Kathryn A. Whitehead

The detection of food soils and cells on stainless steel using industrial methods: UV illumination and ATP bioluminescence.

Open food contact surfaces were subjected to organic soiling to provide a source for transfer of microbial cells. Rapid industrial methods used for the detection of residual cells and soil e.g. ATP (adenosine triphosphate) bioluminescence and an ultraviolet (UV) light detection method were assessed for their ability to detect organic soils, or organic soil-cell mix on surfaces. A range of soils (complex [meat extract, fish extract, cottage cheese extract]; oils [cholesterol, fish oil, mixed fatty acids]; proteins [bovine serum albumin, fish peptones casein]; carbohydrates [glycogen, starch, lactose]); was used. Under UV, oily soils, mixed fatty acids, cholesterol and casein were detected at low concentrations, with detection levels ranging from 1% to 0.001% for different substances. Glycogen was the most difficult substance to detect at lower concentrations. Using UV wavelength bands (lambda) of 330-380 nm, 510-560 nm and 590-650 nm, wavelength bands of 330-380 nm, illuminated most of the soils well, whilst the wavelength band of 510-560 nm illuminated the fish extract, cholesterol and fatty acids; the 590-650 nm wavelength band illuminated the lactose. Soils at all concentrations were detected by the ATP bioluminescence method; the complex soils gave the highest readings. When complex soils were combined with Listeria monocytogenes Scott A or a non-pathogenic Escherichia coli O157:H7, ATP measurements increased by 1-2 logs. For UV illumination, the L. monocytogenes and cheese combination was the most intensely illuminated, with E. coli and meat the least. UV illumination is a simple well established method for detecting food soil, with little change in findings when microorganisms are included. Performance can be enhanced in certain circumstances by altering the wavelength. ATP bioluminescence is a proven system for hygienic assessment being especially useful in the presence of microorganisms rather than organic soil alone.

Factors affecting microbial adhesion to stainless steel and other materials used in medical devices.

The role of biofilm in medical device associated infections is well documented. Biofilms are more resistant to antibiotics than planktonic cells, these are extremely difficult to treat. Prevention strategies include efforts to insert implants under stringent aseptic conditions, and also encompass the development of novel materials which interfere with the initial attachment of microorganisms to the surface of the device. Microbial cells also attach onto hygienic surfaces in the hospital setting, and thereby pose a cross-infection problem. In this case, vigorous cleaning and sanitizing regimes may be employed in addition to any surface modifications. Many factors affect the initial attachment of organisms to inert substrata, and their subsequent retention or removal/detachment, including the physical and chemical nature and location of the substratum, the type of organic material and microorganisms potentially fouling the surface, and the nature of the interface (solid-liquid in the body; solid-air on environmental surfaces). Focusing on one factor, surface topography, it is apparent that many further variables need to be defined in order to fully understand the interactions occurring between the cell and surface. It is therefore important when modifying one substratum surface property in order to reduce adhesion, to also consider other potentially confounding factors.

Microbial retention on open food contact surfaces and implications for food contamination.

Publisher Summary In the food industry, food contact surfaces are generally described as being “open” or “closed”. Closed surfaces are primarily pipework, where wet product or ingredients are contained within a flowing, liquid system. Open surfaces are exposed, with moist or dry food passing along conveyors; thus liquid does not necessarily enclose the food, or cover the surface, and consequently flow is absent. Closed systems present any contaminating microorganisms with a solid–liquid interface for attachment and colonization. Access to these surfaces for cleaning is difficult, thus the opportunity exists for the development of biofilm. Open surfaces present a solid–air–, or a solid–liquid–air interface, where microorganisms attached on the surface may encounter an environment less conducive to growth, encompassing opportunity for dehydration during drying, lack of moisture, and exposure to regular cleaning and disinfection. Thus for hygienic, open, food contact surfaces, the retention and survival of viable microorganisms pre- and postcleaning and disinfection is of key concern.

The retention of bacteria on hygienic surfaces presenting scratches of microbial dimensions

Aims:  To produce surfaces of defined linear topographical features which reflect those found on worn and new stainless steel, to monitor the effect of feature dimensions on the retention of Listeria monocytogenes and Staphylococcus sciuri.

Titanium-coating of stainless steel as an aid to improved cleanability

In the food industry, wear of surfaces provides numerous topographical features in which microorganisms may be retained. We hypothesise that by modifying the surface chemistry, this effect may be decreased. Cellulose acetate sheets softened with acetone were pressed onto both new fine polished and used stainless steel surfaces in order to take impressions of surface features for subsequent visualisation and characterisation in the laboratory, using scanning electron microscopy, atomic force microscopy (R(a)) and white light profilometry (S(a)). The method gives high resolution negative replicas of the surface, can be used quickly, safely and efficiently, and enables investigations into surface wear over time, and the effect of defined topographic features on surface hygiene and cleanability. Subsequently, the retention of microorganisms on fine polished stainless steel and titanium coated fine polished stainless steel was assessed in the presence and absence of a meat conditioning film. The titanium coating discouraged the retention and enhanced the removal of both Escherichia coli and the meat conditioning film.

Initial adhesion of Listeria monocytogenes to solid surfaces under liquid flow.

Some strains of the food borne pathogen Listeria monocytogenes persist in food processing environments. The exact reason behind this phenomenon is not known, but strain differences in the ability to adhere to solid surfaces could offer an explanation. In the present work, initial adhesion of nine strains of L. monocytogenes was investigated under liquid flow at two levels of shear stress on six different surfaces using a flow chamber set-up with microscopy measurements. The surfaces tested were glass and PVC, and glass coated with beef extract, casein, and homogenised and unhomogenised milk. In addition, the effect of prior environmental stress (5% NaCl, low nutrient availability) on initial adhesion was investigated. The hydrophobicity of the investigated surfaces was determined by contact angle measurements and the surface properties of the investigated L. monocytogenes strains were determined using Microbial Adhesion To Solvents (MATS). All surfaces with the exception of PVC were found to be hydrophilic. Strain differences were found to significantly influence the initial adhesion rate (IAR) of all nine strains to all the surfaces (p<0.05) at both low and high shear stress. Furthermore, there was a significant effect of the surfaces tested (p<0.05) in the adhesion ability of almost all strains. The IAR was affected by flow rate (shear stress) as seen by a decrease in adhesion at high shear stress for most strains. A significant effect of interactions between strain-surface and strain-shear stress (p<0.001) was observed but not of interactions between surface-shear stress. No correlation between surface hydrophobicity and IAR was observed. Addition of 5% NaCl during propagation resulted in a decrease in IAR whilst propagation in low nutrient media caused an increase indicating a general change in surface characteristics under these conditions. Known persisting strains did not display general better adherence.

The detection and influence of food soils on microorganisms on stainless steel using scanning electron microscopy and epifluorescence microscopy

A range of food soils and components (complex [meat extract, fish extract, and cottage cheese extract]; oils [cholesterol, fish oil, and mixed fatty acids]; proteins [bovine serum albumin (BSA), fish peptones, and casein]; and carbohydrates [glycogen, starch, and lactose]) were deposited onto 304 2B finish stainless steel surfaces at different concentrations (10-0.001%). Scanning electron microscopy (SEM) and epifluorescence microscopy were used to visualise the cell and food soil distribution across the surface. Epifluorescence microscopy was also used to quantify the percentage of a field covered by cells or soil. At 10% concentration, most soils, with the exception of BSA and fish peptone were easily visualised using SEM, presenting differences in gross soil morphology and distribution. When soil was stained with acridine orange and visualised by epifluorescence microscopy, the limit of detection of the method varied between soils, but some (meat, cottage cheese and glycogen) were detected at the lowest concentrations used (0.001%). The decrease in soil concentration did not always relate to the surface coverage measurement. When 10% food soil was applied to a surface with Escherichia coli and compared, cell attachment differed depending on the nature of the soil. The highest percentage coverage of cells was observed on surfaces with fish extract and related products (fish peptone and fish oil), followed by carbohydrates, meat extract/meat protein, cottage cheese/casein and the least to the oils (cholesterol and mixed fatty acids). Cells could not be clearly observed in the presence of some food soils using SEM. Findings demonstrate that food soils heterogeneously covered stainless steel surfaces in differing patterns. The pattern and amount of cell attachment was related to food soil type rather than to the amount of food soil detected. This work demonstrates that in the study of conditioning film and cell retention on the hygienic properties of surfaces, SEM may not reveal the presence of retained conditioning film, and thus methods such as epifluorescence microscopy should also be used. This is an essential facet to the methodology design of future work carried out in our laboratories on the effectiveness of the removal of cells and conditioning films from surfaces using different cleaning regimes.

The use of physicochemical methods to detect organic food soils on stainless steel surfaces

Food processing surfaces fouled with organic material pose problems ranging from aesthetic appearance, equipment malfunction and product contamination. Despite the importance of organic soiling for subsequent product quality, little is known about the interaction between surfaces and organic soil components. A range of complex and defined food soils was applied to 304 stainless steel (SS) surfaces to determine the effect of type and concentration of soil on surface physicochemical parameters, viz surface hydrophobicity (ΔGiwi ), surface free energy (γs), Lifshitz van der Waals ( ), Lewis acid base ( ), electron acceptor ( ) and electron donor ( ) measurements. When compared to the control surface, changes in and were indicative of surface soiling. However, soil composition and surface coverage were heterogeneous, resulting in complex data being generated from which trends could not be discerned. These results demonstrate that the retention of food soil produces changes in the physicochemical parameters of the surface that could be used to indicate the hygienic status of a surface.

The Effect of Substratum Properties on the Survival of Attached Microorganisms on Inert Surfaces

Biofilm formation is dependent on the surrounding environmental conditions and substratum parameters. Once a biofilm forms many factors may influence cell survival and resistance. Cell adhesion to a surface is a prerequisite for coloniza- tion. However, attached microorganisms may not be able to multiply, and may merely be surviving on the surface, for example at a solid-air interface, rather than forming a biofilm. Retention of attached cells is a key focus in terms of surface hygiene and bio- film control. Factors that affect this retention may differ from those affecting biofilm formed at the solid-liquid interface: the nature of the substratum, presence of organic material, vitality of the attached microorganism, and of course the surrounding envi- ronment. The majority of publications focus on the solid-liquid interface; literature addressing the solid-air interface is considerably less substantial.

The detection of food soils on stainless steel using energy dispersive X-ray and Fourier transform infrared spectroscopy

Organic soiling is a major issue in the food processing industries, causing a range of biofouling and microbiological problems. Energy dispersive X-ray (EDX) and Fourier transform infra red spectroscopy (FT-IR) were used to quantify and determine the biochemical groups of food soils on stainless steel surfaces. EDX quantified organic material on surfaces where oily based residues predominated, but was limited in its usefulness since other food soils were difficult to detect. FT-IR provided spectral ‘fingerprints’ for each of the soils tested. Key soiling components were associated with specific peaks, viz. oils at 3025 cm−1–3011 cm−1, proteins at 1698 cm−1–1636 cm−1 and carbohydrates at 1658 cm−1–1596 cm−1, 783 cm−1–742 cm−1. High concentrations of some soils (10%) were needed for detection by both EDX and FT-IR. The two techniques may be of use for quantifying and identifying specific recalcitrant soils on surfaces to improve cleaning and hygiene regimes.