The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) to assess microbial populations on food contact surfaces

Two rapid methods, direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) on swab resuspension fluids, were compared with the traditional total viable count (TVC) on swab resuspension fluids for their ability to enumerate surface populations of attached bacteria. The degree of error in estimating surface populations was shown to be significantly less with DEM than DEFT followed by TVC. DEM estimated populations in the range 3 x 10(3) to 5 x 10(7) colonies/cm2 whilst DEFT enumerated populations above 3 x 10(4) colonies/cm2 and TVC above 3 x 10(5) colonies/cm2 (as measured by DEM). Swabbing was shown to remove a constant proportion of organisms from the surface populations tested, although below 3 x 10(5) colonies/cm2 most of the organisms remained in the cotton matrix and were difficult to resuspend. DEFT was more able to enumerate swab resuspension fluids obtained from surface populations below 3 x 10(5) colonies/cm2 than was TVC.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk (r = 0.94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0.1 M, pH 3.0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better (r = 0.90) than that using standard 2 ml samples (r = 0.88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk ( r = 0·94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0·1 M, pH 3·0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better ( r = 0·90) than that using standard 2 ml samples ( r = 0·88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

Collaborative trial of the direct epifluorescent filter technique (DEFT), a rapid method for counting bacteria in milk

The direct epifluorescent filter technique (DEFT) is a new rapid method which uses membrane filtration and epifluorescent microscopy for counting bacteria in milk. A collaborative trial of the DEFT was conducted between six laboratories. Each laboratory obtained a highly significant relationship between the DEFT count and plate count with a correlation coefficient generally > 0.9 but there were significant differences between these relationships. The repeatability of the DEFT, although ca 1·5 times worse than that of the plate count, was of a level acceptable in practice. Reproducibility of the DEFT was ca 3 times that of the plate count. This poor reproducibility was probably mainly due to counting errors. Possible reasons for this and ways of reducing counting errors are discussed.     

Use of the direct epifluorescent filter technique for the enumeration of yeasts

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.     

Use of the direct epifluorescent filter technique for the enumeration of yeasts

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.     

Use of the direct epifluorescent filter technique for the enumeration of bacterial spores

Heat treatment at 80°C for 10 min effectively destroyed all vegetative cells (except for Gram-positive cocci) and made easier the counting of bacterial spores, which stained orange, green or rarely transparent/black with a dull green halo, in the direct epifluorescent filter technique. The numbers of both orange- or green-staining spores were lower than the plate count. A variety of physiological conditions were used to investigate the relationship of the different staining patterns with germination status. It was concluded that orange-staining spores had germinated and their number agreed with the plate count after incubation in yeast glucose broth at 30°C for 4 h. This observation was unreliable, however, but it was found that a total spore count in the DEFT gave a good agreement with the plate count.     

Rapid enumeration of microorganisms in foods by the direct epifluorescent filter technique.

Filtration of "stomachered" food suspensions through nylon filters (pore size, 5 microns) removed most of the food debris without affecting the recovery of microorganisms. Two to ten milliliters of these prefiltered suspensions could be filtered in the direct epifluorescent filter technique (DEFT). The technique takes less than 30 min to complete and has a lower sensitivity of less than 60,000 microorganisms per g for all products examined. Vegetative bacterial cells, spores, fungal hyphae, and yeasts could be distinguished with the technique. For fresh meat and fish, the DEFT count of prefiltered suspensions agreed well with the plate count of unfiltered suspensions over the range of 10(4) to 10(10)/g (correlation coefficient of 0.91). For frozen meat and fish and frozen vegetables, the two counting methods had correlation coefficients of 0.87 and 0.66, respectively. The poor correlation for frozen vegetables was due to the inclusion in the DEFT count of nonviable bacteria killed by the blanching process used to inactivate enzymes. Good agreement was obtained between the prefiltered DEFT count and unfiltered plate count for cooked meats, cream doughnut, and whole peppers. Possible reasons for the poor agreement between the DEFT count and plate count for certain products are discussed.     

A modified direct epifluorescent filter technique for the detection and enumeration of yeast in yoghurt

A modified direct epifluorescent filter technique (DEFT) for the detection and enumeration of visible yeast in fruit-flavoured yoghurt is described. The method involves an initial enrichment in oxytetracycline glucose yeast peptone broth (OGYP, 30C/24, 48 h), prefiltration prior to DEFT and use of the vital stain Viablue 1. Additional yoghurt samples were subjected to prolonged incubation at 12C or spiked with Kluyveromyces fragilis . When DEFT was compared with the plate count, regression and correlation coefficients of 1.12 and 0.85 respectively were obtained for values above the sensitivity threshold of DEFT (10³ cells/ml). The use of an enrichment stage (OGYP, 30C/24 h) enabled, by calculation, a theoretical minimum yeast contamination level of 7 yeast cells/g in the original yoghurt to be detected assuming the cells exhibit no lag phase of growth.     

The use of tumors in wild populations of fish to assess ecosystem health

Evidence has linked toxicants in aquatic systems with cancer in fish and population level effects on species. Thus some types of tumors may be useful monitors of ecosystem health, at least as affected by genotoxins and promoters. However, tumors caused by purely genetic mechanisms or by virus would not be good indicators. Only neoplasms which have chemicals as a portion of their etiology (either as initiators or promoters) would be useful in assessing ecosystem health. Lesions which may fit these criteria include liver neoplasms (both biliary and hepatic) and skin lesions in a variety of primarily benthic fishes, and neural lesions in various drum species and in butterfly fish species. Two studies purporting to demonstrate a lack of tumors in fish from polluted areas have been reexamined and found either to have insufficient data on vulnerable species or to actually support a tumor-pollution linkage. Thus certain lesions in vulnerable species or species groups may serve as a mechanism to assess one facet of ecosystem health.     

The use of isoelectric focusing to assess percentage hymenopterous parasitism in aphid populations

The primary parasitoid Aphidius uzbekistanicus Luzhetski and its host, the cereal aphid Sitobion avenae (F.) both showed specific bands for the enzyme malate dehydrogenase (MDH), thereby allowing clear detection of parasitism. The specific profiles of MDH activities remained recognizable through all post-embryonal life-stages, but the intensity of staining depended on the instar and morph subjected to analysis. A calibrated equation, representing the relationship between percentage parasitoid-specific MDH activity and percentage parasitism, was elaborated for third instar S. avenae. This equation was, however, not applicable to field-collected material. Reasons for this failure and the possible use of isolectric focusing (IEF) for other parasitoid: host relationships are discussed.

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Zusammenfassung Die herkömmlichen Methoden zur Bestimmung der Parasitierungsrate bei Blattläusen sind zeit- und arbeitsaufwendig, so daß sich meist nur ein geringer Stichprobenumfang bearbeiten läßt. Wir haben daher untersucht, ob die Parasitierung größerer Blattlauskollektive mittels der isoelektrischen Fokussierung (IEF) schnell und verläßlich zu ermitteln ist, wobei wir die Malatdehydrogenase (MDH) als Enzymsystem wählten.Die Modellpopulationen (der Parasitoid Aphidius uzbekistanicus und die Wirtsblattlaus Sitobion avenae) zeigten in allen Stadien und Morphen spezifische Bandenprofile, die ein Erkennen parasitierter Blattläuse eindeutig ermöglichten. Die Intensität der Färbung hing aber von den untersuchten Larvenstadien ab, d.h. ältere, größere Tiere ergaben quantitativ bedeutendere Enzymaktivitäten als jüngere, kleinere.Bei S. avenae wurde dieser Sachverhalt noch von der jeweiligen Morphenzugehörigkeit überlagert: alatiforme Stadien bewirkten stärkere Färbungsintensitäten als apteriforme. Dieses ist wahrscheinlich auf die Anhäufung von MDH-reichen Mitochondrien in der Flugmuskulatur zurückzuführen.Durch eine densitometerische Auswertung war es uns möglich, den relativen Anteil des parasitoidenspezifischen Peaks einer Probe mit dem jeweiligen (bekannten) Parasitierungsgrad in Beziehung zu setzen. Zwischen dem kleinsten und größten Larvenstadium des Parasitoiden ergab sich dabei eine bestimmte Spanne für einen gegebenen Parasitierungsgrad.Mit diesen Werten haben wir eine auf Feldbedingungen ausgerichtete, simulierte Gleichung errechnet, die wir auf Freilandblattläuse mit bekanntem Parasitierungsgrad anwendeten. Um den Einfluß der Stadienzugehörigkeit auszuschalten, wurden nur Blattläuse im dritten Stadium untersucht.Die Verteilung der Larvenstadien der Parasitoiden erwies sich aber als zu heterogen, so daß die errechneten Werte nur in zwei von neun Proben mit den durch Zuchtansätze ermittelten Werten übereinstimmten. In anderen Wirt-Parasitoid-Systemen mit ausgeprägter Stadienspezifität und demzufolge synchroner Entwicklung könnte die IEF aber durchaus von großem Nutzen sein.

     

Removal and transfer of viruses on food contact surfaces by cleaning cloths

Contamination of food contact surfaces with pathogens is considered an important vehicle for the indirect transmission of food-borne diseases. Five different cleaning cloths were assessed for the ability to remove viruses from food contact surfaces (stainless steel surface and nonporous solid surface) and to transfer viruses back to these surfaces. Cleaning cloths evaluated include two different cellulose/cotton cloths, one microfiber cloth, one nonwoven cloth, and one cotton terry bar towel. Four viral surrogates (murine norovirus [MNV], feline calicivirus [FCV], bacteriophages PRD1 and MS2) were included. Removal of FCV from stainless steel was significantly greater (P ≤ 0.05) than that from nonporous solid surface, and overall removal of MNV from both surfaces was significantly less (P ≤ 0.05) than that of FCV and PRD1. Additionally, the terry towel removed significantly fewer total viruses (P ≤ 0.05) than the microfiber and one of the cotton/cellulose cloths. The cleaning cloth experiments were repeated with human norovirus. For transfer of viruses from cloth to surface, both cellulose/cotton cloths and microfiber transferred an average of 3.4 and 8.5 total PFU, respectively, to both surfaces, and the amounts transferred were significantly different (P ≤ 0.05) from those for the nonwoven cloth and terry towel (309 and 331 total PFU, respectively). There was no statistically significant difference (P > 0.05) in the amount of virus transfer between surfaces. These data indicate that while the cleaning cloths assessed here can remove viruses from surfaces, some cloths may also transfer a significant amount of viruses back to food contact surfaces.     

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the calgary biofilm device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.     

Cleaning and decontamination efficacy of wiping cloths and silver dihydrogen citrate on food contact surfaces

Aims: To test the efficacy of four wipe cloth types (cotton bar towel, nonwoven, microfibre and blended cellulose/cotton) with either quaternary ammonia cleaning solution or silver dihydrogen citrate (SDC) in cleaning food contact surfaces. Methods: Swab samples collected from untreated, cloth‐treated and cloth disinfectant‐treated surfaces were subjected to hygiene monitoring using adenosine triphosphate (ATP) bioluminescence and aerobic total plate counting (TPC) assays. Results: Adenosine triphosphate measurements taken after wiping the surfaces showed poor cleaning by nonwoven cloths (2·89 RLU 100 cm^?2) than the microfibre (2·30 RLU 100 cm^?2), cotton terry bar (2·26 RLU 100 cm^?2) and blended cellulose/cotton cloth types (2·20 RLU 100 cm^?2). The cellulose/cotton cloth showed highest log reduction in ATP‐B RLU values (95%) and CFU values (98·03%) when used in combination with SDC disinfectant. Conclusions: Cleaning effect of wiping cloths on food contact surfaces can be enhanced by dipping them in SDC disinfectant. ATP‐B measurements can be used for real‐time hygiene monitoring in public sector, and testing microbial contamination provides more reliable measure of cleanliness. Significance and Impact of the Study: Contaminated food contact surfaces need regular hygiene monitoring. This study could help to estimate and establish contamination thresholds for surfaces at public sector facilities and to base the effectiveness of cleaning methods.     

The use of muscle near-infrared spectroscopy (NIRS) to assess the aerobic training loads of world-class rowers

The objectives of this study were (1) to characterize the changes in oxygenation derived from muscle near-infrared spectroscopy (NIRS) during aerobic constant-load exercise with intensities close to Maximal Lactate Steady-State (MLSS) and (2) to establish reference values in the world-class rowers, for such workload often included in rowing training programs. Eight senior world-class rowers performed an incremental progressive submaximal exercise test and a 30-minute test on a rowing ergometer. The power corresponding to intensive aerobic training (84±1% of the anaerobic threshold) was adopted as an exercise load in the 30-minute test. The NIRS device was fixed on the vastus lateralis muscle which was active during rowing to record muscle O₂ saturation (SmO₂) and total hemoglobin concentration (THb) at rest and during exercise. Statistically significant increments in blood lactate (LA) and heart rate (HR) were observed, with 1.18±0.61 mmol/l and 10±5 beats/min, respectively, in 30th minute compared to 10th minute in 30-minute test. SmO₂ decreased significantly by 2.9±1.4%, whereas THb did not change. The examinations may suggested the low diagnostic value of THb in constant-load exercise. In each subject, SmO₂ was gradually reduced during the intense aerobic exercise. During workload close to MLSS, the SmO₂ of the vastus lateralis ranged from 14.0±3.13 to 11.1±2.81% in 10 and 30 minutes respectively, with a reduction in muscle oxygenation (ΔSmO₂) exceeding 50%. The non-invasive nature of the NIRS measurement and the continuous monitoring of SmO₂ values are useful in the practice of monitoring training in terms of aerobic training loads.     

The use of NASBA for the detection of microbial pathogens in food and environmental samples

The isothermal amplification method nucleic acid sequence-based amplification (NASBA), which amplifies RNA, has been reported as useful for the detection of microbial pathogens in food and environmental samples. Methods have been published for Campylobacter spp., Listeria monocytogenes and Salmonella enterica ser. Enteritidis in various foods and for Cryptosporidium parvum in water. Both 16S rRNA and various mRNAs have been used as target molecules for detection; the latter may have advantages in allowing specific detection of viable cells. Most of the methods to detect pathogens in foods have employed enrichment in nutrient medium prior to NASBA, as this can ensure sensitivity of detection and encourage the detection of only viable target cells. Although a relatively recent method, NASBA has the potential for adoption as a diagnostic tool for environmental pathogens.