Langmuir 2002, 18, 2343-2346
2343
Use of the Atomic Force Microscope To Determine the Effect of Substratum Surface Topography on Bacterial Adhesion R. D. Boyd and J. Verran* Manchester Metropolitan University, John Dalton Building, Chester Street, Manchester, M1 5GD, United Kingdom
M. V. Jones and M. Bhakoo Unilever Research Port Sunlight, Quarry Road East, Bebington, Wirral, L63 3JW, United Kingdom Received July 23, 2001. In Final Form: November 30, 2001 Changes in surface roughness and topography on the macroscopic scale are known to affect bacterial attachment and retention. Little quantitative information is available as to how changes in surface topography on the micron and submicron scale affect the strength of bacterial attachment to substrata. A novel method is described using the atomic force microscope where a varying shear/lateral force (in nanonewtons) is used to detach individual bacterial cells from various substrata of different surface topographies. Lateral changes of 0.1 µm in the surface topography are sufficient to affect the strength of bacterial attachment. An increase in applied force from 4 to 8 nN was necessary to move bacteria retained in surface defects of approximately 1 µm wide and 0.2 µm deep compared with cells attached on smooth surfaces.
Introduction The initial attachment of microorganisms to a surface is a prerequisite for the colonization of that surface. A reduction in attachment in terms of either the number of cells or the strength of cell-surface interaction could delay surface colonization/biofilm formation in a dynamic (e.g., flowing) situation and could also improve surface hygiene by making cleaning more effective.1,2 Measurement of the amount of attachment has traditionally relied on cell counting methods, either in situ via microscopy/image analysis or radiolabeling of cells or indirectly via quantifying cells removed from the surface.3 The few studies concerned with the strength of attachment have tended to focus on flow cells where a known shear force is applied across the test surface and the cell removal is monitored4,5 or via the passing of an air-liquid interface which has the ability to displace attached bacterial cells by applying large shear forces.6 To measure the force require to displace individual cells, an instrument is required that can image down to the nanometer level with high force resolution. The atomic force microscope (AFM) fulfils both of the requirements. * Corresponding author. Tel: +44 161 247 1206. Fax: +44 161 247 6357. E-mail:
[email protected]. (1) Holah, J. Effective microbiological sampling of food processing environments. Guideline No. 20; Campden and Chorleywood Food Research Association: Chipping, Campden, U.K., 1999. (2) Verran, J.; Hissett, T. In Biofilms in the aquatic environment; Keevil, C. W., Godfree, A., Holt, D., Dow, C., Eds.; Royal Society of Chemistry: Cambridge, U.K., 1999; pp 25-33. (3) Wirtanen, G.; Storgards, E.; Saarela, M.; Salo, S.; MattilaSandholm, T. In Biofilms and Biofouling: Vol. 1, Biofouling in Industry and Process Engineering; Walker, J., Surman, S., Jass, J., Eds.; John Wiley: Chichester, U.K., 2000; pp 175-203. (4) Callow, M. E.; Santos, R.; Bott, J. R. In Microbial biofilms: Formation and control; Denyer, S. P., Gorman, S. P., Sussman, M., Eds.; Society for Applied Bacteriology, Technical Series No. 30; Blackwell Scientific: Oxford, U.K., 1993; pp 247-258. (5) Sjollema, J.; Busscher, H. J.; Weerkamp, A. H. J. Microbiol. Methods 1989, 9, 73-78. (6) Gomez-Suares, C.; Busscher, H. J.; van der Mei, H. C. Appl. Environ. Microbiol. 2001, 67, 2531-2537.
Figure 1. Normally, the AFM is operated with a low tipsurface force (a) so that as the tip scans across the surface it will move in response to changes in surface topography, such as an absorbed bacterium (b) setting up a bending moment. Increasing the tip-surface force causes the AFM to displace any weakly attached particle (bacterium) (c).
The atomic force microscope was invented in 19867 and allows high-resolution, three-dimensional imaging of nonconducting surfaces.8 A very sharp silicon nitride tip (10 nm diameter) is connected to a cantilever. The tip is placed in contact with the surface with a force applied between the tip and the surface so that the cantilever is under tension (Figure 1a).9 Changes in the surface topography cause the cantilever to be deflected setting up a bending moment in the cantilever that is proportional to the tip-surface force (Figure 1b).9 Normally, the height of the tip is adjusted using a piezoelectric scanner, so that the cantilever returns to its original position. However, if the tip-surface force is high enough the bending moment will overcome the adhesion force of a weakly attached particle,10 such as a bacterial cell,11 causing its detachment (7) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 9, 930933. (8) Bowen, W. R.; Donera, T.; Nilal, N.; Wright, C. J. Microsc. Anal. 2001, January, 5-7. (9) Magonov, S. N. Appl. Spectrosc. Rev. 1993, 28, 1-121. (10) Ling, J. S. G.; Leggett, G. J.; Murray, A. J. Polymer 1998, 39, 5913-5921.
10.1021/la011142p CCC: $22.00 © 2002 American Chemical Society Published on Web 02/22/2002
2344
Langmuir, Vol. 18, No. 6, 2002
Boyd et al.
Figure 2. AFM images and cross sections of the bare substrates: (a) polished, (b) unpolished, and (c) abraded stainless steel. The Z-scale is 2 µm in all cases.
(Figure 1c). By use of the AFM in this way, the force required to displace bacteria attached to a surface may be measured. Alteration of the roughness or the surface topography of a sample greatly affects bacterial adhesion and retention.12 This property has been attributed to the provision of a greater surface area for attachment13,14 and protection of bacteria from shear forces.15 The concept of cell mobility has also been introduced,6 where microorganisms diffuse or swim freely across a surface until they encounter a “sticky patch”, where immobilization occurs due to chemical and/or structural heterogeneities in the surface: changes in lateral forces are necessary to facilitate further movement. This paper describes the use of the AFM to quantify cell-surface adhesion forces using surfaces with differing submicron scale topographies.
Instruments (Agoura Hills, CA). The majority of AFM studies are carried out either in air or in a vacuum where a laser beam is reflected off the cantilever and onto a photodiode. When the AFM is used in an optically clear liquid medium, the cantilever is completely submerged in the liquid and, to protect the electronics of the AFM from the medium, a plastic cover fitted with a transparent window (termed a liquid cell) is placed above the cantilever. The laser beam can then pass through the window and is reflected off the cantilever back through the window. Standard triangular silicon nitride cantilevers and tips (NP-20, Veeco Instruments Ltd., Cambridge, U.K.) that are 100 µm long with a spring constant of 0.32 N/m were used. Repeated images of the cell-substrate were obtained by scanning the same area as the tip-surface force was gradually increased from 2 to 10 nN, hence giving an indication of the strength of attachment of cells on the surface.
Experimental Section
AFM images of the bare substrates are shown in Figure 2. The polished and abraded samples show unidirectional surface topography with an average peak to valley distance of 0.04 ( 0.01 µm and 0.30 ( 0.08 µm and average width of 0.96 ( 0.20 µm and 2.8 ( 0.8 µm, respectively. The unpolished stainless steel sample has a characteristic grain structure of size approximately 10 µm with gaps 1.6 ( 0.4 µm wide and 1.2 ( 0.3 µm deep at the grain boundaries. Bacteria were visualized on all three stainless steel substrates, and individual cells were clearly resolved at low tip-surface force (2 nN, Figure 3a). An increase in this force caused some of the bacteria to be displaced (Figure 3b,c). For polished stainless steel samples, a small increase in the tip-surface force from 2 to 4 nN was sufficient to displace most of the attached bacteria. However, for the abraded sample a higher force of 8 nN was required to cause significant displacement of the bacteria, with residual cells being retained along the surface features with the AFM tip being scanned across, not along, the defects. For the 8 and 10 nN images (Figure 3d,e), the AFM tip has scanned part of the bacterial cell before its displacement leading to a characteristic “streak” on the final image. On unpolished stainless steel, bacteria were attached within the large grain boundaries and could not be
Preparation of Stainless Steel Substrates. Three stainless steel samples with different surface finishes were used: polished (stainless steel grade 304), unpolished, and abraded (grade 316 (Avesta, Sheffield, U.K.)).16 Prior to bacterial attachment, all stainless steel samples were cut into approximately 1 cm2 size coupons using a guillotine and cleaned by rinsing in methanol before being air-dried. Bacterial Suspensions. Staphylococcus aureus (Oxford strain) was grown in 10 mL of nutrient broth (Oxoid, Basingstoke, U.K.) for 24 h. Bacteria were then harvested by centrifugation at 1100g for 10 min and resuspended three times in 10 mL of sterile distilled water to a concentration of 1.0 ( 0.1 × 109 cfu/ mL. Two drops of this bacterial suspension from a 50 dropper (0.04 mL) were placed on each stainless steel substrate, which were then dried in air at room temperature. Atomic Force Microscopy. All AFM images were obtained in distilled water using a Resolver AFM manufactured by Quesant (11) Grantham, M. C.; Dove, P. M.; DiChristina, T. J. Geochim. Cosmochim. Acta 1997, 61, 4467-4477. (12) Verran, J.; Boyd, R. D. Biofouling, in press. (13) Holah, J. T.; Thorpe, R. H. J. Appl. Bacteriol. 1990, 69, 599608. (14) Leclercq, M.; Lalande, M. J. Food Eng. 1994, 23, 501-517. (15) Korber, D. R.; Choi, A.; Woolfaardt, G. M.; Ingham, S. C.; Caldwell, D. E. Appl. Environ. Microbiol. 1997, 63, 3352-3358. (16) Verran, J.; Rowe, D. L.; Boyd, R. D. J. Food Prot. 2001, 64, 1183-1187.
Results
Effect of Surface Topography on Bacterial Adhesion
Langmuir, Vol. 18, No. 6, 2002 2345
Figure 4. 40 × 40 µm wide low-force (2 nN) AFM image of S. aureus attached to polished stainless steel taken after imaging at high force. The center of the image shows a 20 × 20 µm area cleared of bacteria using the AFM tip.
unpolished substrata, with 8 nN tip-surface force required to cause the largest reduction in retained cells, compared to 4 nN for the other substrates. The unpolished stainless steel sample also retained a higher percentage of bacterial cells than the polished substrate at lower tip-surface force. Discussion
Figure 3. AFM images of S. aureus attached to polished, unpolished, and abraded stainless steel imaged at (a) 2 nN, (b) 4 nN, (c) 6 nN, (d) 8 nN, and (e) 10 nN. All images are 20 × 20 µm across and 4 µm high.
removed even at high tip-surface force (10 nN, Figure 3e). However, the majority of cells appeared to be attached to the surface of the grains in large “aggregates”. These were removed relatively easily from the surface, although less easily than from the polished stainless steel. It also appears that cells were initially removed from the edges of the aggregates (Figure 3a-c). The cells remaining appeared to be attached to small surface features on the grain boundary surface; these were typically 1.4 µm wide and 0.2 µm deep (comparing parts c and e of Figure 3). When the same area is rescanned at lower magnification and low force (Figure 4), the area cleared of bacteria is clearly visible. There was no buildup of bacteria at the scan area edges, indicating that the AFM tip caused the bacteria to detach from the surface into the liquid rather than being pushed across it. Due to the small area analyzed by the AFM, the number of bacteria varies from scan to scan. To compensate for this and to give consistent results, the proportion of bacteria remaining on the surface after each scan was determined (Figure 5). The abraded sample retained bacteria more effectively than either the polished or
The use of the AFM as a force measuring technique is now well established.8 It has been used to measure cell to surface adhesion by attaching either a fungal spore17 or a group of bacterial cells18-20 to the tip or to coat the tip with a representative layer.21 The force required to detach the cell from the surface is then directly measured. All of these techniques measure the force applied perpendicular to the surface. Little work has been done on the shear (parallel)/lateral force required to move cells,22 which is also more representative of the force applied during any cleaning process. Previous studies have shown that the lateral force required to displace a bacterial cell is considerably smaller than the perpendicular force (up to 10 times smaller) and is affected by changes in surface topography.23 The size of individual S. aureus cells (approximately 1 µm diameter) is comparable to the size of the surface features on the abraded surface. Bacteria were more strongly held on the abraded surface due the maximum area of cell to surface contact. On the unpolished stainless steel surface, features much smaller than the size of individual cells also increased the strength of bacterial adhesion, but to a lesser extent. This agrees with earlier theoretical work where the small lateral force of bacterial cell detachment, compared to the perpendicular force, leads to cell “surface mobility” with the cell migrating (17) Bowen, W. R.; Lovitt, R. W.; Wright, C. J. J. Colloid Interface Sci. 2000, 228, 428-433. (18) Razatos, A.; Ong Y.-L.; Sharma, M. M.; Georgiou, G. Proc. Natl. Acad. Sci. 1998, 19, 11059-11064. (19) Ong, Y.-L.; Razatos, A.; Georgiou, G.; Sharma, M. M. Langmuir 1999, 15, 2719-2725. (20) Lower, S. K.; Tadanier, C. J.; Hochella, M. F. Geochim. Cosmichim. Acta 2000, 64, 3133-3139. (21) Considine, R. F.; Dixon, D. R.; Drummond, C. J. Langmuir 2000, 16, 1323-1330. (22) Dagvolen, G.; Giaver, I.; Pettersen, E. O.; Feder, J. Proc Natl. Acad. Sci. U.S.A. 1999 96, 471-476. (23) Busscher, H. J.; Poortinga, A. T.; Bos, R. Curr. Microbiol. 1998, 37, 319-323.
2346
Langmuir, Vol. 18, No. 6, 2002
Boyd et al.
Figure 5. Graph showing the effect of surface topography on bacterial adhesion. Increasing the shear force causes a greater proportion of the attached bacteria to be displaced. The 100% value is taken to be the number of cells remaining after imaging with a tip-surface force of 2 nN.
across the surface until it encounters areas of low lateral energy, such as a surface defect.6 All the test substrata had Ra values below 0.8 µm, generally deemed to be the cutoff point for a “hygienic” surface. Studies using more traditional microbiological methods to evaluate cleanability of these surfaces were unable to differentiate surfaces in terms of the number of cells remaining postclean, although the pattern of retention was affected by the topographical features.16 Cells were sprayed onto the surfaces and dried before a wipe-clean event. Yet the use of a small, nanonewton, lateral force applied by the AFM probe under liquid discriminated between these surfaces in terms of cell
retention, relating retention to changes in surface topography on the micron and submicron scale. The use of the AFM, by directly assessing the strength of bacterial attachment, has proved to be more sensitive and time efficient than routine microbiological/microscopic procedures. This method shows great potential in areas as diverse as surface hygiene, medical devices, and novel material development. Future work will monitor the effect of conditioning films, cleaning agents, and different substrata on cell retention upon application of shear force. LA011142P
