Direct Epifluorescent Filter Technique Research Articles

Experimental validation and evaluation of electronic sensing techniques for rapid discrimination of electron-beam, γ-ray, and X-ray irradiated dried green onions (Allium fistulosum).

This study focuses on applicability of e-sensing methods in addition to direct epifluorescent filter technique (DEFT), aerobic plate counts (APC), and photostimulated luminescence (PSL) to discriminate e-beam, γ-ray, and X-ray irradiated green onions (scallions) at 1, 4, and 7kGy for each irradiation treatment, along with two confirmatory analysis. Although low dose (1kGy) reduced the APC (6.15-2.69 log CFU/g), no significant difference was observed in DEFT. Scallions with log DEFT/APC ratio of ≥ 5.2 can be suspected as irradiated at at least 1kGy. Both non-irradiated (< 700counts/min) and irradiated (> 5000counts/min) scallions were identified by PSL. Besides distinct volatile compounds profile, cumulative e-nose discrimination of 92.06% from principle component analysis was apparent among irradiation conditions. Sensitivity to changes in perceived taste by e-tongue showed 96.07% variation. The e-sensing capability to discriminate irradiated scallions can be exploited for exposing counterfeit claims as either non-irradiated or irradiated within the recommended standards.

Assessment of Microbial and Radioactive Contaminations in Korean Cold Duck Meats and Electron-Beam Application for Quality Improvement.

Animal-origin food products pose serious threat to public food safety due to high microbial loads. The microbial and radioactive contaminations in commercial cold duck meat products were evaluated. Ten different lots of commercial samples (C1-C10) were classified based on type and smoking process. All samples were highly contaminated (< 4-7 Log CFU/g) with total aerobic bacteria (TAB), yeasts and molds (Y&M), and 7 samples (C1-C7) were positive for coliforms. Furthermore, three samples were contaminated with Listeria monocytogenes (C4-C6) and one with Salmonella typhimurium (C6). No radionuclides (131I, 137Cs, and 134Cs) were detected in any sample. The results of DEFT (direct epifluorescent filter technique)/APC (aerobic plate count), employed to screen pre-pasteurization treatments of products, indicated that smoked samples were positive showing DEFT/APC ratios higher than 4. Notably, the samples showed a serious threat to microbial safety, thus were irradiated with electron-beam (e-beam). The D10 values for S. typhimurium and L. monocytogenes were 0.65 and 0.42 kGy, respectively. E-beam application at 3 and 7 kGy resulted in reduction of initial TAB, Y&M, and coliform populations by 3 and 6 log cycles, respectively. Thus, e-beam was proven to be a good decontamination approach to improve the hygiene of cold duck meat.

Detection of Viable but Nonculturable Cells of Listeria monocytogenes with the Use of Direct Epifluorescent Filter Technique

AbstractDirect epifluorescent filter technique was studied as a method to enumerate Listeria monocytogenes in artificially contaminated cheese. Occurrences of viable but nonculturable (VBNC) cells of L. monocytogenes prompted us to investigate the viability of this pathogen in hard cheese during refrigerated storage under different packaging conditions. This study compared the results of two enumeration methods: epifluorescent microscopic counting and plating. The microscopic enumeration was based on fluorescent staining with carboxyfluorescein diacetate (CFDA) for tracing cells that display metabolic activity. In this study, the L. monocytogenes persisted well throughout a prolonged storage of cheese, regardless of the packaging conditions. Based on a discrepancy between CFDA and plate count results, a dormant fraction of VBNC cells was distinguished, thus greater quantities of VBNC cells were estimated along with storage time. We conclude that direct epifluorescent filter technique may be an attractive approach for assessing bacterial viability, especially for pathogens with the ability to enter a dormant state.Practical ApplicationsListeria monocytogenes is a foodborne pathogen with a remarkable long‐term ability to persist in a variety of ready‐to‐eat foods. The plating method for enumeration of L. monocytogenes in food is well established, but it is time consuming and relies only on cell reproduction, so that injured and nonculturable cells have no chance to be counted. As a solution, we describe a rapid method for the enumeration of L. monocytogenes in cheese using the direct epifluorescent filter technique. This method is based on fluorescent staining of cells with a metabolic activity indicator and cell counting with the help of epifluorescence microscopy. The method presented in this study allows the rapid enumeration of both active and nonculturable cells of L. monocytogenes and could be used by predictive microbiologists for precise estimation of the safety of dairy products with respect to pathogenic bacteria.

Food-borne Pathogens of Animal Origin-Diagnosis, Prevention, Control and Their Zoonotic Significance: A Review

The term food borne diseases or food-borne illnesses or more commonly food poisoning are used to denote gastrointestinal complications that occur following recent consumption of a particular food or drink. Millions of people suffer worldwide every year and the situation is quiet grave in developing nations creating social and economic strain. The food borne pathogens include various bacteria viz., Salmonella, Campylobacter, Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus, Arcobacter, Clostridium perfringens, Cl. botulinum and Bacillus cereus and helminths viz., Taenia. They also include protozoa viz., Trichinella, Sarcocystis, Toxoplasma gondii and Cryptosporidium parvum. The zoonotic potential and the ability to elaborate toxins by many of the microbes causing fatal intoxication are sufficient to understand the seriousness of the situation. The viral agents being host specific their transmission to humans through food of animal origin is not yet confirmed although these animal viruses are similar to that of viruses infecting human. Food-borne bacteria; protozoa and helminthes have complex distribution pattern in the environment and inside the host system. This along with complexity of the maintenance chain and life cycle (of parasites) has made it difficult for epidemiologist and diagnostician to undertake any immediate safety measures against them. Serological and molecular diagnostic tests viz. ELISA, Latex agglutination test, Lateral flow assays, Immunomagnetic separation assays, molecular assays viz. Polymerase Chain Reaction (PCR), multiplex PCR, immuno-PCR, Realtime PCR, Random Amplified Polymorphic DNA (RAPD)-PCR, DNA microarrays and probes are widely used. Along with these LAMP assays, Capillary Electrophoresis-Single Strand Confirmation polymorphism (CE-SSCP); Flow cytometry, FISH, Biosensors, Direct epifluorescent filter technique, nanotechnology based methods and sophisticated tools (ultrasonography, magnetic resonance imaging and chlonangio-pancreatography) have aided in the diagnosis greatly. Most of the food-borne illnesses are self-limiting but in many instances antibiotics are recommended. With the increased drug resistance however use of chicken immunoglobulin, bacteriophage therapy, probiotics and herbs are gaining much importance these days. Adoption of proper prevention and control measures (including cooking procedures; hygiene, strict adherence to HACCP principles, public awareness and disease surveillance and monitoring) are the need of hour. All these have been discussed vividly in this review to help epidemiologists, diagnosticians, clinicians and above all common people so as to enable them avoid negligence regarding such serious issue.

Patterns of survival and volatile metabolites of selected Lactobacillus strains during long-term incubation in milk

The focus of this study was to monitor the survival of populations and the volatile compound profiles of selected Lactobacillus strains during long-term incubation in milk. The enumeration of cells was determined by both the Direct Epifluorescent Filter Technique using carboxyfluorescein diacetate (CFDA) staining and the plate method. Volatile compounds were analysed by the gas-chromatography technique. All strains exhibited good survival in cultured milks, but Lactobacillus crispatus L800 was the only strain with comparable growth and viability in milk, assessed by plate and epifluorescence methods. The significant differences in cell numbers between plate and microscopic counts were obtained for L. acidophilus strains. The investigated strains exhibited different metabolic profiles. Depending on the strain used, 3 to 8 compounds were produced. The strains produced significantly higher concentrations of acetic acid, compared to other volatiles. Lactobacillus strains differed from one another in number and contents of the volatile compounds.

Total count and growth rates of bacteria in saline water of Lake Timsah and hypersaline water of Bardawil Lagoon, Egypt

This study may present first total bacterial counts estimated in Lake Timsah and El-Teloul and Mesafaq Basins of Bardawil Lagoon using the dilution culture technique followed by filtration through 0.2 µm cellulose nitrate filters and examined by epifluorescent microscope after staining with acridine orange. Also, the staining of bacterial cultures with acridine orange in metabolically active and inactive states was investigated using direct epifluorescent filter technique. Generation time of Lake Timsah bacterial cultures was 2.04 generations day −1 and growth rate was 0.049 h −1 . In Bardawil Lagoon doubling time was 3.3 and 1.79 generations day −1 for samples collected from El-Teloul and Mesafaq, respectively. The growth rates were 0.09 and 0.04 h −1 for El-Teloul and Mesafaq Basins, respectively.

Application of the microbiological method DEFT/APC to detect minimally processed vegetables treated with gamma radiation

Marketing of minimally processed vegetables (MPV) are gaining impetus due to its convenience, freshness and apparent health effect. However, minimal processing does not reduce pathogenic microorganisms to safe levels. Food irradiation is used to extend the shelf life and to inactivate food-borne pathogens. In combination with minimal processing it could improve safety and quality of MPV. A microbiological screening method based on the use of direct epifluorescent filter technique (DEFT) and aerobic plate count (APC) has been established for the detection of irradiated foodstuffs. The aim of this study was to evaluate the applicability of this technique in detecting MPV irradiation. Samples from retail markets were irradiated with 0.5 and 1.0 kGy using a 60Co facility. In general, with a dose increment, DEFT counts remained similar independent of the irradiation while APC counts decreased gradually. The difference of the two counts gradually increased with dose increment in all samples. It could be suggested that a DEFT/APC difference over 2.0 log would be a criteria to judge if a MPV was treated by irradiation. The DEFT/APC method could be used satisfactorily as a screening method for indicating irradiation processing.

Automated fluorimetry in quality control of pasteurized and ultra-high temperature-treated starch soup

Summary Automated monitoring of the microbiological quality of heat-processed foods by the resazurin reduction test was applied to microtitration plate incubator-fluorimeter technology. The appearance and disappearance of the fluorescing peak of resorufin was monitored on microtitration trays. Pasteurized or ultra-high temperature-treated starch-based soup was used as the model food system. Bacillus subtilis spores (ultra-high temperature treatment) and vegetative cells of Enterococcus faecalis (pasteurization) were inoculated into the soup before the heat treatment at levels which resulted in some survival. The timing of appearance of maximum fluorescence correlated with the number of bacteria in pre-incubated samples. Automated resazurin-reduction fluorimetry was compared with conventional plating, turbidometry and microcolony count by the direct epifluorescent filter technique. The results of the resazurin test correlated well with those of all the other methods tested. Fluorimetry had the advantage that the results could be read within 1–5h and the reproducibility was superior to the other methods.

Evaluation of two alternative techniques for counting mesophilic aerobic bacteria in raw milk

Traditional techniques of microbiological analysis do not meet the current needs of having immediate analytical results in order to be able to make decisions in the process of food manufacturing, specially in HACCP application systems concerned with quality assurance. The development of modern techniques is showing new alternatives but it is important that the advantages for each of these new methodologies be considered for each application. The objective of this paper was to evaluate the advantages of two microbiological alternative techniques (dry rehydratable film and direct epifluorescent filter technique), applied to enumerating total aerobic mesophilic micro-organisms in refrigerated raw milk supplied by dairy farms, compared to the reference technique (standard plate count). The two alternative techniques under study showed good correlation levels with the standard in plate technique. Dry rehydratable film technique had higher costs but this technique allows the analysis of twice the quantity of samples in the same unit of time compared to the standard plate technique. This compensates for the higher costs. The epifluorescent technique gave the results in shorter time but it is necessary to use sophisticated equipment and trained personnel. Therefore, it is preferred when having the appropriate infrastructure.

Use of the Direct Epifluorescent Filter Technique for the Enumeration of Viable and Total Acetic Acid Bacteria from Vinegar Fermentation

A rapid epifluorescence staining method using the LIVE/DEAD® BacLight™ Bacterial Viability kit was applied to differentiate both viable and total counts of acetic acid bacteria in vinegar fermentation. The results obtained were compared with those from other measurement techniques: 4′,6-diamidine-2phenyl indole (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and colony counts. BacLight total counts were comparable to DAPI (differing by <3.5%). BacLight viable counts were similar to CTC counts but considerable higher than colony-forming cells in plates.

Influence of Fluorescence of Bacteria Stained with Acridine Orange on the Enumeration of Microorganisms in Raw Milk

The staining of Gram-positive and Gram-negative cultures with acridine orange in metabolically active and inactive states was investigated using a Bactoscan, direct epifluorescent filter technique (DEFT), and standard plate count as the reference method.The evaluation of the bacterial cultures in the Bactoscan revealed a linear relationship between Bactoscan counts (pulses) and the quantity of pure culture suspension used. But the proper detection of bacteria with the fluorescence optic methods was dependent on the type of microorganism and the physiological state of the cells. The Bactoscan and DEFT underestimated the bacterial counts of Gram-negative cultures as compared with standard plate counting. When stained with acridine orange, metabolically active bacteria showed more orange fluorescence and a lower percentage of green fluorescent cells as compared with inactive bacteria. Bactoscan pulse height analysis (PHA) diagrams, graphs of the detected pulses and their intensity, showed low pulses of inactive bacteria. Many of these weak pulses were eliminated from counting because of their faint fluorescent staining. In contrast, PHA diagrams of metabolically active microorganisms showed bright staining and, therefore, high pulses. A complete count of these bacteria was possible. These investigations point out that discrepancies between the fluorescence optical counting methods and the standard plate count depend strongly on the staining of the cultures with acridine orange and, therefore, on the type of microorganism and the metabolic state of the cells measured.

Semi-Automated Direct Epifluorescent Filter Technique for Total Bacterial Count in Raw Milk

The Direct Epifluorescent Filter Technique (DEFT) and the reference method of counting total bacterial colonies on Petri dishes were compared. IDF Standards 128 (1985) and 161A (1995) were applied. A total of 496 samples of milk were analyzed. Colony forming units per microL milk were transformed to decimal logarithmic units: log (cfu/microL). The repeatability standard deviation, Sr = 0.114, was typical for a routine microbiological method. To study the carryover at different levels of bacteria, 3 tests were performed on milk samples of approximately 100, 700, and >1000 cfu/microL. For the first 2 experiments, no carryover was detected; in the milk sample with >1000 cfu/microL, the carryover was <0.12%. When the DEFT counts were regressed versus the reference method, the values of the slope and intercept were 0.92 and 0.17, respectively; the correlation coefficient was r = 0.84; and the residual standard deviation was Syx = 0.287. The paired t-test showed that the reference method and DEFT do not give significantly different results (p = 0.05).

Direct enumeration of Escherichia coli and enteric bacteria in water, beverages and sprouts by 16S rRNA in situ hybridization

A fluorescent oligonucleotide probe complementary to a published 16S ribosomal RNA sequence ofEscherichia coli was used for in situ hybridization on membrane filters for direct enumeration of cells. The direct epifluorescent filter technique (DEFT) was modified by performing a 2-h oligonucleotide hybridization on intact cells collected on a membrane filter surface (‘oligo-DEFT’), instead of the traditional acridine orange staining. The filter was examined by epifluorescence microscopy, and digital image analysis was used for the cell enumeration. Oligo-DEFT counts were linear over the range of 103–106cells ml−1and correlated with DEFT counts (r=0·99). The oligo-DEFT detection limit was approximately 1 cell ml−1. Oligo-DEFT counts correlated with standard coliform counts by membrane filtration or plating on selective media for analysis of water and beverages artificially inoculated with E. coli. Heat inactivation of the cells did not destroy their reactivity in the oligo-DEFT. Natural coliform populations in aquarium water were countable in the oligo-DEFT after a 90-min incubation of the filter on nutrient agar before the hybridization step, which allowed the dormant cells to increase their rRNA content. Natural populations in bean sprouts were countable directly, without having to perform the incubation before hybridization.

Methodology for detection and typing of foodborne microorganisms

Over the past decade many improvements have been seen in both conventional and modern methods for the detection of pathogenic bacteria in foods. Modifications and automation of conventional methods in food microbiology include sample preparation, plating techniques, counting and identification test kits. ATP bioluminescence techniques are increasingly used for measuring the efficiency of cleaning surfaces and utensils. Cell counting methods, including flow cytometry and the direct epifluorescent filter technique are suitable techniques for rapid detection of microorganisms, especially in fluids. Automated systems based on impedimetry are able to screen high numbers of samples based on total bacterial counts within 1 day. Immunoassays in a wide range of formats make rapid detection of many pathogens possible. Recently, there have been important developments in the use of nucleic acid-based assays for the detection and subtyping of foodborne pathogens. The sensitivity of these methods has been significantly increased by the use of the polymerase chain reaction and other amplification techniques. Alternative and rapid methods must meet several requirements concerning accuracy, validation, speed, automation, sample matrix, etc. Both conventional and rapid methods are used within hazard analysis critical control point programs. Further improvements especially in immunoassays and genetic methods can be expected, including the use of biosensors and DNA chip technology.

Development and characterization of a fluorescent-bacteriophage assay for detection of Escherichia coli O157:H7.

In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coli O157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 10(4) cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 10(2) and 10(3) cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coli O157:H7 in broth cultures.

Determination of Viable Bacterial Populations in Raw Milk within 20 Minutes by Using a Direct Epifluorescent Filter Technique

The results from a shortened procedure for the direct epifluorescent filter technique (DEFT) determination of viable bacterial populations in raw milk were compared to standard plate counts. Shortening the prefiltration trypsin-Triton X-100 incubation period from 10 to 3 min enabled the completion of the analysis within 20 min. The short DEFT method results had a correlation coefficient (r) of 0.81 with plate counts. With respect to precision, the average difference between values of duplicate plate count analyses was 0.16 log units; that of the short DEFT was 0.14 log units. The slopes of the regressions equations were less than 1, indicating that a direct correlation is not achieved. Short DEFT values were 0.17 log units higher than those of plate counts on milk samples containing less than 10,000 CFU/ml. For milk samples containing counts over 10,000 CFU/ml, short DEFT values averaged only 0.05 log units above plate count readings. Daily preparation of the stain appears unnecessary since acridine orange solutions stored for up to 2 days at 4°C did not produce results significantly (P > 0.05) different from those obtained with fresh solutions. The short DEFT method has potential for the assessment of the bacteriological quality of raw milk in tanker deliveries.

Microbial quality of lamb carcasses during processing and the acridine orange direct count technique (a modified DEFT) for rapid enumeration of total viable counts

This study was designed to set up a hazard analysis and critical control points (HACCP) system for sheep slaughtering operations at four different plants in Ireland and to determine the differences plants in terms of microbial contamination. A single carcass area, the abdomen, was examined by swabbing and a microbiological profile was determined at different stages along the slaughter line. The level of contamination was assessed from the total bacteria counts, Enterobacteriaceae and Listeria spp. For the total counts, a modified direct epifluorescent filter technique (acridine orange direct count technique (AODC)) was developed and tested. No significant differences were found among plants in the level of bacterial contamination. This was observed for all groups of organisms. The rapid direct technique (AODC) was found to be very successful. A correlation coefficient of 0.87 was obtained for this method and the standard plate count. Each test could be carried out in about 10–15 min and could be used to predict the standard plate count.

Developments in rapid methods for detection of agents of foodborne disease

The application of techniques such as the direct epifluorescent filter technique (DEFT), electrical impedance or conductance measurements, hydrophobic grid membrane filters (HGMFs), genetically engineered luminescent bacteria, DNA probes, polymerase chain reaction, monoclonal antibodies, and identification kits and systems, to the detection of foodborne microbial pathogens is described.

Detection of irradiated deep‐frozen foodstuffs by comparison of DEFT and APC counts

The suitability of a microbiological method based on the comparison of an aerobic plate count (APC) with a count obtained with the direct epifluorescent filter technique (DEFT) for the detection of irradiation of deep‐frozen foodstuffs was evaluated. The study was carried out on parsley, mechanically deboned poultry meat (MDPM) and liquid egg white. The foodstuffs were stored at—18°C and examined after storage for 0, 4 and 12 months.The results showed that this method is discriminating during the whole storage period. Nevertheless, it has been observed that for non‐irradiated liquid egg white the difference between DEFT and APC tends to increase with the time of storage. This phenomenon which did not limit the interest of the test, was not observed for the other products tested.

Fluorescent Antibodies Applied to Direct Epifluorescent Filter Technique for Microscopic Enumeration of Escherichia coli 0157:H7 in Milk and Juice

In a modification of the direct epifluorescent filter technique (DEFT), direct fluorescent antibody staining was used for the rapid (<1 h), specific enumeration of foodbome Escherichia coli 0157:H7 by epifluorescence microscopy. Cell counts obtained by this method (Ab-DEFT) correlated well with DEFT counts obtained by acridine orange staining and with viable plate counts ranging from 108 to 101 cells per ml for pure cultures in buffer. Ab-DEFT also was effective for counting E. coli 0157:H7 cells inoculated into milk and juice; the sensitivity limit was about 103 for milk. The highly specific nature of the technique was demonstrated by enumeration of E. coli 0157:H7 cells in the presence of large numbers of indigenous spoilage microorganisms in milk. This is the first known demonstration of the combination of DEFT and antibody probe technology for the specific enumeration of a microbe directly in food without a growth or enrichment step.