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Comparison of adhesion of the food spoilage bacterium Shewanella putrefaciens to stainless steel and silver surfaces

2002; Oxford University Press; Volume: 92; Issue: 5 Linguagem: Inglês

10.1046/j.1365-2672.2002.01609.x

1365-2672

M. Hjelm, Lisbeth Rischel Hilbert, Per Møller, Lone Gram,

Antimicrobial agents and applications

Journal of Applied MicrobiologyVolume 92, Issue 5 p. 903-911 Free Access Comparison of adhesion of the food spoilage bacterium Shewanella putrefaciens to stainless steel and silver surfaces M. Hjelm, M. Hjelm Department of Manufacturing Engineering and Management, Technical University of Denmark, Lyngby, Denmark,Search for more papers by this authorL.R. Hilbert, L.R. Hilbert Department of Manufacturing Engineering and Management, Technical University of Denmark, Lyngby, Denmark,Search for more papers by this authorP. Møller, P. Møller Department of Manufacturing Engineering and Management, Technical University of Denmark, Lyngby, Denmark,Search for more papers by this authorL. Gram, L. Gram Danish Institute for Fisheries Research, Department of Seafood Research, c/o Technical University of Denmark, Lyngby, DenmarkSearch for more papers by this author M. Hjelm, M. Hjelm Department of Manufacturing Engineering and Management, Technical University of Denmark, Lyngby, Denmark,Search for more papers by this authorL.R. Hilbert, L.R. Hilbert Department of Manufacturing Engineering and Management, Technical University of Denmark, Lyngby, Denmark,Search for more papers by this authorP. Møller, P. Møller Department of Manufacturing Engineering and Management, Technical University of Denmark, Lyngby, Denmark,Search for more papers by this authorL. Gram, L. Gram Danish Institute for Fisheries Research, Department of Seafood Research, c/o Technical University of Denmark, Lyngby, DenmarkSearch for more papers by this author First published: 23 April 2002 https://doi.org/10.1046/j.1365-2672.2002.01609.xCitations: 13 M. Hjelm Danish Institute for Fisheries Research, Department of Seafood Research, c/o Technical University of Denmark, Building 221, DK-2800 Kgs. Lyngby, Denmark (e-mail: meh@dfu.min.dk). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: To compare the number of attached Shewanella putrefaciens on stainless steel with different silver surfaces, thus evaluating whether silver surfaces could contribute to a higher hygienic status in the food industry. Methods and Results: Bacterial adhesion to three types of silver surface (new silver, tarnished silver and sulphide-treated silver) was compared with adhesion to stainless steel (AISI 316) using the Malthus indirect conductance method to estimate the number of cfu cm−2. The number of attached bacteria on new silver surfaces was lower than on steel for samples taken after 24 h. However, this was not statistically significant (P > 0·05). The numbers of attached bacteria were consistently lower when tarnished silver surfaces were compared with stainless steel and some, but not all, experiments showed statistical significance (P < 0·05). Treating new silver with sulphide to reproduce a tarnished silver surface did not result in a similar lowering of adhering cells when compared with steel (P > 0·05). Conclusions: New or tarnished silver surfaces caused a slight reduction in numbers of attached bacteria; however, the difference was only sometimes statistically significant. Significance and Impact of the Study: The lack of reproducibility in differences in numbers adhering to the different surfaces and lack of statistical significance between numbers of adhered viable bacteria do not indicate that the tested silver surfaces can be used to improve hygienic characteristics of surfaces in the food industry. INTRODUCTION An important quality parameter for food and beverage products is to ensure that they do not contain bacteria in amounts that could cause disease in humans or lead the food to perish. Bacteria are a natural part of the raw materials for food production but can also reside in food-processing equipment where they can (re)contaminate food products (20; 8). Ready-to-eat foods are potentially high risk products since they do not receive further heat treatment before consumption. This makes them vulnerable to (re)contamination and, if storage conditions allow further growth of bacteria, they may cause disease in humans (6). Bacteria that reside in food-processing equipment may adhere to surfaces. After being attracted to a surface, the bacteria will produce extracellular polysaccharides (EPS) and will, if not removed, become embedded in a so-called biofilm consisting of EPS, water, cells and nutrients (4). The removal of bacterial biofilms from surfaces can be difficult. Dead ends, crevices, corners, gaskets and joints are vulnerable areas where bacterial biofilms may accumulate because of difficult access during cleaning (18). Inactivation of bacteria is achieved using disinfecting agents. However, attached bacteria, and bacteria embedded in biofilms, are more resistant to disinfectants and heat than planktonic bacteria (10; 33) making clean-in-place disinfecting procedures almost ineffective. Adhesion and biofilm formation of bacteria also cause problems in medical applications. Infections due to the use of invasive medical devices account for nearly half of all hospital-related infections (28). Colonization on the surface of a medical device is a precursor to clinical infection, with possible fatal consequences, and a substantial amount of research has dealt with coating device surfaces with antimicrobial substances such as silver (5). Silver is used alone or in combination with antibiotics on, for example, bladder, vascular or peritoneal catheters and heart valve sewing rings in an attempt to prevent infection (5). Silver and its compounds have been used as antimicrobial agents since 1000 BC (25). In 1967, silver sulphadiazene (AgSD) was introduced as an antiseptic for the treatment of burns (9). AgSD is the most important antimicrobial agent currently in use despite suggestions that other compounds, such as silver acetate, silver nitrate and silver protein, are effective (19). Furthermore, silver derivatives have been used to prevent inflammation of the eyes of new-born babies (25), for disinfecting drinking and swimming pool water (15) and have been incorporated into paint to prevent biofilm formation in water systems (24). Silver is also used in the food industry to maintain purity and freedom from metallic taste and silver equipment is used for handling essential oils, syrups and fruit juices (27). The mechanism of the antimicrobial activity of silver surfaces is unknown. However, it is established that silver ions are toxic to bacteria (12). The minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of electrically generated Ag ions in nutrient broth range from 0·03 to 1·25 μg ml−1 (MIC) and from 0·48 to 10·05 μg ml−1 (MBC) for some bacterial strains (3). In view of these considerations, we decided to investigate the efficacy of silver surfaces in reducing the number of adhered viable bacteria. If effective, silver could substitute stainless steel in food-processing areas where cleaning is difficult. This could lead to improved hygiene and subsequently to a higher quality of food products. The majority of studies on the potential role of silver as an antimicrobial coating relate to medical applications. The aim of this study was to assess the adherence of the food spoilage bacterium Shewanella putrefaciens to different silver surfaces and to compare these results with the more commonly used stainless steel. The study focused on conditions in the food industry where stagnant water occurs, like a dead end, and the model system was designed to reflect these conditions. MATERIALS AND METHODS Stainless steel and silver surfaces Stainless steel surfaces. Stainless steel plates (10 × 20 × 1 mm) were made of sheet SS 2343 (AISI 316) with surface finish 2 B (cold rolled, annealed, pickled and lightly rolled). The composition was as follows (wt %): C, 0·03; Si, 0·49; Mn, 1·7; P, 0·032; S, 0·001; Cr, 16·7; Ni, 10·7 and Mo, 2·5. New silver surfaces. Stainless steel plates were silver plated with a layer (thickness 15 ± 5 μm) of Ag by the following procedure. The plates were cathodically degreased in a cyanide bath (1000 A m−2 for 2 min), activated in dry acid for 20–30 s, wood nickel plated (500 A m−2 for 75–90 s; however, for the initial 15–20 s, an anodic current of 50 A m−2 was applied), plated with a strike layer of silver (100 A m−2 for 60 s) in a silver bath (composition: AgCN, 7 g l−1; KCN, 80 g l−1; K2CO3, 30 g l−1), then bright silver plated in Engbright S bright silver-plating bath (100 A m−2 for 20 min) and finally dried by pressurized air. Bath temperatures were 20–25°C and plates were thoroughly rinsed in distilled water between each bath. In order to obtain an even material deposition and layer thickness the plates were agitated during the processes and, in the bright silver bath, turned over periodically to change the point of contact. The layer thickness and adhesion of the plating were controlled after each run. The surface obtained can be characterized as a dense, pure silver surface. Tarnished silver surfaces. Tarnished silver surfaces were obtained through repeated usage of plates coated with new silver. Thus, tarnish resulted from the general use of plates in adhesion experiments and especially from measurements in the Malthus glass cells, as described below, followed by autoclaving. Cleaning the silver plates, as described below, also added to the tarnishing process. Sulphide-treated silver surfaces. To obtain a sulphide-treated silver surface, silver-plated specimens were cathodically degreased in a cyanide bath, rinsed in distilled water and exposed under agitation to a 5% (NH4)2S solution for 60 s at 40°C. This treatment resulted in a thin silver sulphide layer corresponding to an energy dispersive X-ray analysis (EDX) of approx. 2·5% S. This analysis was mainly for semiquantitative comparisons since the results were affected by a signal from the silver substrate. The rings used as holders for plates during adhesion and biofilm experiments were made from polypropylene to avoid a possible galvanic current between silver and steel plates. Scanning electron microscopy and energy dispersive X-ray analysis of silver surfaces Scanning electron microscopy (SEM) studies and EDX were applied to characterize the physical appearance and composition of the metal surfaces and deposits. In general, weight percentages are given with an accuracy of ± 10%. The EDX analysis values were corrected for penetration depth, absorption and reflection of the different elements. Elements with atomic weights lower than sodium, e.g. carbon and oxygen, cannot be detected. Signals from the substrate affect the analysis for thin deposits (few μm) and, therefore, analyses were carried out at 10 kV to reduce this effect. It was not possible to correctly analyse small heterogeneous deposits. However, a semiquantitative analysis and comparison of the composition of deposits was possible. Adhesion of Shewanella putrefaciens in batch or flow system Adhesion experiments were conducted as described by 2). Cleaning of surfaces and assembly of batch system for adhesion. Stainless steel plates were soaked overnight in 10% Deconex® (Borer Chemie AG, Zuchwil, Switzerland) solution and autoclaved at 121°C for 15 min. Deconex could not be used for silver surfaces as it caused a significant oxidation of the silver. Silver-coated plates were cleaned in a 3% dilution of detergent (Decon Neutracon®, Decon Laboratories Ltd, Hove, UK) suitable for metals that may be corroded by acids or alkaline cleaning agents. Plates were soaked for 1 h, autoclaved at 121°C for 15 min and rinsed immediately. Finally, all plates were degreased in acetone for 30 min. After drying, 20 plates of steel or silver were positioned vertically in a round polypropylene rack in a glass beaker and autoclaved. Preparation of bacterial culture. Shewanella putrefaciens A2 (22) was grown on Iron Agar Lyngby (CM964; Oxoid). One colony was inoculated in 4 ml Tryptone Soya Broth (TSB; CM 129; Oxoid) and, after 24 h at 25°C, 100 μl were transferred to 50 ml TSB and cells grown for 24 h at 25°C (250 rev min−1). Cells were harvested by centrifuging at 1450 g for 10 min and resuspended (without washing) in 4 ml phosphate-buffered saline (PBS; NaCl, 8·0 g; KCl, 0·2 g; Na2HPO4.2H2O, 1·80 g; KH2PO4, 0·24 g suspended in 0·8 l water; pH adjusted to 7·4 and refilled to 1·0 l) leading to a cell concentration of approx. 1 × 1011 cfu ml−1. Adhesion in batch system with buffer. Tryptone Soya Broth (250 ml of one-seventh strength) was added to the beaker and left for a minimum of 30 min (250 rev min−1) to obtain a conditioning layer of organic material on the metal surfaces. The holder and plates were transferred to a new beaker and 250 ml PBS with resuspended S. putrefaciens were added. A magnetic stirrer in the centre of the round rack allowed for slow circulation in the suspension and free circulation around the metal surfaces was ensured by the set-up. The adhesion on metal surfaces was investigated using different initial cell concentrations in the suspension (102–106 cfu ml−1) and plates were sampled at regular intervals between 1 and 170 h to estimate the number of adhering bacteria. The cell concentration in the suspension was determined by total colony counts (cfu ml−1) on Iron Agar Lyngby. Adhesion in batch system with nutrient broth. For experiments in nutrient broth, the same procedure was followed as described in the previous paragraph, except that no initial preconditioning film was used on the plates and 250 ml of a one-seventh strength TSB were added instead of PBS. All experiments were conducted at ambient temperature (20–25°C). Adhesion in flow system with buffer. Adhesion under laminar flow conditions was investigated using a modified Robbins device (MRD; Tyler Research Co-operation, Edmonton, Canada). The set-up was as described by 2). Experiments were conducted with a flow of 10 ml min−1 (equalling 0·006 m s−1). Silver and stainless steel surfaces were inserted on every second place in the MRD and conditioned with one-seventh strength TSB for 30 min. The PBS suspension was recirculated for up to 7 d with an initial cell concentration of 1 × 108 cfu ml−1. Detection of bacterial cells on steel and silver surfaces using the Malthus® instrument Each plate sampled was rinsed gently with 5–10 ml PBS and placed on sterile absorbent paper to remove excess drops and poorly attached bacteria. Care was taken not to swab or rub the plates. The plate was transferred to 3 ml TSB in a Malthus® (2000 instrument; Malthus Instrumental Ltd, West Sussex, UK) glass tube and the number of bacteria on surfaces estimated using the Malthus indirect conductance technique at 25°C. An inner plastic tube in the Malthus glass tube contained 500 μl 0·1 mol l−1 NaOH and electrodes immersed in this solution recorded changes in conductance as a function of time as detected when CO2 from respiring bacteria reacted with OH−. Thus, the conductance of the NaOH solution would decrease as bacteria on the plate proliferated and CO2 was liberated and scavenged in the NaOH solution. The time from the start of the experiment until a detectable change occurred in the conductance (detection time, DT) is inversely proportional to the initial number of bacteria in the Malthus glass tube, i.e. on the plate. The DT was defined when the conductance changed with 2·5 μS h−1 followed by a change of 0·1 μS h−1. A standard curve was made from 10-fold diluted planktonic bacteria, measuring the DT of each individual dilution and plotting DT against log (cfu (Malthus glass)−1). Subsequently, the DT for a metal plate in a Malthus glass tube could be used to estimate the total number of bacteria on the 4-cm2 surface. All samples except one were processed in triplicate. Statistical analysis Cell numbers were log transformed and the difference between numbers on silver surfaces and stainless steel was calculated for each sampling point. Each adhesion experiment was tested for statistical significance of the difference between the two surfaces with the following t-test: where n represents the number of tested paired observations, Δmean is the mean of the differences between pairs of observations (log transformed) and sΔ is the S.D. of the differences. P < 0·05 was considered statistically significant. Numbers of adhering bacteria on the two surfaces were, for selected points, compared by testing for the equality of the means assuming the same variance (13). RESULTS Bacterial adhesion to silver surfaces and stainless steel Shewanella putrefaciens adhered readily to silver surfaces and stainless steel. Four experiments were conducted with low initial cell numbers in the buffer suspension (102–103 cfu ml−1) but, despite limited nutrients, several log increases were observed over 1–2 d. The term `adhesion' is here used to describe the attachment of a monolayer of bacteria on surfaces (typically between 103 and 106 bacteria cm−2). These low levels often occur on food-processing equipment but still have biotransfer potential (14). `Biofilm' is more often used to describe a thick multilayer of bacteria (typically above 108 bacteria cm−2). Bacterial adhesion to new silver surfaces The number of bacteria attached to conditioned steel and new silver surfaces was between 3·3 and 4·9 log (cfu cm−2) in a cell suspension of 106 cfu ml−1, depending on the time the metal surfaces were exposed in the batch system. The numbers of adhered S. putrefaciens (log (cfu cm−2)) were 0·3 log units higher on new silver surfaces than on stainless steel during the first 4 h in the batch system (Fig. 1). After 24 h a change was observed, as the number of bacteria on new silver surfaces was 0·4 log units lower than on steel. The difference in numbers of adhered bacteria on the two types of surface was not, however, statistically significant (P > 0·05). Figure 1Open in figure viewerPowerPoint Number of attached Shewanella putrefaciens on conditioned stainless steel (□) and new silver (•) in phosphate-buffered saline buffer with an initial cell concentration of 1 × 106 cfu ml−1. Experiments were conducted at 20–25°C. Data points are the mean of six measurements from two separate experiments. Bars show S.D. The difference in numbers of attached bacteria is not statistically significant (P > 0·05) Bacterial adherence to new silver was also tested in laminar flow (0·006 m s−1) using an MRD over a period of 7 d (Table 1). The difference between the two materials varied, having a maximum of 1·5 log (cfu cm−2), but this was not statistically significant (P > 0·05). However, the number of attached bacteria on silver and steel surfaces was several log units lower than in the batch system, despite a higher initial cell concentration in the MRD buffer suspension of 1 × 108 cfu ml−1. Table 1. Adhesion of Shewanella putrefaciens to conditioned stainless steel and new silver surfaces in a modified Robbins device using phosphate-buffered saline buffer at 20–25°C and a laminar flow of 0·006 m s−1 Bacterial adhesion to tarnished silver surfaces The silver surface changed from new shiny silver (Fig. 2a) to a silver surface scattered with brown spots or stripes as the silver oxidized through one to two cycles of cleaning and usage in the adhesion experiments (Fig. 2b and c). The whole surface tarnished (Fig. 2d) after further use (three to four cycles). The tarnished surface was believed to consist of silver oxide and/or silver sulphide. Energy dispersive X-ray analysis showed a sulphur content between 0·3 and 0·4 wt % on surfaces with spots and 1·5 and 2·6 wt % on blackened surfaces. These results should be taken as semi-quantitative and are not necessarily representative for the whole plate since the EDX only measured a small area. Figure 2Open in figure viewerPowerPoint Silver surfaces used in batch system to estimate bacterial adhesion. (a) New silver; (b) and (c) silver scattered with brown spots and stripes (approx. 0·3–0·4 wt % sulphur); (d) whole silver surface tarnished (approx. 1·5–2·6 wt % sulphur) and (e) sulphide-treated silver (approx. 2·5 wt % S) The tendency towards fewer adhered bacteria on silver surfaces compared with steel became more pronounced as the silver tarnished through consecutive cleaning and usage in the adhesion experiments. The numbers of S. putrefaciens on conditioned steel and tarnished silver surfaces varied from 1·6 log (cfu cm−2) to 7·1 log (cfu cm−2) (Fig. 3a). The numbers of adhering bacteria were typically 0·1−2·1 log units lower on tarnished silver than on steel from 7 h and up to 170 h. The differences were, however, not consistent within an adhesion experiment and were only statistically significant in one of two experiments (P < 0·05) (Fig. 3a). Figure 3Open in figure viewerPowerPoint Number of attached Shewanella putrefaciens on stainless steel (□) and tarnished silver (▴) surfaces. Experiments were conducted at 20–25°C. Bars show S.D. (a) Conditioned plates in phosphate-buffered saline (PBS) buffer with an initial cell concentration of 4 × 103 cfu ml−1 peaking after 120 h to 7 × 106 cfu ml−1 (–––) or an initial cell concentration of 3 × 103 cfu ml−1 (– – –). The difference in the number of attached bacteria is statistically significant (P < 0·05) for the 170-h experiment (–––). At the 24-h sampling point P < 0·01 for the short-time experiment (– – –). (b) Non-conditioned plates in PBS buffer with an initial cell concentration of 4 × 102 cfu ml−1 peaking after 72 h to 4 × 105 cfu ml−1. The difference in the number of attached bacteria is statistically significant (P < 0·05) The first organic layer on a surface may be important for bacterial adhesion (2) and may change the surface charge (7). When the conditioning film (Fig. 3b) on tarnished silver and steel was omitted, a statistically significant lower number of bacteria (P < 0·05) attached to tarnished silver. Immersing steel and tarnished silver plates in nutrient broth (one-seventh TSB) instead of PBS buffer allowed for the rapid growth of bacteria in the substrate and the numbers of bacteria on surfaces increased from less than 0·2 log (cfu cm−2) to 6·2 log (cfu cm−2) after 48 h (Fig. 4). Except for one sampling point after 8 h, approximately the same number of bacteria were detected on the two types of surface. Interestingly, despite the same cell concentration in the one-seventh TSB (Fig. 4) and PBS buffer (Fig. 3a, 170 h experiment), a lower number of bacteria (1·5 log units for steel and 1·9 for silver) adhered to surfaces from one-seventh TSB, although this could only be observed during the first 4 h. Figure 4Open in figure viewerPowerPoint Number of attached Shewanella putrefaciens on stainless steel (□) and tarnished silver (▴) in nutrient broth (one-seventh strength Tryptone Soya Broth) with an initial cell concentration of 2 × 103 cfu ml−1 peaking after 48 h to 2 × 108 cfu ml−1. The experiment was conducted at 20–25°C. Bars show S.D. The difference in the number of attached bacteria is not statistically significant (P > 0·05) Bacterial adhesion to sulphide-treated silver surfaces The increasing difference in adhesion, resulting in fewer bacteria on silver surfaces compared with stainless steel, was believed to be caused by the gradual tarnishing/oxidizing of silver. The apparent difference in sulphur content on new silver vs tarnished silver surfaces led us to produce a sulphide-treated silver surface (Fig. 2e), thus creating a deliberately tarnished surface that could be reproduced. The number of bacteria adhering to the sulphide-treated silver surface (approx. 2·5 wt % sulphur) was not different from the number adhering to stainless steel (P > 0·05) (data not shown). The number of bacteria adhering to the sulphide surfaces was 0·1–0·2 log units higher than on steel at samplings after 4 and 24 h and 0·4 log units lower than on steel at 7 and 48 h. There was a tendency to a greater number of bacteria attaching to the silver surface than to stainless steel within the first 4 h for any silver surface (new, tarnished or sulphide treated). In general, however, bacterial numbers were lower on silver surfaces than on steel at longer adhesion times. Scanning electron microscopy of silver surfaces New silver surfaces appeared smooth and homogeneous but with small contours due to the deposition process (Fig. 5a). After exposure for 48 h in PBS with an initial cell concentration of 106 cfu ml−1 followed by the Malthus procedure and autoclaving, the surface appeared slightly pitted and with minor grooves. The fine pits almost formed a network covering the entire surface (Fig. 5b). Repeated use of the silver surfaces (five cycles of cleaning and adhesion experiment) led to a completely tarnished surface, as shown in 5Fig. 5c. This tarnished surface was characterized by increased micro pitting and deposition of corrosion products leading to an increased surface roughness and surface area. The silver surface treated in sulphide solution appeared smooth with minor grooves (Fig. 5d) but fine pits did not appear. Figure 5Open in figure viewerPowerPoint Scanning electron microscopy of silver surfaces (original magnification, 1250×). (a) New silver surface; (b) new silver surface after 48 h in phosphate-buffered saline with 106 cfu ml−1 followed by measurement in Malthus glass tubes and autoclaving; (c) completely tarnished silver after approx. five cycles of cleaning and adhesion experiment and (d) sulphide-treated silver surface (approx. 2·5 wt % S) DISCUSSION As expected, S. putrefaciens adhered readily to stainless steel surfaces (21; 2). In general, we detected a lower number of viable bacteria on silver surfaces compared with steel surfaces. However, the difference was, on average, less than 1 log unit and, in most cases, not statistically significant (P > 0·05). A lower, but not statistically significant, bacterial adhesion was also seen on silver-coated polymeric fixation pins as compared with stainless steel (17). 32) reported a statistically significant difference in numbers of adhered bacteria on steel and silver-coated fixation pins. After 2 h, four of five bacterial strains adhered in lower numbers to silver surfaces than to stainless steel (detected as extracted ATP) and one strain showed enhanced adhesion to silver surfaces. The effect of silver coating on bacterial adhesion has been compared for a variety of materials. Silver coating of urinary catheter material (latex) prevented formation of a Pseudomonas aeruginosa biofilm which formed easily on non-coated latex (16). 1) similarly found that several bacterial species adhered in higher numbers on polypropylene than on polypropylene coated with pure silver, silver oxide or silver chloride. Bacteria were counted using SEM and maximum differences were within 1 log unit, e.g. a minimum of 2·5 × 103 and a maximum of 14 × 103 cfu mm−2. In contrast to this, bacterial adhesion was not affected if silver was deposited using ion beam-assisted deposition (1). Scanning electron microscopy showed that bacterial cells were present, i.e. that they had attached, but that they were collapsed and lysed. Thus, use of the Malthus procedure, where viable cells were detected, would probably have demonstrated a lower number. In four of six experiments (1, 3data shown for four experiments; Figs 1 and 3a), a higher number of bacteria was seen on silver surfaces than on stainless steel during the first 4 h of adhesion. This would indicate that the silver surfaces do not inhibit or reduce adherence per se (when compared with steel) and that any subsequent reduction of bacterial numbers results from either a repelling of the bacteria from the surface or a bactericidal/bacteriostatic activity of the silver on the surface-adhered bacteria. As this study quantified undisturbed viable (CO2-producing) bacteria adhered onto the surfaces by the Malthus indirect conductance method, we cannot directly distinguish between these two phenomena. Ultrasonic removal of cells and subsequent quantification using plate counts confirmed the cell numbers as determined by the Malthus method (2). DAPI (4′,6′-diamidino-2-phenylindole) (Sigma) staining consistently resulted in a slightly higher bacterial number on both types of surface (data not shown). The tendency to fewer viable bacteria attaching to silver surfaces compared with stainless steel was particularly noted on tarnished silver in PBS buffer (Fig. 3). This could be due either to a direct antiadhesive effect or to an increase in the antibacterial activity of silver. 11) studied the antibacterial activity of silver compounds in agar diffusion assays and reported that pure silver metal, or silver carefully cleaned, had no or decreased activity compared with silver oxide (cooled in air) or silver treated with nitric acid. In contrast to this, 1) did not detect any differences in adhesion to surfaces coated with oxidized silver as compared with pure (new silver). However, in the study of 1), adhesion only took place for 2 h, which is much shorter than the time intervals in the present study and the time span used in growth experiments by 11). Also, bacteria vary in their sensitivity to silver compounds (3) and differences could exist between the Serratia marcescens used by 1) and our S. putrefaciens. It could be hypothesized that the slightly lower numbers of bacteria on silver surfaces could result from a