73± 22PTFE 0:49± 0:23 1:37± 0:12 72± 31PMMA o=ra 0:02± 0:01 2:2± 0:03Unpolished stainless steel 0:11± 0:08 0:63± 0:01 75± 29Polished stainless steel o=ra 0:06± 0:02 79± 0:6Glass o=ra 0:05± 0:01 1:6± 0:2Porcelain 1:16± 0:21 6:69± 2:27 216± 26aOut of range.

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Fig. 1. AFM (a) and SEM (b) images of polished stainless steel; for polished stainless steel, the AFM better visualised surface defects, and generatesRa values.

Fig. 2. AFM (a) and SEM (b) images of unpolished stainless steel; for unpolished stainless steel grain boundaries are apparent using both methods.

pro�lometric methods used to obtain Ra values were rel-atively crude, and did not facilitate discrimination of sur-faces di�ering only slightly in Ra value. The AFM hasbeen used to examine and measure the topography of wornsurfaces, and of surfaces of new materials used in thefood industry, providing data and information not hithertoavailable on a comparable measurement scale. Ra valueson a nanometer scale, below that given for a hygienicsurface, were generated for a range of in use surfaces, en-abling discrimination between surfaces, and providing the

opportunity for future investigation of the true identity ofa hygienic surface.There have been many problems in this area. In general,

there has been a lack of good correlation between rough-ness measurements and parameters such as cleanability,and microbial adhesion=retention on surfaces (Boulange-Petermann, 1996; Garry et al., 1995; Langeveld et al.,1972; Masurovsky and Jordan, 1958; Taylor et al., 1998b,Verran and Maryan, 1997; Verran et al., 1991). Rough-ness measurements, particularly Ra are statistical values

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Fig. 3. AFM images of impressions of new brushed stainless steel. The surfaces have comparable Ra values.

Fig. 4. AFM images of impressions of used brushed stainless steel. The surfaces have comparable Ra values, but the worn surface has a less evenappearance and more debris is evident.

describing a surface, thus are only truly representative ifthere is a regular topography, as for polished and brushedstainless steel, but not for worn surfaces whose abrasionstend to be of an random nature. This feature is also of im-portance if the area examined is considered, since defectsmay be included or excluded depending on the samplingmethod and area. If wear occurs on a microbiologicalscale, then only the AFM would be able to both measureand visualise the relevant topographic features, and po-tentially relate surface features to fouling and cleanability.

However, it must also be remembered that the Ra rangeof interest will vary with the application under study.For oral surfaces, higher Ra values may be relevant(Quirynen, 1998; Taylor et al., 1998b; Verran and Maryan,1997; Verran, 1998). For water distribution systems, sig-ni�cant microbiological accumulations form on major de-fects easily visible to the naked eye. For hygienic surfaces,with low Ra values, further information on the surface to-pography, such as the depth and width of indentationsrather than the Ra; or the use of cross-power spectrum

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Fig. 5. AFM image of worn stainless steel, produced from direct exam-ination of a work surface in a catering hall.

Table 2Range of Ra values (in nm) for surfaces created in vitro for use as“standard” worn surfaces in subsequent fouling and cleanability studies

Roughening agent Ra value

(1 �m) Diamond paste 1:9± 0:1(3 �m) Diamond paste 3:0± 0:7(9 �m) Diamond paste 14± 2p1200 wet and dry (15 �m) 199± 54p600 wet and dry (26 �m) 188± 23p240 wet and dry (60 �m) 500± 60

Fig. 6. Epi uorescence microscopy of microorganisms retained onstainless-steel surfaces after a gentle washing procedure. Test surfaceswere immersed in a standardised cell suspension for 1 h prior to re-moval. Cells (Staphylococcus aureus) on unpolished 316 stainless steel.There is some evidence of retention along grain boundaries, and a sur-face scratch is also evident.

analysis to mathematically describe the dominant surfacefeatures (Boyd et al., 2000) may assist in determiningfeatures which a�ect hygiene and cleanability.One might postulate that there is a threshold for rough-

ness (type and degree) below which no fouling occurs(Freeman et al., 1990). In oral microbiology, it has beenstated that below an Ra of 0:2 �m, a further decrease inroughness does not result in an additional reduction in

Fig. 7. Epi uorescence microscopy of microorganisms retained onstainless-steel surfaces after a gentle washing procedure. Test surfaceswere immersed in a standardised cell suspension for 1 h prior to re-moval. Cells on stainless steel abraded with P240 grade emery paper.Retention in surface defects is evident.

plaque formation (Bollen et al., 1996; Quirynen et al.,1996), suggestive of a “threshold surface” roughnessabove which bacterial adhesion will be facilitated (Bollenet al., 1997). The AFM allows us to extend these �nd-ings, and investigate the problem on the nanometer scale,to begin to determine the features of a true hygienic sur-face.

Acknowledgements

This work was funded via the MAFF-LINK programme,Advanced Food Hygiene and Manufacture. Partners areUnilever Research, Zeneca, British Nuclear Fuels Limited,Pilkington Tiles and John L Lord. The work is carriedout with Campden and Chorleywood Food Research As-sociation, with whom in situ samples were taken. Someanalytical work is subcontracted to the Centre for SurfaceMaterials Analysis, Manchester.

References

Allen, S., Davies, M.C., Roberts, C.J., Tendler, S.J.B., Williams, P.M.,1997. Atomic force microscopy in analytical biotechnology. Tibtech15, 101–105.

Anon, 1988. BSI 1134Anon, 1990. BSI 1134Anon, 1995. ASME B46. 1Arnold, J.W., 1998. Development of bacterial bio�lms during poultryprocessing. Poultry and Avian Biology 9, 1–9.

Bollen, C.M.L., Papaioannou, W., Van Eldere, J., Schepers, E.,Quirynen, M., van Steenberghe, D., 1996. The in uence of abutmentsurface roughness on plaque accumulation and peri-implant mucositis.Clinical Oral Implants Research 7, 201–211.

Bollen, C.M.L., Lambrechts, P., Quirynen, M., 1997. Comparison ofsurface roughness of oral hard materials to the threshold surfaceroughness for bacterial plaque retention: a review of the literature.Dental materials 13, 258–269.

Boulange-Petermann, L., 1996. Processes of bioadhesion on stainlesssteel surfaces and cleanability: a review with special reference to thefood industry. Biofouling 10, 275–300.

J. Verran et al. / International Biodeterioration & Biodegradation 46 (2000) 99–105 105

Boulange-Petermann, L., Rault, J., Bellon-Fontaine, M.-N., 1997.Adhesion of Streptococcus thermophilus to stainless steel withdi�erent surface topography and roughness. Biofouling 11, 201–216.

Boyd, R.D., Cole, D., Rowe, D.L., Verran, J., Coultas, S.J., Paul, A.J.,West, R.H., Goddard, D.T., 2000. Surface characterisation of glassand poly(methylmethacrylate) soiled with a mixture of fat, oil andstarch. Journal of Adhesion Science and Technology, in press.

Flint, S.H., Bremer, P.J., Brooks, J.D., 1997. Bio�lms in dairymanufacturing plant – description, current concerns and methods ofcontrol. Biofouling 11, 81–97.

Freeman, W.B., Middis, J., Muller-Steinhangen, 1990. In uence ofaugmented surfaces and of surface �nish on particulate fouling indouble pipe heat exchangers. Chemical Engineering Processes 27, 1–11.

Garry, P., Andersen, T., Vendeuvre, J.L., Bellon-Fontaine, M.-N., 1995.In uence de la rugosite de surfaces en polyurethane sur l’adhesionde Bacillus subtilis et Bacillus cereus. In: Bellon-Fontaine, M.-N.,Fourniat, J. (Eds.), Adhesion des microorganismes aux surfaces:Bio�lms – Nettoyage – Desinfection. Technique et Documentation,Paris, pp. 21–30.

Langeveld, L.P.M., Bolle, A.C., Vegter, J.E., 1972. The cleanability ofstainless steel with di�erent degrees of surface roughness. NetherlandMilk Dairy Journal 42, 149–154.

Lee, J., 1998. Bacterial bio�lms less likely on electropolished steel.Agricultural Research February, 19–21.

Masurovsky, E.B., Jordan, W.K., 1958. Studies on the relative bacterialclenability of milk-contact surfaces. Journal of Dairy Science 41,1958.

Matilla-Sandholm, T., Wirtanen, G., 1992. Bio�lm formation in theindustry: a review. Food Reviews International 8, 573–603.

Mettler, E., Carpentier, B., 1998. Variations over time of microbial loadand physicochemical properties of oor materials after cleaning infood industry premises. Journal of Food Protection 61, 57–65.

Notermans, S., Dormans, J.A.M.A., Mead, G.C., 1991. Contribution ofsurface attachment to the establishment of microorganisms in foodprocessing plants: a review. Biofouling 5, 21–36.

Percival, S.L., Knapp, J.S., Wales, D.S., Edyvean, R., 1998. Physicalfactors in uencing bacterial fouling on type 304 and 316 stainlesssteels. British Corrosion Journal 33, 121–129.

Quirynen, M., 1998. Intra-oral de novo plaque formation. In: Busscher,H.J., Evans, L.V. (Eds.), Oral Bio�lm and Plaque Control. HarwoodAcademic Publishers, Amsterdam, pp. 193–204.

Quirynen, M., Bollen, C.M.L., Papaioannou, W., Van Eldere, J.,Van Steenberghe, D., 1996. The in uence of titanium abutmentssurface roughness on plaque accumulation and gingivitis. Short termobservation. The International Journal of Oral and MaxillofacialImplants 11, 169–178.

Rowe, D.L., Boyd, R.D., Cole, D., Verran, J., 1999. Determination ofthe resolving potential of impression materials using Atomic ForceMicroscopy. Journal of Dental Research 78, 1071.

Smith, A., 1999. Atomic force microscopy. Microbiology Today 26, 54–55.

Taylor, J.H., Holah, J.T., 1996. A comparative evaluation with respect tothe bacterial cleanability of a range of wall and oor surface materialsused in the food industry. The Journal of Applied Bacteriology 81,262–266.

Taylor, R.L., Verran, J., Lees, G.C., Ward, A.J.P., 1998a. Thein uence of substratum topography on bacterial adhesion topolymethylmethacrylate. Journal of Materials Science: Materials inMedicine 9, 17–22.

Taylor, R.L., Maryan, C., Verran, J., 1998b. Retention of oralmicroorganisms on cobalt–chromium alloy and dental acrylic resinwith di�erent surface �nishes. The Journal of Prosthetic Dentistry 80,592–597.

Verran, J., Boyd, R., Rowe, D., Cole, D., 1999. Topography of foodcontact surfaces and soil retention. In: Wimpenny, J, Gilbert, P,Walker, J, Brading, M, Bayston, R (Eds.), Bio�lms: the good, thebad and the ugly. Cardi�: Bioline, pp. 287–293.

Verran, J., Lees, G., Shakespeare, A.P., 1991. The e�ect of surfaceroughness on the adhesion of Candida albicans to acrylic. Biofouling3, 183–192.

Verran, J., 1998. Denture plaque, denture stomatitis and the adhesionof Candida albicans to inert materials. In: Busscher, H.J., Evans,L.V. (Eds.), Oral Bio�lm and Plaque Control. Harwood AcademicPublishers, Amsterdam, pp. 175–191.

Verran, J., Maryan, C., 1997. Retention of Candida albicans on acrylicand silicone of di�erent surface topography. Journal of ProstheticDentistry 77, 535–539.