Direct Epifluorescence Microscopy Research Articles

P157 Entomophthoromycosis: a rare fungal infection case series

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesTo present a case series of entomophthoromycosis (a rare fungal infection of the skin and subcutaneous tissue with a protracted and chronic clinical evolution), which would aid in the early recognition and management of this condition.Materials and MethodsA total of 7 laboratory-confirmed cases of entomophthoromycosis, diagnosed and treated between May 2018 and March 2022 at our center, were included in the series. Diagnostic criteria were based on the results of conventional mycological diagnostic methods, including direct epifluorescence microscopy and visual identification of isolates obtained by culture on solid media. Clinical histories were collected retrospectively by chart review and correlated with laboratory findings.ResultsOf the seven patients, six were male and one was female, with ages ranging from 2.5 to 42 years. Most of the patients presented with chronic progressive nasal or rhinofacial swelling and nasal obstruction. Aseptate or pauciseptate hyphae were observed in direct epifluorescence microscopy of tissue samples from all the patients. Conidiobolus spp was isolated from samples from five patients, and Basidiobolus spp from the remaining two. Although growth in culture was obtained several days after sample inoculation, specific antifungal treatment for entomophthoromycosis (using a combination of potassium iodide and itraconazole) was initiated early based on concurrence between clinical features, typical fungal elements seen in direct microscopy and histopathological findings. All the patients improved rapidly and made full recoveries.ConclusionEntomophthoromycosis is a rare condition whose identification requires a high index of clinical suspicion. The treatment for this condition is specific and different from the treatment for other clinically significant invasive fungal infections, but confirmation of diagnosis by culture may take weeks. Therefore, early diagnosis supported by a combination of suggestive clinical features as well as mycological and histopathological evidence, is vital to ensure successful outcomes.

P275 Dematiaceous fungi as a rare cause of fungal sinusitis in a tertiary care center

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM ObjectivesTo discuss the occurrence and diagnosis of dematiaceous fungi as a causative organisms of fungal sinusitis in patients at a tertiary care center in North India. Since there is limited data on its prevalence, this study was aimed to know the non-Aspergillus causes of fungal sinusitis focusing on the dematiaceous fungi.MethodsA total of 451 nasal biopsy samples, from the department of pulmonary medicine ward and ICU were received over a period of 3 years, from January 2019 to December 2021. The samples were subjected to conventional mycological diagnostic techniques including direct epifluorescence and light microscopy, culture on solid media and visual identification of growth in culture using lactophenol cotton blue mounts.ResultsOut of 451 samples, no fungi were isolated from 299 samples (66.29%), Aspergillus spp from 63 samples (13.96%), dematiaceous fungi from 10 samples (2.21%), and other fungi from 79 samples (17.5%). Among the dematiaceous fungi isolated, 7 isolates were identified as Alternaria spp. (70%) and 3 isolates were identified as Curvularia species (30%) and described.ConclusionMost reported cases of allergic and invasive sinusitis were attributed to Aspergillus spp. However, in the current study, dematiaceous ‘black’ fungi like Alternaria and Curvularia, were also identified as causes of fungal sinusitis in both immunocompromised and immunocompetent individuals, showing an increasing pathogenic spectrum. Hence a high index of clinical suspicion and appropriate laboratory diagnosis assists in initiating appropriate treatment such as surgical debridement, reducing immunosuppression, and antifungal treatment with newer azoles.

Removal of Listeria monocytogenes biofilms on stainless steel surfaces through conventional and alternative cleaning solutions

Conventional treatments are not effective enough to completely remove Listeria monocytogenes biofilms from surfaces, thus implying the presence of certain persistent bacterial forms. In this study, eleven treatments (i.e. two enzymatic agents applied at two different temperatures and concentrations, two alkaline cleaners and one acid detergent) were used to remove mature L. monocytogenes S2-bac biofilms. A combined treatment was then selected for its application to four different L. monocytogenes strains (i.e. CECT 5672, CECT 935, S2-bac, EDG-e). Effectivity of the treatments was evaluated quantitatively using TEMPO and qualitatively by direct epifluorescent microscopy (DEM). Bacterial detachment obtained after the application of acid, alkaline and chlorinated alkaline treatments were 6.03, 6.24 and 4.76 Log CFU/cm2, respectively. Enzymatic treatments applied at 50 °C obtained the greatest detachment and biocidal activity. The results derived from the observation of the remaining biofilm structure by DEM proved that conventional treatments were unable to completely remove conformed structures with the potential risk this entails. Last, the application of a combined treatment using a chlorinated alkaline cleaner followed by an enzymatic treatment enhanced the dispersal of the bacterial cells from surfaces, thus consolidating this as a good option to recommend for the 5-step cleaning procedure.

Pathogenic mono-species biofilm formation on stainless steel surfaces: Quantitative, qualitative, and compositional study

The aims of this study were, first, to determine Listeria monocytogenes, Salmonella enterica, ser. Typhimurium, and Staphylococcus aureus biofilm counts, structure, and their composition in macromolecules by direct epifluorescence microscopy (DEM); and second, to evaluate the distribution of the components of the produced biofilms through 3D representations obtained by confocal laser scanning microscopy (CLSM). Results showed that all assessed strains have a high capacity to generate biofilms, with counts greater than 7 log CFU cm⁻2 in all cases. The highest and lowest survival percentages were obtained by S. aureus with 94.13 ± 1.77% and S. Typhimurium with 60.31 ± 4.45%, respectively. Biofilm matrix composition in macromolecules was as follows: (a) L. monocytogenes: proteins 83.35 ± 5.81%, polysaccharides 10.98 ± 5.77%, and extracellular DNA 5.67 ± 2.5%; (b) S. Typhimurium: 83.37 ± 3.35%, 0.68 ± 0.77%, and 15.95 ± 3.39%, respectively; and (c) S. aureus: 82.55 ± 2.39%, 8.48 ± 2.07%, and 8.97 ± 2.27%, respectively. The qualitative analysis of the 3D representations of the biofilms formed by the pathogens modelized showed no homogeneity and/or ordered distribution of their components within the biofilm architecture. These findings could lead to the development of addressed cleaning and disinfection alternatives.

Evaluation of bacterial population using multiple sampling methods and the identification of bacteria detected on supermarket food contact surfaces

This study tested multiple methods for assessing microbial populations and identified bacteria detected on supermarket food-contact surfaces. The results for culture medium–based conventional methods, i.e., swabbing, direct count, sampling discs, and Petrifilm, showed that surface cleaning was satisfactory. However, direct epifluorescence microscopy (DEM) and bioluminescence detection of adenosine triphosphate (ATP) showed that surface cleaning was unsatisfactory. The surfaces of the fruit and vegetables section were the most contaminated, followed by the sections for cured meats and cheeses, fresh seafood, the aisles of the sections tested, meat, and yogurts and desserts. Likewise, significantly higher microbiological counts were observed on painted wood than on stainless steel, plastic, and painted epoxy surfaces (p < 0.05). Room-temperature food storage areas had higher bacterial counts than refrigerated areas (p < 0.05). Although no Salmonella spp., Listeria monocytogenes, or Staphylococcus aureus were detected, opportunistic pathogenic bacteria such as coagulase-negative staphylococci, Listeria grayi, and Pseudomonas spp. were isolated. Furthermore, the identification of the opportunistic and emerging foodborne pathogen Cronobacter sakazakii and the indicator of bacterial contamination Escherichia coli demonstrates potential risks to consumer health. Moreover, the Enterobacteriaceae count was low; however, the high total microbiological count detected by DEM indicates that cleaning and disinfection procedures should be improved. Compared to conventional methods, the use of DEM with stainless-steel disc surfaces as sampling support could increase the accuracy of detecting and enumerating microbial populations on surfaces by identifying residual bacteria after inadequate disinfection procedures.

Effect of an enzymatic treatment on the removal of mature Listeria monocytogenes biofilms: A quantitative and qualitative study

Biofilms increase the probability of microorganism survival and potentiate subsequent food product cross-contamination. The search for alternatives capable of eliminating the matrix produced by these communities, and accessing to the cells that made them up, is of real interest. This study aimed to evaluate the effectiveness of an enzymatic product on the detachment of mature Listeria monocytogenes biofilms formed on stainless steel surfaces. Biofilms were produced for one week with washes and replenishment of nutrients and, when formed, enzymatic treatments were applied at 50 °C for 20 min. The cells remaining on the surface were quantified by TEMPO and qualitatively evaluated by direct epifluorescent microscopy. The results showed that the maximum reduction obtained was approximately 6.9 log CFU/cm2 for the strain L. monocytogenes 5672, with a mature biofilm removal between of 85–99% for the different L. monocytogenes strains. Furthermore, the results showed that the detachment of mature biofilms may be directly related to the strain of L. monocytogenes, which could be because some strains can generate structures with a more robust matrix, making their detachment more difficult. Enzymatic cleaning treatment could be exploited as a potent technology to combat mature biofilms in the food industry.

Quantitative and Compositional Study of Monospecies Biofilms of Spoilage Microorganisms in the Meat Industry and Their Interaction in the Development of Multispecies Biofilms.

Food spoilage is a serious problem in the food industry, since it leads to significant economic losses. One of its main causes is the cross-contamination of food products from industrial surfaces. Three spoilage bacterial species which are highly present in meat and the gastrointestinal tract of chickens were selected: Pseudomonas fragi, Leuconostoc gasicomitatum, and Lactobacillus reuteri. The dual aim was to determine their ability to form monospecies biofilms and to examine how they interact when they coexist together. To do so, mature monospecies biofilms were produced statically for seven days at a temperature of 30 °C. L. gasicomitatum was also used to investigate the behavior of P. fragi and L. reuteri in the formation of multispecies biofilms. The structure and composition of the monospecies biofilms were evaluated by direct epifluorescence microscopy, and the multispecies biofilms were evaluated by plate counting. Both L. gasicomitatum and L. reuteri were able to form biofilms, with counts of approximately 7 Log CFU/cm2 and a defined structure. However, P. fragi obtained counts to the order of 4 Log CFU/cm2, which is significantly different from the previous species (P < 0.05), and it had no network of cell conglomerates. The content of the L. gasicomitatum and L. reuteri biofilm matrices were 70–80% protein, unlike P. fragi, which presented a higher polysaccharide content (P < 0.05). In the multispecies biofilms, the presence of P. fragi did not affect the growth of L. gasicomitatum, which remained at between 5.76 to 6.1 Log CFU/cm2. However, L. reuteri was able to displace L. gasicomitatum growth after 24 h of coexisting in a mixed biofilm, presenting differences in counts of approximately 2 Log CFU/cm2. The study of the biofilms constructed by food industry resident microbiota can help to understand the ecological relations that exist between species, characterize them, and propose strategies to eliminate them. The name of genes and species should be written in italic.

Coomassie Blue G250 for Visualization of Active Bacteria from Lake Environment and Culture.

Bacteria play a fundamental role in the cycling of nutrients in aquatic environments. A precise distinction between active and inactive bacteria is crucial for the description of this process. We have evaluated the usefulness of Coomassie Blue G250 for fluorescent staining of protein containing potentially highly active bacteria. We found that the G250 solution has excitation and emission properties appropriate for direct epifluorescence microscopy observations. It enables fast and effective fluorescent visualization of living, protein-rich bacteria, both in freshwater environment and culture. Our results revealed that the number of G250-stained bacteria from eutrophic lake was positively correlated with other standard bacterial activity markers, like number of bacteria containing 16S rRNA, bacterial secondary production or maximal potential leucine-aminopeptidase activity. In case of the E. coli culture, the percentage of bacteria visualized with G250 was similar to that of bacteria which accumulated tetracycline. Compared to other common methods utilizing fluorogenic substances for bacteria staining, the approach we evaluated is inexpensive and less hazardous (for example mutagenic) to the environment and researchers. It can be regarded as an additional or alternative method for protein rich, active bacteria staining.

Virus abundance in alluvial-sod soil determined by direct epifluorescence microscopy

Virus abundance in alluvial-sod soil determined by direct epifluorescence microscopy

Ultraviolet germicidal coil cleaning: Decreased surface microbial loading and resuspension of cell clusters

Cooling coil surfaces within building ventilation systems are ideal sites for biofilm formation due to the presence of adequate nutrients (deposited particles) and moisture (condensate). In this study, a heating, ventilation, and air-conditioning (HVAC) test apparatus was built consisting of two parallel ducts, each with its own cooling coil. One coil was exposed to ultraviolet germicidal coil cleaning (UVG-CC) while the other was the comparison control to investigate the impact of UVG-CC on surface microbial loading and bacterial attachment. Surface samples were collected by swabbing a uniform area of coil surface and airborne samples were collected isokinetically with sterile funnel filters. All samples were quantified via direct epifluorescent microscopy. Prior to irradiating, higher concentrations of surface microbial loading were found on the downstream side of both cooling coils under condensing conditions. Conversely, under dry surface conditions with downstream UV irradiance, surface concentrations were higher upstream. UVG-CC (at an average 200 uW/cm2 on the coil surface) reduced surface microbial loading by 55% on average during condensing conditions and inhibited bacterial attachment causing clusters of bacterial matter to become airborne downstream of the cooling coil. Additionally, it was found that desiccation also inhibited surface microbial loading and yielded cluster detachment but to a lesser degree than UVG-CC treatment.

Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces

One source of foodborne listeriosis is related to a virulent strain established in the food-processing environment. We used direct epifluorescence microscopy (DEM) to evaluate the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces, in wet conditions at room temperature. Biofilms that were allowed to develop for 72 h were subsequently treated with different concentrations of phage (5, 6, 7, and 8 log PFU/ml). L. monocytogenes was monitored up to 48 h, using both DEM and cultivation method. When L. monocytogenes was monitored using a cultivation method, the first significant reduction was observed after phage treatment with 7 and 8 log PFU/ml at 8 h. Subsequently, these treatments achieved undetectable levels of pathogen at 48 h, with a mean reduction of 5.29 log CFU/cm2. Conversely, when samples were evaluated using DEM in treatments with 6, 7, and 8 log PFU/ml, although disaggregation of biofilms could be observed after 8 h, viable cells were still present up to 48 h (maximal reduction 1.5 log units). The phage titer remained stable or increased up to 2.59 log units during the study period. In conclusion, phage P100 may provide an adjuvant measure to control L. monocytogenes biofilm on stainless steel surfaces. However, phage treatment must be used in combination with other hygienization measures to increase efficacy. In this study, DEM was a good tool to quickly and accurately assess the real effect of phage P100 on L. monocytogenes biofilms.

Temporal and spatial distribution of virioplankton in large shallow freshwater lakes

Virioplankton dynamics at seven sample sites (from two freshwater lakes) with different trophic states were monitored over the period of a year. Water was sampled monthly from August 2004 to July 2005, and the abundances of viruses and bacteria were measured using direct epifluorescence microscopy counting. Results indicated that both natural and anthropogenic factors could influence the distribution of virioplankton. Temporally, viral abundance was significantly correlated to bacterial density and water temperature. Spatially, viral abundance was significantly correlated to trophic state. This in turn indicated that viral abundance was directly dependant on host abundance in eutrophic lakes, while trophic state and temperature could drive the distribution of virioplankton. The virus-bacteria ratio was significantly lower in less productive water-bodies. The result implied that the control of virioplankton on their hosts may change according to the host density.

Potential use of copper as a hygienic surface; problems associated with cumulative soiling and cleaning

It has been suggested that antibacterial copper could be used in place of stainless steel to help reduce the occurrence of hospital-acquired infections. The antibacterial activity of copper has been clearly demonstrated when using cell suspensions held in prolonged contact with copper or copper alloys. The aim of this study was to evaluate the antimicrobial properties of copper in comparison with stainless steel in a generally dry environment. Three stainless steels of varying surface finish and polished copper were soiled with Staphylococcus aureus suspended in a protein-based organic soil (bovine serum album), dried rapidly, and then incubated for 24 h. Surfaces were then wiped clean using a standardised wiping procedure with two cleaning agents recommended by UK National Health Service guidelines. This soiling/cleaning procedure was carried out daily over five days. After each cleaning cycle the amount of residual soil and live cells was assessed using direct epifluorescence microscopy. All materials were easily cleaned after the first soiling episode but a build-up of cells and soil was observed on the copper surfaces after several cleaning/wiping cycles. Stainless steel remained highly cleanable. Accumulation of material on copper is presumably due to the high reactivity of copper, resulting in surface conditioning. This phenomenon will affect subsequent cleaning, aesthetic properties and possibly antibacterial performance. It is important to select the appropriate cleaning/disinfecting protocols for selected surfaces.

Effect of different environmental conditions on the bacteria survival on stainless steel surfaces

The effect of different environmental conditions on the survival of Enterobacter cloacae, Pseudomonas aeruginosa and Staphylococcus aureus on stainless steel surfaces was determined using direct epifluorescence microscopy (DEM) and plate count method. A strong linear relation ( R 2 = 0.97; P < 0.01) between DEM and plate counts over the range of 10 4–10 7 CFU/cm 2 was observed when counts by DEM were performed over viable microorganisms. However, when microorganisms were subjected to adverse conditions, such as slow air-drying, the correlation between both methods diminished considerably ( R 2 = 0.225). This may be due to microorganisms subjected to adverse conditions can be injured, so some bacteria can not be detected by cultivation, but they still present a danger. Microorganisms on wet surfaces had the ability to produce biofilm after 24 h. However, air-drying on surfaces, reduced considerably the bacterial count. E. cloacae was the most susceptible to slow air-drying on surfaces at room temperature however, S. aureus, remained viable for at least 72 h. The characteristics of an organism and its surrounding environment are among the important factors that may affect the survival of bacterial cells on surfaces.

Impact of Environmental Factors on Efficacy of Upper-Room Air Ultraviolet Germicidal Irradiation for Inactivating Airborne Mycobacteria

This study evaluated the efficacy of an upper-room air ultraviolet germicidal irradiation (UVGI) system for inactivating airborne bacteria, which irradiates the upper part of a room while minimizing radiation exposure to persons in the lower part of the room. A full-scale test room (87 m3), fitted with a UVGI system consisting of 9 louvered wall and ceiling fixtures (504 W all lamps operating) was operated at 24 and 34 degrees C, between 25 and 90% relative humidity, and at three ventilation rates. Mycobacterium parafortuitum cells were aerosolized into the room such that their numbers and physiologic state were comparable both with and without the UVGI system operating. Airborne bacteria were collected in duplicate using liquid impingers and quantified with direct epifluorescent microscopy and standard culturing assay. Performance of the UVGI system degraded significantly when the relative humidity was increased from 50% to 75-90% RH, the horizontal UV fluence rate distribution was skewed to one side compared to being evenly dispersed, and the room air temperature was stratified from hot at the ceiling to cold at the floor. The inactivation rate increased linearly with effective UV fluence rate up to 5 microW cm(-2); an increase in the fluence rate above this level did not yield a proportional increase in inactivation rate.

Ambient bioaerosol indices for indoor air quality assessments of flood reclamation

An air quality study was conducted in arid-region residences that were cleaned and reoccupied following a major regional flood (Arkansas River, Colorado, USA). This demonstration study leveraged a suite of aerosol measurements to assess the effects of common flood reclamation practices on indoor air quality. These assays included (i) optical counting (OPC) of airborne particulate matter (0.3– 5 μ m optical diameter), (ii) composite observations of volatile organic compounds (VOC), (iii) culturing and direct microscopic counts of airborne bacteria and fungi, and (iv) air-exchange rate measurements. As judged by OPC, most of the flood damaged homes surveyed had higher concentrations of airborne particulate matter indoors than outdoors; the same trend was observed for selected VOC. When compared to large literature databases, culturing from air samples collected in houses reclaimed from flood damage had significantly higher airborne microorganism levels than in houses where no flood damage had occurred—in many cases this difference was between two and three orders of magnitude. As determined by direct epifluorescence microscopy, total airborne microorganism concentrations were 3–1000 times higher than those recovered by conventional culturing. In flood damaged homes, biological particles averaged 52% of the total particles measured indoors, and 18% of the total particles measured immediately outdoors. Relative differences between the indoor and outdoor concentrations of airborne particulate matter, microorganisms, and associated VOCs, suggested that flood-impacted building materials were sustaining high aerosol bioburdens and contributing to poor indoor air quality more than 3 months after the structures had been reclaimed from flood damage.

The influence of hydrogen bubble formation on the removal of Pseudomonas fluorescens biofilms from platinum electrode surfaces

Hydrogen bubble formation on the surface of platinum electrodes as a means of removing biofilms was studied. Biofilms of Pseudomonas fluorescens of different ages were grown on platinum electrodes and challenged with hydrogen bubbles formed at the surface of the electrodes, by cycling the potential at −2.0 V. The removal of the biofilms from the surfaces was assessed by direct epifluorescence microscopy. The removal of the biofilm from the surface was dependent on the biofilm age. As the biofilm became older, the duration of bubble formation needed to achieve complete removal changed, but in some cases, it was not possible to obtain a completely clean surface. An enhancement of biofilm removal was obtained if the potential was cycled between −0.5 and 1.0 V for 30 min prior to bubble generation, probably due to the weakness of the forces established between the surface and the biofilm and within the biofilm.

In situ assessment of active Thiobacillus species in corroding concrete sewers using fluorescent RNA probes

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of concrete corrosion in sanitary sewers. Thiobacillus species are often considered the major representative of the acid-producing bacteria in these environments, and members of the genus Acidiphilium have been implicated to support their growth. Active populations of selected Thiobacillus, Leptospirillum, and Acidiphilium species were compared to total bacterial populations growing on the surfaces of corroding concrete using three oligonucleotide probes that have been confirmed to recognize unique sequences of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and Thiobacillus thiooxidans (probe: Thio820), Leptospirilium ferrooxidans (Probe: Lept581) and members of the genus Acidiphilium (probe: Acdp821). With these genetic probes, fluorescent in situ hybridizations (FISH) were used to identify and enumerate selected bacteria in homogenized biofilm samples taken from the corroding crowns of concrete sewer collection systems operating in Houston, Texas, USA. Direct epifluorescent microscopy demonstrated the ability of FISH to identify significant numbers of active acidophilic bacteria among concrete particles, products of concrete corrosion (e.g. CaSO 4), and other mineral debris. As judged by FISH analyses with the species-specific probe Thio820, and a domain-level probe that recognizes all Bacteria (Eub338), T. ferrooxidans and T. thiooxidans comprised between 12% and 42% of the total active Bacteria present in corroding concrete samples. Although both Acidiphilium and Leptospirillum have also been postulated to have ecological significance in acidic sulfur-oxidizing environments, neither genera was detected using genus-specific probes (Lept581 and Acdp821).

Microbial activity in soils following steam treatment.

Steam enhanced extraction (SEE) is an aquifer remediation technique that can be effective at removing the bulk of non-aqueous phase liquid (NAPL) contamination from the subsurface, particularly highly volatile contaminants. However, low volatility compounds such as polynuclear aromatic hydrocarbons (PAHs) are less efficiently removed by this process. This research evaluated the effects of steam injection on soil microbial activity, community structure, and the potential for biodegradation of contaminants following steam treatment. Three different soils were evaluated: a laboratory-prepared microbially-enriched soil, soil from a creosote contaminated field site, and soil from a chlorinated solvent and waste oil contaminated field site. Results from field-scale steaming are also presented. Microbial activity before and after steam treatment was evaluated using direct epifluorescent microscopy (DEM) using the respiratory activity dye 5-cyano-2,3, ditolyl tetrazolium chloride (CTC) in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) to yield a quantitative assessment of active and total microbial numbers. DEM results indicate that steamed soils that were analyzed while still hot exhibited microbial activity levels that were below detection. However, soil samples that were slowly cooled, more closely reflecting the conditions of applied SEE, exhibited microbial activity levels that were comparable to presteamed soils. Samples from a field-site where steam was applied continuously for 6 weeks also showed high levels of microbial activity following cooling. The metabolic capabilities of the steamed communities were investigated by measuring cell growth in enrichment cultures on various substrates. These studies provided evidence that organisms capable of biodegradation were among the mesophilic populations that survived steam treatment. Fluorescent in situ hybridization (FISH) analysis of the soils with domain-level rRNA probes suggest that both Archaea and Bacteria survived steam exposure.

The Detection of Microorganisms and Organic Material on Stainless Steel Food Contact Surfaces

Few methods are available for the differentiation of microorganism and organic material on surfaces, although such mixtures are commonplace, particularly in the food industry, where food debris (soil) and microorganisms frequently foul food contact surfaces and pose challenges in terms of hygiene and cleanability. It would be of value to discern any differences in removal or persistence on surfaces. This review considers some methods which are available. Direct epifluorescence microscopy (DEM) enables visual differentiation, but traditional microbiological culture methods cannot detect organic soil. Atomic force microscopy (AFM) provides images of the surface on the nanometer scale, with minimal preparation, and is able to visualise both cellular and acellular components of the mixture, particularly prior to cleaning. Confocal laser scanning microscopy (CLSM) also has potential in this area. Surface sensitive methods such as X-Ray photoelectron spectroscopy (XPS) and Time-of-flight secondary ion mass spectrometry (ToFSIMS) provide information on chemical species present on a surface. Those chemical species more likely on microbial cells may be differentiated from those more likely in a specified organic soil, thus comparisons may be made as to differential removal of the organic soil and microbial cells. These methods may be of value in studies on the fouling and cleanability of surfaces.