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 times 10³ to 5 times 10⁷ colonies/cm² whilst DEFT enumerated populations above 3 times 10⁴ colonies/cm² and TVC above 3 times 10⁵ colonies/cm² (as measured by DEM). Swabbing was shown to remove a constant proportion of organisms from the surface populations tested, although below 3 times 10⁵ 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 times 10⁵ colonies/cm² than was TVC.     

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.     

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 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.     

Analysis of high-quality pharmaceutical grade water by a direct epifluorescent filter technique microcolony method

A modified direct epifluorescent filter technique (DEFT) was used to assess the microbial quality of water. Sterile, pharmaceutical grade water was inoculated with Pseudomonas cepacia. Microcolonies were grown and counted with a Bio-Foss System 3 DEFT apparatus. In this way it was possible to achieve a sensitivity of 10 cfu per 100 ml. Results were obtained within 1 working day.     

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.     

Semi-automated counting of bacteria and somatic cells in milk using epifluorescence microscopy and television image analysis

An epifluorescent microscope fitted with a 'Chalnicon' closed circuit television camera linked to an Optomax System III image analyser was used to count bacteria and somatic cells on membrane filters prepared from milk by the direct epifluorescent filter technique (DEFT). For both bacteria and somatic cells, the semi-automated DEFT count of low count milk exceeded the visual DEFT count but this difference became proportionately less as the count increased. There was close agreement between the semi-automated and visual DEFT counts over the range 10⁵-5 times 10⁶/ml for bacteria and 3 times 10⁵-5 times 10⁶/ml for somatic cells. The semi-automated DEFT count of bacteria and somatic cells correlated well with the plate count and Coulter count respectively, with correlation coefficients of 0.83 and 0.81.     

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.     

Microbial production and consumption of dimethyl sulfide (DMS) in a sea grass (Zostera noltii)-dominated marine intertidal sediment ecosystem (Bassin d'Arcachon, France)

The relation between net dimethyl sulfide (DMS) production and changes in near surface (0-5 mm) oxygen concentrations in a sea grass (Zostera noltii Hornem)-covered intertidal sediment ecosystem was examined during a diel cycle. Sediment covered with Zostera was found to be more oxygenated than uncovered sediment during the period of photosynthesis. This phenomenon was probably caused by radial oxygen loss of the Zostera root-rhizome system. The population sizes of the three functional groups of microbes mainly responsible for the concentration of DMS, the dimethylsulfoniopropionate (DMSP)-demethylating, DMSP-cleaving and DMS-oxidizing bacteria, were quantified by most probable number (MPN) methodologies. Sediments with Zostera supported substantially higher populations of both aerobic (149x10(6) cm(-3) DMSP-utilizing and 0.4x10(6) cm(-3) DMS-oxidizing) and anaerobic (43x10(6) cm(-3) DMSP-utilizing and 0.4x10(6) cm(-3) DMS-oxidizing) microorganisms than sediments without Zostera (DMSP-utilizing aerobes and anaerobes both 2x10(6) cm(-3) and DMS-oxidizing aerobes and anaerobes both 0.2x10(6) cm(-3)). Experiments conducted with sediment cores and sediment slurries suggested that the net production of DMS in these sediments was significantly lower during oxic periods than during anoxic periods. Intact sediment cores with and without Zostera produced DMS when incubated under anoxic/dark conditions (97.0 and 53.6 nmol DMS m(-2) h(-1), respectively), while oxic/light-incubated cores did not produce detectable amounts of DMS. In addition, kinetic parameter values (V(max) and K(m)) for DMSP degradation in cell suspensions of isolated DMSP-demethylating and DMSP-cleaving bacteria were measured and compared to documented values for other strains. Both V(max) and K(m) values for DMSP-demethylating organisms were found to be relatively low (14.4-20.1 nmol DMSP mg protein(-1) min(-1) and 4.1-15.5 μM, respectively) while these parameter values varied widely in the group of the DMSP-cleaving organisms (6.7-1000 nmol DMSP mg protein(-1) min(-1) and 2-2000 μM, respectively). It was hypothesized that a diel rhythm in DMS emission occurred, with a relatively low net production during the day and a high net production during the night. Environmental changes which result in increased anoxic conditions in coastal sediments, such as an increase in eutrophication, may therefore result in increased atmospheric DMS emission rates.     

Hydrophobic Grid-Membrane Filters: New Approach to Microbiological Enumeration

A square grid pattern, printed in hydrophobic material on conventional membrane filters, subdivided the surface into a plurality of areas. This separated colonies from one another and prevented lateral growth, spreading, and confluence. Very high colony-packing densities were achieved (2,500/in(2); ca. 1.61 x 10(5)/cm(2)) reducing the need to dilute samples. Recovery of organisms was better than on conventional filters, particularly at high inoculum levels. At the same time, visibility of normally pale or diffuse colonies was improved, since colonies grew upwards instead of sideways, and counting was facilitated because colonies grew in orderly arrays.     

Detection of low numbers of osmophilic yeasts in creme fondant within 25 h using a pre-incubated DEFT count

The results presented show that the direct epifluorescent filter technique (DEFT) can be used to detect low numbers of osmophilic yeasts in creme fondant after pre-incubation, or high numbers without pre-incubation, and within 30 min. Pretreatment with trypsin and Triton X-100 was necessary to achieve efficient filtration of the fondant. The detection limits for osmophilic yeasts in the pre-incubated DEFT count, as extrapolated from the initial plate count, were approximately 1/g after 25 h and 1/10 g after 49 h.     

Environmental factors affecting tissue regeneration of the reef--building coral Montastraea annularis (Faviidae) at Los Roques National Park,Venezuela

In this study, the rates of tissue regeneration and recovery from injuries that emulated the bites of either butterfly or parrotfish on colonies of Montastraea annularis exposed to different sedimentation regimesp were determined. Two small reef patches were chosen close to key Dos Mosquises, north of the Venezuclan mainland. Sixteen colonies (8 treatments + a single replicate) were artificially damaged at each patch and their recovery was monitored for three months by photographic means. The lesions were inflicted using two different techniques: scratching the polyps with a hard-nylon brush to resemble parrotfish (Scaridae) damages (Lesions Type 1) or jetting out the tissue with a syringe to simulate butterflyfish (Chaetondontidae) bites (Lesions Type 2). The diameter of the wounds ranged from 5 (small lesion) to 8 cm (large lesions) and both kinds were inflicted on the top and bottom of the colonies, with a single replicate for each treatment. The main factors affecting the recovery of the colonies' surface were lesion features (type, position and size), turbidity and chiefly, the sedimentation rate. While lesion recovery was slow where sedimentation and resuspension rates were high, tissue regeneration was improved under low sedimentation and resuspension conditions. Moreover, lesions located at the bottom of colonies regenerated completely, whereas sediments frequently covered top lesions and limited their recovery. More than 60% of the colonies with small lesions recovered almost completely in less than 90 days, whereas those with larger injuries frequently showed extensions of their damage and increased mortality. Tissue-only lesions (LT2) regenerated two to three times faster than those involving both tissue and skeletal damage (LT1). Other variables not controlled in this study, such as diseases, encrusting organisms overgrowth and herbivory introduced further variability to the regeneration rates.     

Susceptibility of Ixodes scapularis (Acari: Ixodidae) to Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) using three exposure assays in the laboratory

An emulsifiable concentrate (EC) and granular (G) formulation of the entomopathogenic fungus, Metarhizium brunneum strain F52 (formerly Metarhizium anisopliae strain F52) were tested against unfed adults and nymphs of Ixodes scapularis Say in the laboratory. Three exposure methods; dip, surface contact, and direct spray application, and three exposure time intervals (3, 30, and 300 min) were used to evaluate the EC formulation. Application rates ranged from 2.6 x 10(2) to 2.6 x 10(8) conidia/cm2. The surface treatment was used for granular formulation with concentrations ranging from 2.3 x 10(5) to 2.3 x 10(7) conidia/cm2 for same three exposure times. Both the EC and G formulations of this fungus were highly pathogenic against I. scapularis adults and nymphs. Logistic regression analysis found formulation, spore concentration, time of exposure, and observation period were significant or highly significant factors influencing tick mortality. For adult I. scapularis, the spray application with the EC formulation of M. brunneum F52 resulted in a lower LC50 (5.9 x 10(4) conidia/cm2) at 30 min than surface exposure to the EC (LC50 = 1.3 x 10(6) conidia/cm2) or G formulation (LC50 = 8.1 x 10(5) conidia/cm2). At higher concentrations, fungal activity was evident in adult I. scapularis held at 5 degrees C suggesting the fungus may provide control in the cooler fall season. While the observed pathogenicity of a fungus against ticks can be dependent upon the bioassay assessment, we found nymphs and adults of I. scapularis to be highly susceptible to M. brunneum F52, regardless of the exposure method used.     

Rapid diffusion of spectrin bound to a lipid surface.

Human erythrocyte spectrin was labelled with the probe 5, 5'-disulfato-1-(6-hexanoic acid N-hydroxysuccinimide ester)-1'-ethyl-3,3,3',3'-tetramethylindocarbocyanine (Cy3). Cy3-spectrin was bound to the outer surface of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and its diffusion measured by fluorescence recovery after photobleaching (FRAP). It was found that at 30 degrees C, above the lipid gel to liquid-crystalline phase transition of the lipids, Cy3-spectrin had an unexpectedly high diffusion coefficient D=(2.1+/-0.6)x10(-7)) cm2/s. At the phase transition, diffusion of Cy3-spectrin was only slightly lower; D=(1.3+/-0.3)x10(-7) cm2/s, whereas at 14 degrees C, well below the lipid phase transition, diffusion was found to be much slower with D=(3.1+/-0.12)x10(-9) cm2/s. The fast diffusion of Cy3-spectrin on the lipid surface implies that the individual bonds which bind spectrin to the lipid surface must rapidly be made and broken. In the light of these results, spectrin-lipid interactions alone appear unlikely to have any significant role in supporting the cell membrane. Probably, the interactions serve only to localise the spectrin at the inner lipid surface in order to facilitate formation of the cytoskeleton.     

Application of antisera raised against sulfate-reducing bacteria for indirect immunofluorescent detection of immunoreactive bacteria in sediment from the German Baltic Sea.

Polyclonal rabbit antisera raised against sulfate-reducing bacteria (SRB) could detect several distinct populations of bacteria in sediment from the German Baltic Sea. The depth distribution of immunoreactive bacteria was determined by an indirect immunofluorescence filter method. Anti-Desulfovibrio desulfuricans DSM 1926 serum showed maximum bacterial numbers at a depth of 18 cm, with a concentration of 60 x 10(6) cells cm-3. With anti-Desulfovibrio baculatus DSM 2555 serum, counts were highest at the same depth, approaching 0.7 x 10(6) cells cm-3. Other significantly smaller populations were observed. Anti-SRBStrain 1 (lactate,vibrio) maxima were at 0 to 4 cm and at 17 to 18 cm. Anti-SRBStrain 2 (lactate,vibrio) serum showed several local maxima. Anti-SRBStrain 3 (lactate,oval) serum detected one single peak at a depth of 10 to 12 cm. Also determined were rates of sulfate reduction, total bacterial counts by acridine orange staining, and the viable counts by dilution series on anaerobic lactate medium. The total bacterial counts were highest (180 x 10(6) cells cm-3) at 3 to 4 cm and dropped to 24 x 10(6) cells cm-3 at 10 to 11 cm but showed additional local maxima reaching 140 x 10(6) cells cm-3 at a depth of 17 to 18 cm. Viable counts probable number) were above 10(5) CFU cm-3 at 0 to 3.6 cm but remained below 10(3) CFU at 7.2 to 18 cm. The sulfate reduction rate was maximal (107 nmol cm-3 day-1) at a depth of 1 to 2 cm, dropped to 10 nmol cm-3 day-1 at 12 to 13 cm, and reached 38 nmol cm-3 day-1 at 17 to 18 cm.     

Antimicrobial activity of argon fluoride (ArF) excimer laser on gram-negative bacteria

The objective of this study was to evaluate the antibacterial activity of argon fluoride (ArF) excimer laser radiation on clinically important strains of gram-negative bacteria. The antibacterial activity of ArF excimer laser radiation was evaluated on two Acinetobacter baumannii, one Enterobacter cloacae, three Escherichia coli, two Helicobacter pylori, one Klebsiella pneumoniae and two Pseudomonas aeruginosa strains. The strains were isolated from clinical specimens and typed by the usual biochemical procedures. Square agar plates of 12 x 12 cm were divided into rectangular (2 x 3 cm) regions and spread with 0.5x 10(4) colony forming units (CFU)/ml of bacterial suspension. The excess liquid was removed and the plates were allowed to dry for 30 min. A total of 96 rectangular (2x3 cm) regions were used for each strain, in order to test an equal number of laser parameters. Each rectangular region was irradiated with different laser parameters, using a 193 nm ArF excimer laser, linked with a simple Galilean afocal system and a rectangular diaphragm of the same dimensions as the original laser beam cross-section, at a distance of 10 cm from the irradiated surface. This system was used in order to keep the laser pulse energy under 80 mJ and to cut-out the non-transverse electromagnetic mode branches of the laser beam. We then studied the bacterial survival ratio versus the number of laser pulses, the repetition frequency and the total laser beam fluence. Our results showed that the total laser beam fluence was the most important parameter to consider in evaluating the bactericidal effect of ArF excimer laser radiation. A critical value of the total fluence was determined for each strain, such that, for laser beam fluences greater than this critical value, no colonies appeared to survive while, for laser fluences less than this critical value, the survival ratio did not exceed 2 x 10(7) CFU (2 x 10(-5)%). These critical values were found to vary between 8 J/cm2 and 16 J/cm2 for the bacterial species studied. Under these conditions, ArF laser irradiation is promising for the sterilisation of hard surfaces and for in situ application.     

Evaluation of a commercial fluorochromic system for the rapid detection and estimation of wine lactic acid bacteria by DEFT

A commercial fluorochromic system was evaluated for the rapid detection of lactic acid bacteria in fortified wines by the epifluorescent filter technique (DEFT). The viability test used, employing the fluorescence dyes SYTO 9 and propidium iodide, was able to detect and clearly differentiate viable from non-viable cells (killed with a 50% v/v ethanol solution). A good overall agreement ( r ₌ 0·92) was obtained between the DEFT count and the plate count in the range studied (5 × 10²–4 × 10⁹ cells ml⁻¹). Wine components which might otherwise interfere with the method could be removed by including simple wash steps in the protocol. This measure proved critical to the success of the procedure. For practical purposes, the rapid method studied seems to be a good alternative to the traditional cultural methods as part of quality control programmes in wine making. It may also be useful when studying the efficacy of certain treatments in the elimination of wine bacterial contaminants.