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Bacterial Adherence: Its Consequences in Food Processing
Can. Inst. Sci. Technol. J. Vo!. 24. No. 3/4, pp. 113-117.1991
REVIEW
Bacterial Adherence: Its Consequences in Food Processing R.D. Pontefract Microbiology Research Division Bureau of Microbial Hazards Health Protection Branch Health and Welfare Canada Ottawa, Ontario KIA OL2
Abstract
rapides, fiables et sensibles pour la detection et I'enumeration de bacteries viables sur des surfaces a contact alimentaire est important si ('on veut effectuer le controle bacteriologique des procedures de nettoyage et d'hygiene en industrie alimentaire durant un traitement alimentaire et non pas considerablement longtemps plus tard.
The adherence of bacteria to various substrates is well known. The results of thi: attachment of contaminating bacteria to glass, plastic or metal surfaces in food processing equipment can have serious consequences during a food processing run and with any subsequent cleaning and sanitizing procedures. If these procedures are inadequate, considerable numbers of viable bacteria may remain. Even with cleaning procedures which appear satisfactory. studies using epifluorescent microscopy and scanning electron microscopy (SEM) have shown that low numbers of organisms can survive and remain attached. Standard bacteriological methods. which are used to evaluate the efficiency of a cleaning procedure, may not be capable of detecting low numbers of organisms adhering to a surface. This has been demonstrated by epifluorescent microscopy on food contact surfaces. Bacteriological techniques such as swabs or contact plates are slow in yielding results compared to such procedures as direct epifluorescent microscopy, which enables enumeration and demonstration of viability of organisms remaining on a food contact surface in less than thirty minutes. The development of fast, reliable and sensitive methods for the detection and enumeration of viable bacteria on food contact surfaces is important, so that bacteriological monitoring of cleaning and sanitizing procedures in the food processing industry can be maintained during, and not considerably after, a food processing run.
The ability of bacteria to adhere to solid surfaces has been recognized for many years. In 1943, Zobell reported the results of extensive studies on the attachment of marine bacteria to various surfaces, which included glass, metal and plastics. Other investigators have reported on the bacterial attachment to plant tissue (Gibbons, 1977; Characklis, 1981) animal tissue (Characklis, 1981; FirstenbergEden, 1981) and mucosal surfaces (Beachey, 1980, 1981). In addition, a number of articles (Fletcher and Floodgate, 1973; Fletcher, 1977; Duguid and Old, 1980; Characklis, 1981; Firstenberg-Eden, 1981) and books (Elwood et al., 1979; Beachey, 1980; Berkeley et al., 1980) have been published on the subject of bacterial adherence to both living and inert substrates. Because of the voluminous literature which exists on attachment in general, this review will focus on the attachment and growth of microorganisms on inert surfaces (e.g., metal, glass and plastic), which are found in food plants and processing systems, and the application of techniques such as scanning electron microscopy to study the nature of the attachment of bacteria to these substrates. Studies on the effect of cleaning and sanitizing agents on attached bacteria will be reviewed and the ability of commonly employed bacteriological sampling techniques to determine the efficacy of such procedures will be discussed. In relation to these standard bacteriological tests, methods such as the direct epifluorescent filter technique (DEFT) and the direct epifluorescent method (DEM) will be compared as to their ability to detect and enumerate bacteria found on food contact surfaces.
Resume 11 est bien connu que les bacteries peuvent adherer a differents substrats. La presence de bacteries adherentes aux surfaces en verre, en plastique ou en metal des equipements alimentaires peut avoir des consequerrces serieuses au cours d'un traitement alimentaire ou lors de toute procedure subsequente de nettoyage et d'hygiene. Si ces procedures ne sont pas adequates, il restera de nombreuses bacteries viables. Meme avec des procedures de lavage apparemment satisfaisantes, il a ete demontre a I'aide de la microscopie epifluorescente et de la microscopie electronique par balayage que quelques bacteries peuvent encore survivre et demeurer attachees aux surfaces. Pour deceler la presence de quelques bacteries adherant a une surface, on ne peut pas se fier aux methodes bacteriologiques couramment utilisees en evaluation de I'efficacite d'une procedure de nettoyage. Ceci a ete demontre par la microscopie epifluorescente sur des surfaces venant en contact avec des aliments. Les techniques bacteriologiques comme les plaques d'ecouvillons ou de contact sont longues par comparaison a la microscopie epifluorescente directe. Celle-ci permet I'enumeration et la demonstration de la viabilite d'organismes sur une surface a contact alimentaire en moins de trente minutes. Le developpement de methodes
Copyright © 1991 Canadian Institute of Food Science and Technology
113
The majority of studies on bacterial adherence to solid surfaces have been concerned with the manner in which bacteria attach to the surface and how the organisms can maintain their attachment with subsequent growth. A number of theories have been advanced. The major forces believed to induce contact with a surface are the London-van-der-Waals attractive energies between two surfaces (Marshall et al., 1971). A secondary and more secure form of attachment, involving the secretion of a mucilaginous holdfast, was proposed by Zobell (1943). Fletcher and Floodgate (1973) demonstrated that an acidic polysaccharide layer was involved in the adherence of bacteria to surfaces as did Herald and Zottola (1988) who studied the involvement of acid mucopolysaccharide, in the attachment of microorganisms to stainless steel. Costerton et al. (1978) suggested that bacteria adhere to surfaces by secreting a mass of tangled fibres or glycocalyx. Others have suggested that flagella or fimbria (Notermans and Kampelmacher, 1974; Beachey, 1981; Notermans and Kampelmacher, 1983) may play an important role in bacterial attachment. However, there is some controversy on this theory since Rogers (1979) and Rutter and Vincent (1980) found that organisms without these structures could also adhere to various substrates. The literature on bacterial adherence and theories of attachment is extensive and beyond the scope of this review. More recently, the scanning electron microscope (SEM) has been employed to visually examine the attachment of bacteria to solid surfaces. FirstenbergEden et al. (1981) and Zottola (1986), using the SEM to study bacterial attachment to skin and inert substrates, observed connecting strands of material between the microorganisms and the surface to which they were attached. Others have employed SEM to study bacterial attachment to stainless steel and glass surfaces (Zoltai et al., 1981; Speers et al., 1984; Stone and Zottola, 1985). These workers were concerned with the contamination of the surfaces of holding tanks by raw milk, which could lead to subsequent contamination of other milk processing equipment. They found that any surface could serve as an attachment site for organisms such as Pseudomonas spp., Micrococcus spp. (Speers et al., 1984), Staphylococcus aureus, Streptococcus cremoris, (Zoltai et al., 1981) as well as Pseudomonas jragi (Stone and Zottola, 1985). Speers et al. (1984) found that after a 1 h exposure to contaminated milk at 25°C, followed by varying incubation times up to 72 h, Pseudomonas and Micrococcus species formed small colonies around the original attached bacterium. SEM studies have also shown that while the originally attached bacteria appear to bind to surfaces by extracellular fibrils (Zoltai et al., 1981; Schwach and Zottola, 1982) the organisms underwent cell division to form microcolonies, which appeared to attach more to each other than to the surface (Speers et al., 1984). Since it had been demonstrated that microor114 / Pontefract
ganisms can attach themselves to solid surfaces and subsequently form colonies, it became important to the food industry to obtain information on the ability of these attached bacteria to withstand the cleaning and sanitizing processes used in a food processing plant. A number of studies have been carried out to analyze the efficiency of various cleaning systems (Maxcy, 1969; Baldock, 1974; Dunsmore et al., 1980; Dunsmore and Thomson, 1981; Mabeya et al., 1982; Powell and Slater, 1982; Mrozek, 1985; Stone and Zottola, 1985; Lopez, 1986). Because of the extended turn around time and the risk of damage occurring upon dismantling of food processing equipment for cleaning, one of the most commonly used practices in cleaning and sanitizing equipment used in processing fluid food is the clean-in-place (CIP) system (Barron, 1986). A number of different steps may be employed with this procedure but the method usually includes a pre-wash rinse to remove the bulk of the soil, a detergent wash, a post-detergent rinse and a pre-soil sanitizer which leaves a film of liquid sanitizer on the food contact surface (Gibbons, 1977; Mitchel, 1981). Different chemicals and various combinations of detergents are used in various CIP procedures (Gibbons, 1977; Barron, 1986). The ultimate goal is proper cleaning and sanitizing of the food contact surfaces of food processing equipment. CIP systems cannot be used with surfaces such as conveyor belts and preparation tables; however, these surfaces are usually cleaned with detergents and a suitable sanitizing agent. The two-fold benefit obtained from properly cleaned equipment is the increased preservation of the product and, most importantly from a health point of view, the minimization of the risks of food-borne illness. The methods of determining the efficiency of cleaning procedures are varied. The most commonly employed include the swab-rinse, agar contact, and direct surface plating. The use of all these techniques requires the subsequent culture of the sample in a suitable medium to determine viable organisms and, therefore, the adequacy of the cleaning process (Baldock, 1974; Dunsmore et al., 1980; Dunsmore and Thomson, 1981). Conditions of sampling determine the use of some of these methods. The direct surface agar plating method is not suitable for plant sampling and the agar contact methods (RODAC PLATE) are only useful for smooth, flat, surfaces. Since dilution is not possible, only small numbers of contaminants can be enumerated and, therefore, RODAC PLATES are not suitable for heavily contaminated surfaces (Patterson, 1971; Baldock, 1974). The most widely used technique is the swab-rinse method whereby a sterile swab (usually moistened with a sterile fluid) is rubbed over the surface of the area to be sampled. The main problem with the swab-rinse method is that the swabbing procedures vary per individual, Le., in some cases the pressure on the swab is heavy, in other instances light, and swabbing may be fast, or slow. The technique is not reproducible and this can lead J. Inst. Can. Sci. Technol. Aliment. Vo!. 24, No. 3/4. 1991
to variable microbial counts and incorrect estimates of the numbers of organisms present on the swabbed area. Further, the cotton retains some of the microorganisms causing reduced counts (Barnes, 1952; Holah et al., 1988). To eliminate this latter problem, soluble alginate swabs were developed which dissolve and thus release all organisms collected on the swab. More recently, to determine the effectiveness of CIP and other cleaning procedures in removing bacteria from various surfaces, the scanning electron microscope (SEM) has been used in a number of studies. One study used stainless steel strips which were attached inside a system where CIP could be carried out (Stone and Zottola, 1985). In testing the effect of a commonly used sanitizing agent such as sodium hypochlorite, Schwach and Zottola (1984) observed that bacteria attached to stainless steel were not removed even after the use of 150 ppm hypochlorite, although the viability of the attached microorganisms was not determined. Stone and Zottola (1985) found that considerable numbers of bacteria became attached to the internal surfaces of the processing equipment if the interval between cleanings exceeded 8 h. They also found that if CIP procedures are reduced (dilution of cleaning chemicals greater than recommended by the manufacturer or the lowering of the temperature suggested for proper cleaning), viable attached cells may remain and fresh product coming into the system may be contaminated. These findings become particularly important when one considers the recent trends to longer production runs in the food industry, with a short interval for cleaning and sanitizing; a practice which can lead to inadequate cleaning and sanitation by the CIP system. The principal advantage of SEM examination of surfaces for bacterial contamination is that specimen samples 3 or 4 cm 2 can be quickly scanned with high resolution, and three or four organisms per cm 2 can be detected. Sample test strips of stainless steel, glass, or plastic can be located at strategic areas in food processing plants or even mounted in food processing equipment (Holah et al., 1987), and removed at suitable intervals to ascertain the development of bacterial contamination. The processing of such samples for SEM takes approximately 40 min. The SEM is, however, basically a research tool and is not suitable for general microbiological testing in food plants, although such an instrument could be employed in a regional testing facility to examine samples sent in from outlying processing plants. As discussed previously, traditional methods of assessing surface hygiene in food processing plants and equipment, such as swabs, rinses and contact techniques require long incubation periods (1-4 d) before the presence of bacteria can be determined and counts can be made. Because of this time delay, the next food processing runs are already underway when the results are obtained and, therefore, these techniques will only detect trends in microbial levels Can. Inst. Food Sci. Technal. J. Vol. 24, No. 3/4, 1991
in processing plants and can only provide a history of the plant hygiene (Patterson, 1971; Holah et al., 1987). Modern developments in the food industry have stressed the need for a fast turnaround in cleaning procedures which necessitates the rapid assessment of microbial contamination of food contact surfaces to allow process control during production periods (Holah et al., 1988). A rapid technique for the enumeration of organisms using the light microscope, the Direct Epifluorescent Filter Technique (DEFT) has been developed by Pettipher et al. (1980). The technique was designed to ascertain the bacteriological quality of raw milk. The method involves the filtration of a milk sample through a membrane filter and bacteria concentrated on the filter are stained with acridine orange, which causes them to fluoresce a bright red-orange when viable cells are exposed to ultraviolet or blue-violet light. This technique enables a bacterial count to made from milk samples in less than 25 min. Shaw et al. (1987) have modified DEFT so it can be used with other food products and Holah et al. (1987), by removing organisms from a swab with a vortex mixer before filtration and staining, have modified the method so it can be used to examine bacterial contamination on solid surfaces. Modifications of DEFT have been made that allow the enumeration of microbial surface populations to be made in about 15-20 min (Holah et al., 1987, 1988). This techniques does not use swabbing methods but uses test strips of stainless steel and other materials mounted in or on food processing equipment such as conveyor belts, milk holding tanks, etc. At suitable intervals these test strips are removed, stained and examined with epifluorescence microscopy. This technique, termed Direct Epifluorescent Method (DEM), enables direct examination of contaminated surfaces to be carried out using the same fluorescent stain employed in DEFT, allowing the operator to determine viability of the attached organisms. Studies by these workers indicate that the DEM compares favourably with DEFT and the total viable count determined by the swab method on an equal area of surface. They also determined that the DEM can detect very low numbers of microorganisms that can not be picked up with DEFT, because many organisms were trapped in the fibres of the swab and were not released for growth in culture media or on the membrane filter. Alginate swabs, which would eliminate this problem, were not suitable for DEFT since they tend to clog the filter used in the procedure. The use of DEM (Holah et al., 1987) has also demonstrated that bacterial adherence can occur on many types of surfaces and with different food products such as baked beans or fish. Analytical techniques such as DEFT and DEM and the use of SEM have demonstrated that in such cases where low numbers of viable bacteria may remain attached to supposedly clean and sanitized surfaces, traditional methods such as swabbing or contact Pontefract / 115
plates may not always present a true picture of the microbiological population present. Besides, these latter methods are subject to a considerable time lag before they yield results. This brief review is not meant to cover all the literature on such a broad subject but it is sufficient to show that there are unresolved problems in applying standard bacteriological sampling techniques in a realistic time frame for modern food processing procedures. Further studies, employing techniques such as epifluorescence microscopy and utilizing research instruments such as the scanning electron microscope, are required to obtain additional information on problems of bacterial attachment in food processing equipment. Also, additional information is needed to determine efficiency of standard sampling techniques in relation to these biofilms. Such studies as the direct examination of contaminated surfaces to determine the viability of organisms and their resistance to various cleaning procedures could provide valuable information on bacterial survival in food processing equipment. The use of test strips at various sites in food processing plants using DEM, would permit almost continuous monitoring of the bacterial contamination level in the plant and on some food processing equipment. The results of these and other studies which may help in the development of fast, accurate, bacteriological test procedures can be important in the assessment of hygienic practices in the food industry.
References Baldock, J.P. 1974. Microbiological monitoring of thefood plant: methods to assess bacterial contamination on surfaces. J. Milk Food Technol. 37:361. Barnes, 1.M. 1952. The removal of bacteria from glass surfaces with calcium alginate, gauze and absorbent cotton wool swabs. Proc. Soc. Appl. Bacteriol. 15:34. Barron, W. 1986. CIP: Change and challenge for the dairy engineer. Dairy Ind. Int. 51:14. Beachey, E.H. 1980. (Ed.). Bacterial Adherence. Chapman and Hall, London, Methuen, NY. Beachey, E.H. 1981. Bacterial Adherence: adhesin receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143:325. Berkeley R.C.W., Lynch, J.M., Smelling, J., Rutter, P.R. and Vincent, B. 1980. (Eds.). Microbial Adhesion to Surfaces. Ellis Horwood. Chichester, UK. Characklis, W.G. 1981. Fouling biofilm development: A process analysis. Biotech. Bioeng. 23:1923. Costerton, 1.W., Geesly, G.G. and Cheng, K.J. 1978. How bacteria stick. Sci. Amer. 238:86. Duguid, J.P. and Old, D.C. 1980. Adhesive properties of Enterobacteriacea. In: Bacterial Adherence. E.H. Beachey (Ed.). p. 184. Chapman and Hall. London, Methuen, NY. Dunsmore, D.G., Westwood, D.A., Jay, D.B. and Embling, M. 1980. Simulator technique for assessing the bacteriological control of food equipment surfaces by cleaning systems. J. Food Prot. 43:850. Dunsmore, D.G. and Thomson, M. 1981. Bacteriological control of food equipment surfaces by cleaning systems. 2 Sanitizer effects. 1. Food Prot. 44:21. Dunsmore, D.G., Twomey, A., Whittlestone, W. G. and Morgan, H. W. 1981. Design and performance of systems for cleaning product-contact surfaces of food equipment. A review. J. Food Prot. 44:220.
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Elwood, D.C., Mulling, J. and Rutter, P. 1979. (Eds.). Adhesion of Microorganisms to Surfaces. Academic Press, London, UK. Firstenberg-Eden, R. 1981. Attachment of bacteria to meat surfaces. A review. J. Food Prot. 44:602. Fletcher, M. 1977. The effects of culture concentration, and age, time and temperature on bacterial attachment to polystyrene. Can J. Microbiol. 23: I. Fletcher, M. and Floodgate, G.D. 1973. An electron microscopic demonstration of an acidic polysaccharide involved in the adhesion of a marine bacterium to solid surfaces. J. Gen. Microbiol. 74:324. Gibbons, R.J. 1977. Adherence of bacteria to host tissue. In: Microbiology. D. Schlessinger (Ed.). p. 395. American Society for Microbiology. Washington, DC. Herald, P.J. and Zottola, E.A. 1988. The use of transmission electron microscopy to study the composition of Pseudomonas jragi attachment material. Food Microstruct. 7:53. Holah, J.T., Betts, R.B. and Thorpe, R.H. 1987. The use of epifluorescent microscopy to determine surface hygiene. Proc. VII Int. Biodeteriation Symposium. Cambridge. UK. Holah, J.T., Betts, R.B. and Thorpe, R.H. 1988. The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) to assess microbial populations on food contact surfaces. J. Appl. Bacteriol. 65:215. Lopez. J.A. 1986. Evaluation of dairy and food plant sanitizers against Salmonella typhimurium and Listeric monocytogenes. J. Dairy Sci. 69:51. Mabeya, R.C., Castillo, M.M., Contreras, E.A., Bonaad, L. and Bandian, V. 1982. Destruction and removal of microorganisms from food equipment and utensil surface, by detergents. 11 Staphylococcus aureus. Philippine J. Sci. 1II:17. Marshall, K.C., Stout, R. and Mitchell, R. 1971. Mechanism of initial events in the sorption of marine bacteria to surfaces. J. Gen. Microbiol. 68:337. Maxcy, R.B. 1969. Residual micro-organisms in cleaned-in-place systems for handling milk. J. Milk Food Technol. 32: 140. Mitchel, T. 1981. Cleaning of CIP systems. Dairy Food Sanit. 1:292. Mrozek, H. 1985. Detergency and disinfection. J. Soc. Dairy Technol. 38:119. Notermans, S. and Kampelmacher, E.H. 1974. Attachment of some bacterial strains to the skin of broiler chickens. Br. Poultry Sci. 15:573. Notermans, S. and Kampelmacher, E.H. 1983. Attachment of bacteria in meat processing. Fleischwirtschaft 63:73. Patterson, J.T. 1971. Microbiological assessment of surfaces. J. Food Technol. 6:63. Pettipher, G.L., Mansell, R., McKinnon, C.H. and Cousins, C.M. 1980. Rapid membrane filtration epifluorescent microscopy technique for direct enumeration of bacteria in raw milk. Appl. Environ. Microbiol. 39:423. Powell, M.S., and Slater, N.K.H. 1982. Removal rates of bacterial cells from glass surfaces by fluid shear. Biotech. Bioeng.24:2527. Rogers, H.J. 1979. Adhesion of microorganisms to surfaces. Some general considerations on the role of the envelope. In: Adhesion of Microorganisms to Surfaces. D.C. Elwood, J. Melling, and P. Rutter. (Eds.). p. 29. Academic Press, London, UK. Rutter, P., and Vincent, B. 1980. The adhesion of microorganisms to surfaces. In: Microbial Adhesion to surfaces. R.C. W. Berkeley, J.M. Lynch, J. Melling, P.R. Rutter and B. Vincent. (Eds.). p. 79. Ellis Horwood. Chichester, UK. Schwach, T.S. and Zottola, E.A. 1982. Use of scanning electron microscopy to demonstrate microbial attachment to beef and beef contact surfaces. J. Food Sci. 47:1401. Schwach, T.S. and Zottola, E.A. 1984. Scanning electron microscope study on some effects of sodium hypochlorite on attachment of bacteria to stainless steel. J. Food Prot. J. Inst. Can. Sci. Techno/. Aliment. Vol. 24, No. 3/4, 1991
47:756. Shaw, B.G., Harding, C.D., Hudson, W.H. and Farr, L. 1987. Rapid estimation of microbial numbers on meat and poultry by the Direct Epifluorescent Filter Technique. J. Food Prot. 50:652. Speers, J.G.S., Gilmour, A., Fraser, T.W. and McCaIl, R.D. 1984. Scanning electron microscopy of dairy equipment surfaces contaminated by two milk-borne organisms. J. Appl. Bacteriol. 57: 139. Stone, L.S. and Zottola, E.A. 1985. Effects of cleaning and sanitizing on the attachment of Pseudomonas jragi to stainless steel. J. Food Sci. 50:951.
Can. Inst. Food Sci. Technol. J. Vo!. 24. No. 3/4, 1991
ZobeIl, C.E. 1943. The effect of solid surfaces on bacterial activity. J. Bacteriol. 46:39. Zoltai, P.T., Zottola, E.A. and McKay, L.L. 1981. Scanning electron microscopy of microbial attachment to milk contact surfaces. J. Food Prot. 44:204. Zottola, E.A. 1986. Bacterial attachment: its importance in cleaning and sanitizing. J. Food Prot. 49:856.
Submitted April 10, 1989 Revised December 5, 1990 Accepted December 8, 1990
Pontefract / 117
REVIEW
Bacterial Adherence: Its Consequences in Food Processing R.D. Pontefract Microbiology Research Division Bureau of Microbial Hazards Health Protection Branch Health and Welfare Canada Ottawa, Ontario KIA OL2
Abstract
rapides, fiables et sensibles pour la detection et I'enumeration de bacteries viables sur des surfaces a contact alimentaire est important si ('on veut effectuer le controle bacteriologique des procedures de nettoyage et d'hygiene en industrie alimentaire durant un traitement alimentaire et non pas considerablement longtemps plus tard.
The adherence of bacteria to various substrates is well known. The results of thi: attachment of contaminating bacteria to glass, plastic or metal surfaces in food processing equipment can have serious consequences during a food processing run and with any subsequent cleaning and sanitizing procedures. If these procedures are inadequate, considerable numbers of viable bacteria may remain. Even with cleaning procedures which appear satisfactory. studies using epifluorescent microscopy and scanning electron microscopy (SEM) have shown that low numbers of organisms can survive and remain attached. Standard bacteriological methods. which are used to evaluate the efficiency of a cleaning procedure, may not be capable of detecting low numbers of organisms adhering to a surface. This has been demonstrated by epifluorescent microscopy on food contact surfaces. Bacteriological techniques such as swabs or contact plates are slow in yielding results compared to such procedures as direct epifluorescent microscopy, which enables enumeration and demonstration of viability of organisms remaining on a food contact surface in less than thirty minutes. The development of fast, reliable and sensitive methods for the detection and enumeration of viable bacteria on food contact surfaces is important, so that bacteriological monitoring of cleaning and sanitizing procedures in the food processing industry can be maintained during, and not considerably after, a food processing run.
The ability of bacteria to adhere to solid surfaces has been recognized for many years. In 1943, Zobell reported the results of extensive studies on the attachment of marine bacteria to various surfaces, which included glass, metal and plastics. Other investigators have reported on the bacterial attachment to plant tissue (Gibbons, 1977; Characklis, 1981) animal tissue (Characklis, 1981; FirstenbergEden, 1981) and mucosal surfaces (Beachey, 1980, 1981). In addition, a number of articles (Fletcher and Floodgate, 1973; Fletcher, 1977; Duguid and Old, 1980; Characklis, 1981; Firstenberg-Eden, 1981) and books (Elwood et al., 1979; Beachey, 1980; Berkeley et al., 1980) have been published on the subject of bacterial adherence to both living and inert substrates. Because of the voluminous literature which exists on attachment in general, this review will focus on the attachment and growth of microorganisms on inert surfaces (e.g., metal, glass and plastic), which are found in food plants and processing systems, and the application of techniques such as scanning electron microscopy to study the nature of the attachment of bacteria to these substrates. Studies on the effect of cleaning and sanitizing agents on attached bacteria will be reviewed and the ability of commonly employed bacteriological sampling techniques to determine the efficacy of such procedures will be discussed. In relation to these standard bacteriological tests, methods such as the direct epifluorescent filter technique (DEFT) and the direct epifluorescent method (DEM) will be compared as to their ability to detect and enumerate bacteria found on food contact surfaces.
Resume 11 est bien connu que les bacteries peuvent adherer a differents substrats. La presence de bacteries adherentes aux surfaces en verre, en plastique ou en metal des equipements alimentaires peut avoir des consequerrces serieuses au cours d'un traitement alimentaire ou lors de toute procedure subsequente de nettoyage et d'hygiene. Si ces procedures ne sont pas adequates, il restera de nombreuses bacteries viables. Meme avec des procedures de lavage apparemment satisfaisantes, il a ete demontre a I'aide de la microscopie epifluorescente et de la microscopie electronique par balayage que quelques bacteries peuvent encore survivre et demeurer attachees aux surfaces. Pour deceler la presence de quelques bacteries adherant a une surface, on ne peut pas se fier aux methodes bacteriologiques couramment utilisees en evaluation de I'efficacite d'une procedure de nettoyage. Ceci a ete demontre par la microscopie epifluorescente sur des surfaces venant en contact avec des aliments. Les techniques bacteriologiques comme les plaques d'ecouvillons ou de contact sont longues par comparaison a la microscopie epifluorescente directe. Celle-ci permet I'enumeration et la demonstration de la viabilite d'organismes sur une surface a contact alimentaire en moins de trente minutes. Le developpement de methodes
Copyright © 1991 Canadian Institute of Food Science and Technology
113
The majority of studies on bacterial adherence to solid surfaces have been concerned with the manner in which bacteria attach to the surface and how the organisms can maintain their attachment with subsequent growth. A number of theories have been advanced. The major forces believed to induce contact with a surface are the London-van-der-Waals attractive energies between two surfaces (Marshall et al., 1971). A secondary and more secure form of attachment, involving the secretion of a mucilaginous holdfast, was proposed by Zobell (1943). Fletcher and Floodgate (1973) demonstrated that an acidic polysaccharide layer was involved in the adherence of bacteria to surfaces as did Herald and Zottola (1988) who studied the involvement of acid mucopolysaccharide, in the attachment of microorganisms to stainless steel. Costerton et al. (1978) suggested that bacteria adhere to surfaces by secreting a mass of tangled fibres or glycocalyx. Others have suggested that flagella or fimbria (Notermans and Kampelmacher, 1974; Beachey, 1981; Notermans and Kampelmacher, 1983) may play an important role in bacterial attachment. However, there is some controversy on this theory since Rogers (1979) and Rutter and Vincent (1980) found that organisms without these structures could also adhere to various substrates. The literature on bacterial adherence and theories of attachment is extensive and beyond the scope of this review. More recently, the scanning electron microscope (SEM) has been employed to visually examine the attachment of bacteria to solid surfaces. FirstenbergEden et al. (1981) and Zottola (1986), using the SEM to study bacterial attachment to skin and inert substrates, observed connecting strands of material between the microorganisms and the surface to which they were attached. Others have employed SEM to study bacterial attachment to stainless steel and glass surfaces (Zoltai et al., 1981; Speers et al., 1984; Stone and Zottola, 1985). These workers were concerned with the contamination of the surfaces of holding tanks by raw milk, which could lead to subsequent contamination of other milk processing equipment. They found that any surface could serve as an attachment site for organisms such as Pseudomonas spp., Micrococcus spp. (Speers et al., 1984), Staphylococcus aureus, Streptococcus cremoris, (Zoltai et al., 1981) as well as Pseudomonas jragi (Stone and Zottola, 1985). Speers et al. (1984) found that after a 1 h exposure to contaminated milk at 25°C, followed by varying incubation times up to 72 h, Pseudomonas and Micrococcus species formed small colonies around the original attached bacterium. SEM studies have also shown that while the originally attached bacteria appear to bind to surfaces by extracellular fibrils (Zoltai et al., 1981; Schwach and Zottola, 1982) the organisms underwent cell division to form microcolonies, which appeared to attach more to each other than to the surface (Speers et al., 1984). Since it had been demonstrated that microor114 / Pontefract
ganisms can attach themselves to solid surfaces and subsequently form colonies, it became important to the food industry to obtain information on the ability of these attached bacteria to withstand the cleaning and sanitizing processes used in a food processing plant. A number of studies have been carried out to analyze the efficiency of various cleaning systems (Maxcy, 1969; Baldock, 1974; Dunsmore et al., 1980; Dunsmore and Thomson, 1981; Mabeya et al., 1982; Powell and Slater, 1982; Mrozek, 1985; Stone and Zottola, 1985; Lopez, 1986). Because of the extended turn around time and the risk of damage occurring upon dismantling of food processing equipment for cleaning, one of the most commonly used practices in cleaning and sanitizing equipment used in processing fluid food is the clean-in-place (CIP) system (Barron, 1986). A number of different steps may be employed with this procedure but the method usually includes a pre-wash rinse to remove the bulk of the soil, a detergent wash, a post-detergent rinse and a pre-soil sanitizer which leaves a film of liquid sanitizer on the food contact surface (Gibbons, 1977; Mitchel, 1981). Different chemicals and various combinations of detergents are used in various CIP procedures (Gibbons, 1977; Barron, 1986). The ultimate goal is proper cleaning and sanitizing of the food contact surfaces of food processing equipment. CIP systems cannot be used with surfaces such as conveyor belts and preparation tables; however, these surfaces are usually cleaned with detergents and a suitable sanitizing agent. The two-fold benefit obtained from properly cleaned equipment is the increased preservation of the product and, most importantly from a health point of view, the minimization of the risks of food-borne illness. The methods of determining the efficiency of cleaning procedures are varied. The most commonly employed include the swab-rinse, agar contact, and direct surface plating. The use of all these techniques requires the subsequent culture of the sample in a suitable medium to determine viable organisms and, therefore, the adequacy of the cleaning process (Baldock, 1974; Dunsmore et al., 1980; Dunsmore and Thomson, 1981). Conditions of sampling determine the use of some of these methods. The direct surface agar plating method is not suitable for plant sampling and the agar contact methods (RODAC PLATE) are only useful for smooth, flat, surfaces. Since dilution is not possible, only small numbers of contaminants can be enumerated and, therefore, RODAC PLATES are not suitable for heavily contaminated surfaces (Patterson, 1971; Baldock, 1974). The most widely used technique is the swab-rinse method whereby a sterile swab (usually moistened with a sterile fluid) is rubbed over the surface of the area to be sampled. The main problem with the swab-rinse method is that the swabbing procedures vary per individual, Le., in some cases the pressure on the swab is heavy, in other instances light, and swabbing may be fast, or slow. The technique is not reproducible and this can lead J. Inst. Can. Sci. Technol. Aliment. Vo!. 24, No. 3/4. 1991
to variable microbial counts and incorrect estimates of the numbers of organisms present on the swabbed area. Further, the cotton retains some of the microorganisms causing reduced counts (Barnes, 1952; Holah et al., 1988). To eliminate this latter problem, soluble alginate swabs were developed which dissolve and thus release all organisms collected on the swab. More recently, to determine the effectiveness of CIP and other cleaning procedures in removing bacteria from various surfaces, the scanning electron microscope (SEM) has been used in a number of studies. One study used stainless steel strips which were attached inside a system where CIP could be carried out (Stone and Zottola, 1985). In testing the effect of a commonly used sanitizing agent such as sodium hypochlorite, Schwach and Zottola (1984) observed that bacteria attached to stainless steel were not removed even after the use of 150 ppm hypochlorite, although the viability of the attached microorganisms was not determined. Stone and Zottola (1985) found that considerable numbers of bacteria became attached to the internal surfaces of the processing equipment if the interval between cleanings exceeded 8 h. They also found that if CIP procedures are reduced (dilution of cleaning chemicals greater than recommended by the manufacturer or the lowering of the temperature suggested for proper cleaning), viable attached cells may remain and fresh product coming into the system may be contaminated. These findings become particularly important when one considers the recent trends to longer production runs in the food industry, with a short interval for cleaning and sanitizing; a practice which can lead to inadequate cleaning and sanitation by the CIP system. The principal advantage of SEM examination of surfaces for bacterial contamination is that specimen samples 3 or 4 cm 2 can be quickly scanned with high resolution, and three or four organisms per cm 2 can be detected. Sample test strips of stainless steel, glass, or plastic can be located at strategic areas in food processing plants or even mounted in food processing equipment (Holah et al., 1987), and removed at suitable intervals to ascertain the development of bacterial contamination. The processing of such samples for SEM takes approximately 40 min. The SEM is, however, basically a research tool and is not suitable for general microbiological testing in food plants, although such an instrument could be employed in a regional testing facility to examine samples sent in from outlying processing plants. As discussed previously, traditional methods of assessing surface hygiene in food processing plants and equipment, such as swabs, rinses and contact techniques require long incubation periods (1-4 d) before the presence of bacteria can be determined and counts can be made. Because of this time delay, the next food processing runs are already underway when the results are obtained and, therefore, these techniques will only detect trends in microbial levels Can. Inst. Food Sci. Technal. J. Vol. 24, No. 3/4, 1991
in processing plants and can only provide a history of the plant hygiene (Patterson, 1971; Holah et al., 1987). Modern developments in the food industry have stressed the need for a fast turnaround in cleaning procedures which necessitates the rapid assessment of microbial contamination of food contact surfaces to allow process control during production periods (Holah et al., 1988). A rapid technique for the enumeration of organisms using the light microscope, the Direct Epifluorescent Filter Technique (DEFT) has been developed by Pettipher et al. (1980). The technique was designed to ascertain the bacteriological quality of raw milk. The method involves the filtration of a milk sample through a membrane filter and bacteria concentrated on the filter are stained with acridine orange, which causes them to fluoresce a bright red-orange when viable cells are exposed to ultraviolet or blue-violet light. This technique enables a bacterial count to made from milk samples in less than 25 min. Shaw et al. (1987) have modified DEFT so it can be used with other food products and Holah et al. (1987), by removing organisms from a swab with a vortex mixer before filtration and staining, have modified the method so it can be used to examine bacterial contamination on solid surfaces. Modifications of DEFT have been made that allow the enumeration of microbial surface populations to be made in about 15-20 min (Holah et al., 1987, 1988). This techniques does not use swabbing methods but uses test strips of stainless steel and other materials mounted in or on food processing equipment such as conveyor belts, milk holding tanks, etc. At suitable intervals these test strips are removed, stained and examined with epifluorescence microscopy. This technique, termed Direct Epifluorescent Method (DEM), enables direct examination of contaminated surfaces to be carried out using the same fluorescent stain employed in DEFT, allowing the operator to determine viability of the attached organisms. Studies by these workers indicate that the DEM compares favourably with DEFT and the total viable count determined by the swab method on an equal area of surface. They also determined that the DEM can detect very low numbers of microorganisms that can not be picked up with DEFT, because many organisms were trapped in the fibres of the swab and were not released for growth in culture media or on the membrane filter. Alginate swabs, which would eliminate this problem, were not suitable for DEFT since they tend to clog the filter used in the procedure. The use of DEM (Holah et al., 1987) has also demonstrated that bacterial adherence can occur on many types of surfaces and with different food products such as baked beans or fish. Analytical techniques such as DEFT and DEM and the use of SEM have demonstrated that in such cases where low numbers of viable bacteria may remain attached to supposedly clean and sanitized surfaces, traditional methods such as swabbing or contact Pontefract / 115
plates may not always present a true picture of the microbiological population present. Besides, these latter methods are subject to a considerable time lag before they yield results. This brief review is not meant to cover all the literature on such a broad subject but it is sufficient to show that there are unresolved problems in applying standard bacteriological sampling techniques in a realistic time frame for modern food processing procedures. Further studies, employing techniques such as epifluorescence microscopy and utilizing research instruments such as the scanning electron microscope, are required to obtain additional information on problems of bacterial attachment in food processing equipment. Also, additional information is needed to determine efficiency of standard sampling techniques in relation to these biofilms. Such studies as the direct examination of contaminated surfaces to determine the viability of organisms and their resistance to various cleaning procedures could provide valuable information on bacterial survival in food processing equipment. The use of test strips at various sites in food processing plants using DEM, would permit almost continuous monitoring of the bacterial contamination level in the plant and on some food processing equipment. The results of these and other studies which may help in the development of fast, accurate, bacteriological test procedures can be important in the assessment of hygienic practices in the food industry.
References Baldock, J.P. 1974. Microbiological monitoring of thefood plant: methods to assess bacterial contamination on surfaces. J. Milk Food Technol. 37:361. Barnes, 1.M. 1952. The removal of bacteria from glass surfaces with calcium alginate, gauze and absorbent cotton wool swabs. Proc. Soc. Appl. Bacteriol. 15:34. Barron, W. 1986. CIP: Change and challenge for the dairy engineer. Dairy Ind. Int. 51:14. Beachey, E.H. 1980. (Ed.). Bacterial Adherence. Chapman and Hall, London, Methuen, NY. Beachey, E.H. 1981. Bacterial Adherence: adhesin receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143:325. Berkeley R.C.W., Lynch, J.M., Smelling, J., Rutter, P.R. and Vincent, B. 1980. (Eds.). Microbial Adhesion to Surfaces. Ellis Horwood. Chichester, UK. Characklis, W.G. 1981. Fouling biofilm development: A process analysis. Biotech. Bioeng. 23:1923. Costerton, 1.W., Geesly, G.G. and Cheng, K.J. 1978. How bacteria stick. Sci. Amer. 238:86. Duguid, J.P. and Old, D.C. 1980. Adhesive properties of Enterobacteriacea. In: Bacterial Adherence. E.H. Beachey (Ed.). p. 184. Chapman and Hall. London, Methuen, NY. Dunsmore, D.G., Westwood, D.A., Jay, D.B. and Embling, M. 1980. Simulator technique for assessing the bacteriological control of food equipment surfaces by cleaning systems. J. Food Prot. 43:850. Dunsmore, D.G. and Thomson, M. 1981. Bacteriological control of food equipment surfaces by cleaning systems. 2 Sanitizer effects. 1. Food Prot. 44:21. Dunsmore, D.G., Twomey, A., Whittlestone, W. G. and Morgan, H. W. 1981. Design and performance of systems for cleaning product-contact surfaces of food equipment. A review. J. Food Prot. 44:220.
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Submitted April 10, 1989 Revised December 5, 1990 Accepted December 8, 1990
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