Gun Wirtanen

Biofilm formation in the industry: A review

Abstract Biofilm and biofouling refer to biological deposits on any surface. Biofilms consist of both microbes and their extracellular products, usually polysaccharides. The purpose of biofilm is to protect the microbes from hostile environments or to act as a trap for nutrient acquisition. Biofilm formation causes problems in many branches of industry, such as in industrial water systems and the medical and process industries. Besides causing problems in cleaning and hygiene, biofilm may cause energy losses and blockages in condenser tubes, cooling fill materials, water and wastewater circuits, and heat exchange tubes, and on ship hulls. Biofilm can also present microbial risks due to the release of pathogens from cooling towers or by reducing water quality in drinking water distribution systems. In the medical industry biofilm is referred to as glycocalyx when diseases of the lungs or the gastrointestinal or urinary tract are involved.

Microbial evaluation of the biotransfer potential from surfaces with Bacillus biofilms after rinsing and cleaning procedures in closed food-processing systems.

The biotransfer potential in food processing is defined as the ability of the microorganisms present on equipment surfaces both before and after cleaning procedures to contaminate products during processing. Growth of Bacillus biofilms was detectable on both stainless-steel and Teflon© surfaces after all the cleaning procedures. B. cereus and B. subtilis cells adhered more firmly to unsoiled than to soiled surfaces, whereas B. thuringiensis adhered most firmly to soiled surfaces. The results showed that the removal of biofilms from surfaces was more difficult from stainless steel because the microorganisms were attached more tenaciously to rough surfaces. Biofilms were cleaned most effectively from unsoiled surfaces with a simple rinsing procedure; thus the mechanical forces of the flow are very important in the cleaning. The chemical and heat treatments used for cleaning had the greatest impact on the abundance of living microorganisms. All the procedures tested did not remove biofilm material effectively from the surfaces. Significantly more cells were removed when the alkaline phase in the alkaline-acid treatment of the cleaning-in-place (CIP) procedure was prolonged, and ethylene diaminetetraacetic acid (EDTA) was used.

Methodologies for the characterization of microbes in industrial environments: a review

Abstract. There is growing interest in research and development to develop novel tools to study, detect, and characterize microbes and their communities in industrial environments. However, knowledge about their validity in practical industrial use is still scarce. This review describes the advantages and limitations of traditional and molecular methods used for biofilm and/or planktonic cell studies, especially those performed with Listeria monocytogenes, Bacillus cereus, and/or Clostridium perfringens. In addition, the review addresses the importance of isolating the microorganisms from the industrial environment and the possibilities and future prospects for exploiting the described methods in the industrial environment.

Microbiological methods for testing disinfectant efficiency on Pseudomonas biofilm.

Biofilms of the Gram-negative bacteria Pseudomonas aeruginosa and Pseudomonas fragi were grown on stainless steel surfaces (AISI 304, 2B) for 4 days in slime broth. These biofilms were treated with four commercial disinfectants. The disinfectants were alcohol-based, tenside-based, peroxide-based and chlorine-based products, covering most disinfectant types used in the food industry. The effects of the disinfectants on the bacterial cells were first investigated in suspension using the permeabilisation test, which is based on fluorescence assessment of hydrophobic 1-N-phenyl-naphtylamine (NPN). The surfaces covered with disinfectant-treated biofilms were investigated using conventional cultivation, impedimetry and epifluorescence microscopy in combination with image analysis of preparations stained with the DNA-stain acridine orange and with the metabolic indicator system CTC-DAPI. The results showed that the tenside-based and peroxide-based disinfectants permeabilised the cells in suspension. The overall biofilm results showed that of the agents tested, the peroxide-based and chlorine-based disinfectants acted most effectively on cells in biofilms.

Microbial testing methods for detection of residual cleaning agents and disinfectants-prevention of ATP bioluminescence measurement errors in the food industry.

The ATP luminescence measurement is based on the presence of an enzymatic reaction and may significantly be affected by cleaning agents and disinfectants. In addition, disinfectants can also reduce the activity of the luciferase enzyme and also act as ATP-releasing agents. The agents disrupt the cell walls but preserve ATP in measurable form, and therefore correlation with culture methods can be poor. Therefore, if a rapid method is used to detect ATP, a control must be used for reliable results. The possible effect of disinfectants can be eliminated with a rapid test to minimize sources of error. In the present study a microbiological residue testing method that is nonspecific for residues was developed. The effects of a total of 38 commercial cleaning agents and disinfectants of various types were assessed using two microbiological methods, the Vibrio fischeri photobacteria test and Micrococcus luteus inhibition zone technique. The results show that the V. fischeri photobacteria test is very sensitive. This test can therefore be used for testing cleaning agent residues on surfaces in very small amounts. A small study was also carried out in a food factory to show applicability in processing facilities. The study showed, that a need for this type of method exists in food processing.

Evaluation of epifluorescence image analysis of biofilm growth on stainless steel surfaces

Biofilm growth of Bacillus subtilis, Pseudomonas fragi, Pediococcus inopinatus and Listeria monocytogenes was studied on stainless steel surfaces at room and low temperatures to evaluate the results of traditional hygiene measures. The results were compared with those of image analysis of stainless steel surfaces in an epifluorescence microscope. Statistical analyses were carried out to determine the variations between the conventional cultivation swab method, the glycocalyx amount obtained using swabbing, and the values of the areas of the biofilm, slime and cells. As a general rule, old biofilms showed total counts at approximately the same levels as the young biofilm. The results showed that temperature affected the results for all strains except B. subtilis. The strains of Pe. inopinatus and Ps. fragi showed increased attachment at 6°C and L. monocytogenes at 25°C. The biofilm slime was more easily detached than the cells. The results indicated that the traditional swab method is not reliable for the measurement of biofilm formation on surfaces.

Ultrasonic cleaning of conveyor belt materials using Listeria monocytogenes as a model organism.

Persistent Listeria monocytogenes contamination of food industry equipment is a difficult problem to solve. Ultrasonic cleaning offers new possibilities for cleaning conveyors and other equipment that are not easy to clean. Ultrasonic cleaning was tested on three conveyor belt materials: polypropylene, acetal, and stainless steel (cold-rolled, AISI 304). Cleaning efficiency was tested at two temperatures (30 and 45 degrees C) and two cleaning times (30 and 60 s) with two cleaning detergents (KOH, and NaOH combined with KOH). Conveyor belt materials were soiled with milk-based soil and L. monocytogenes strains V1, V3, and B9, and then incubated for 72 h to attach bacteria to surfaces. Ultrasonic cleaning treatments reduced L. monocytogenes counts on stainless steel 4.61 to 5.90 log units; on acetal, 3.37 to 5.55 log units; and on polypropylene, 2.31 to 4.40 log units. The logarithmic reduction differences were statistically analyzed by analysis of variance using Statistical Package for the Social Sciences software. The logarithmic reduction was significantly greater in stainless steel than in plastic materials (P < 0.001 for polypropylene, P = 0.023 for acetal). Higher temperatures enhanced the cleaning efficiency in tested materials. No significant difference occurred between cleaning times. The logarithmic reduction was significantly higher (P = 0.013) in cleaning treatments with potassium hydroxide detergent. In this study, ultrasonic cleaning was efficient for cleaning conveyor belt materials.

Prevalence of Listeria monocytogenes in, and Microbiological and Sensory Quality of, Rainbow Trout, Whitefish, and Vendace Roes from Finnish Retail Markets

The prevalence of Listeria monocytogenes in retail roe, as well as the microbiological and sensory qualities of the roe, were studied for three fish species under three different storage conditions. A total of 147 Finnish rainbow trout (Oncorhynchus mykiss), whitefish (Coregonus lavaretus), vendace (Coregonus albula), and burbot (Lota lota) roe samples were bought fresh, frozen, or frozen-thawed from Finnish retail markets. The overall prevalence of L. monocytogenes was 5%; however, the prevalence of the pathogen in fresh roe was 18%. Fresh-bought roe tested positive for Listeria spp. and for L. monocytogenes, respectively, 5 and 20 times as often as did frozen and frozen-thawed roe products combined. The microbiological quality (analyzed as total aerobic heterotrophic bacteria and coliform bacteria) of 78% of the roe samples was unacceptable. Frozen roe samples were found to have the best microbiological quality. According to the results of a sensory evaluation, at least one sensory attribute (appearance, odor freshness, texture, and freshness of taste) was unacceptable for 29% of the roe samples studied. The sensory quality of roe samples bought fresh was better than that of roe samples bought frozen or frozen-thawed. From the results of this study, it is concluded that both the microbiological and the sensory qualities of roe at the retail level need to be improved.

Penetration of bacteria through microholes in semirigid aseptic and retort packages

The penetration of vegetative cells of Enterobacter aerogenes , Bacillus subtilis , Micrococcus varians , and Leuconostoc mesenteraides through laser-drilled holes of diameter 10 to 20 μm in the lid of commercially manufactured semirigid aseptic cups filled with milk chocolate pudding and retort trays filled with spaghetti in meat sauce (only E. aerogenes ) was studied. The ability of bacteria to pass via leakages was determined using an immersion biotest method. The threshold leakage diameter determined for both products tested was about 10 μm. The contamination rate of leaking packages was much higher for aseptic cups than retort trays. The results indicated that the factors increasing the rate of bacterial penetration into test packages are large hole size, small hole length, small bacteria diameter and leakage channels filled with liquid instead of dried foodstuff and/or bacterial suspension. The results are useful as a guideline for the requirements of on-line, nondestructive, package-integrity testers for testing packages.

Measurement of biofilm of Pediococcus pentosaceus and Pseudomonas fragi on stainless steel surfaces

Abstract Biofilm was grown on stainless steel surfaces (AISI 304 and AISI 316) for 2, 5 and 10 days in slime broth inoculated with Pediococcus pentosaceus and Pseudomonas fragi . The steel surfaces on which biofilm had developed were investigated using conventional plating, quantitative glycocalyx determination and epifluorescence microscopy with image analysis. The results showed that after 10 days of growth the cells of P. fragi were difficult to cultivate from the surface and the growth was detected better by microscopy and image analysis. The slime produced by P. pentosaceus could be demonstrated by epifluorescence image analysis and quantitative glycocalyx measurement. The methods for detecting bacterial growth on surfaces are discussed with reference to conventional plating, image analysis and biofilm formation.