Rapid preparation of cyanobacterial DNA for real-time PCR analysis

AIMS: To develop a rapid preparation method for real-time PCR analysis of cyanobacteria from cultures or field samples. METHODS AND RESULTS: Field samples and cultures containing Anabaena circinalis, Cylindrospermopsis raciborskii or Microcystis aeruginosa were subjected to three cell disruption treatments: (i) heating during thermocycling, (ii) microwave irradiation in the presence of detergent and (iii) probe sonication. Treated samples were directly added to the PCR reaction and analysed on two different real-time devices. A statistically significant difference was evident in the cycle thresholds for each of the treatments in all but one culture and one environmental sample, sonication and microwave treatments performing better than direct addition. The microwave treatment was also compared to the Qiagen DNA Mini kit and performance was equivalent when treated samples were analysed as above. CONCLUSIONS: Whilst microwave treatment was slightly less effective than probe sonication across all samples, it was more amenable to processing multiple samples and significantly better than heat treating the sample during thermocycling. SIGNIFICANCE AND IMPACT OF THE STUDY: The microwave method described here is a simple, rapid and effective preparation method for cyanobacterial DNA that can be easily deployed in the field, making the most of the speed and flexibility offered by fixed and portable real-time PCR devices.     

Survival of enterobacteria in liquid cultures during microwave radiation and conventional heating

Bacteria in food have been reported to survive in larger numbers after processing by microwave radiation than after conventional processing. The bactericidal effect of a domestic microwave oven (SHARP R-7280) on certain pathogenic enterobacteria species was investigated in vitro, in comparison with conventional heating (boiling). The death rates of different nosocomial strains of Escherichia coli, Salmonella sofia, Salmonella enteritidis, Proteus mirabilis and Pseudomonas aeruginosa were tested. The microwave oven and the conventional heating system used were both calibrated in order to calculate temperatures from exposure times. For each strain duplicate samples of 25 ml of pure culture with concentrations at least 10(6) cfu/ml were exposed to microwave radiation. An equal number of samples of the same volume and concentration were exposed to conventional heating. Subsequently all samples were examined qualitatively and quantitatively following standard microbiological procedures. The results indicate that microwaves have an efficient bactericidal effect on the enterobacteria in liquid cultures.     

Combined effect of mild heat and acetic acid treatment for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an asparagus puree

AIMS: This study was conducted to validate combined heat and acid treatments for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an acidified brine containing, or pickled, asparagus model food. METHODS AND RESULTS: A mixture of three strains of E. coli O157:H7, L. monocytogenes and S. typhimurium were inoculated onto pickled asparagus samples. Combinations of various concentrations of acetic acid [0%, 0.25%, 0.5%, 0.75%, 1%, 1.5% and 2% (v/v)] and various temperatures (40 degrees C, 50 degrees C, 60 degrees C and 75 degrees C) were investigated. Following treatment, asparagus samples were stored at room temperature and enumerated at 0, 0.5, 1, 2 and 3 days. Heat and acetic acid treatments were synergistic. The inhibitory effects of these combined treatments on the tested foodborne pathogens were also effective during storage. Loss of green colour in the pickled asparagus significantly increased with increasing concentrations of acetic acid. CONCLUSIONS: Using a combination of mild heat and acetic acid treatments can successfully control E. coli O157:H7, L. monocytogenes and S. typhimurium in pickled asparagus, combinations of heat and acid are synergistic and effective treatments can be selected to reduce adverse effect on colour which occur during product storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Mild heating plus acetic acid treatment are synergistic, so combined treatments can be developed, which would lower the temperature and amount of acetic acid required for minimally processed vegetables while maintaining pathogen control.     

The effect of prior heat shock on the thermoresistance of Salmonella thompson in foods

The resistance of Salmonella thompson to heating at 54° or 60°C in tryptone soya broth, liquid whole egg, 10% or 40% reconstituted dried milk or minced beef was increased if cells were held at 48°C for 30 min before heating at the higher temperatures. Induction of thermotolerance by mild heat shock is thus not confined to cells grown and heated in broth systems. The heat shock phenomenon may therefore have implications for the safety of foods given marginal heat treatment.     

Microwave oven and boiling waterbath extraction of hepatotoxins from cyanobacterial cells

Low-cost, straightforward methods for the extraction of microcystins and nodularins from cyanobacterial cells were developed using a microwave oven and boiling waterbath. The use of organic solvents, such as methanol, which can interfere with sensitive analytical procedures, e.g. immunoassays, can thus be avoided. Analysis by protein phosphatase inhibition assay and high performance liquid chromatography indicated that purified microcystin-LR was unaffected by the microwave oven and boiling waterbath treatments. Four microcystins of differing hydrophobicities were successfully extracted from Microcystis PCC 7813 by both treatments at yields equivalent to those obtained by longer protocols using methanol. Assessment of the microwave oven and boiling waterbath extraction methods with laboratory strains and environmental samples of cyanobacteria showed good correlation with results from lyophilisation and methanol extraction, when extracts were analysed by high performance liquid chromatography with diode array detection (R(2)>/=0.92). The microwave and boiling waterbath extraction methods also sterilised the environmental bloom samples, as evidenced by the abolition of heterotrophic bacterial growth.     

Effect of microwave radiation on Bacillus subtilis spores

AIMS: To compare the killing efficacy and the effects exerted by microwaves and conventional heating on structural and molecular components of Bacillus subtilis spores. METHODS AND RESULTS: A microwave waveguide applicator was developed to generate a uniform and measurable distribution of the microwave electric-field amplitude. The applicator enabled the killing efficacy exerted by microwaves on B. subtilis spores to be evaluated in comparison with conventional heating at the same temperature value. The two treatments produced a similar kinetics of spore survival, while remarkably different effects on spore structures were seen. The cortex layer of the spores subjected to conductive heating was 10 times wider than that of the untreated spores; in contrast, the cortex of irradiated spores did not change. In addition, the heated spores were found to release appreciable amounts of dipicolinic acid (DPA) upon treatment, while extracellular DPA was completely undetectable in supernatants of the irradiated spores. These observations suggest that microwave radiation may promote the formation of stable complexes between DPA and other spore components (i.e. calcium ions); thus, making any release of DPA from irradiated spores undetectable. Indeed, while a decrease in measurable DPA concentrations was not produced by microwave radiation on pure DPA solutions, a significant lowering in DPA concentration was detected when this molecule was exposed to microwaves in the presence of either calcium ions or spore suspensions. CONCLUSIONS: Microwaves are as effective as conductive heating in killing B. subtilis spores, but the microwave E-field induces changes in the structural and/or molecular components of spores that differ from those attributable only to heat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the effect of microwaves on B. subtilis spore components.     

Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures.

The thermal inactivation of 11 strains of Mycobacterium paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. paratuberculosis per ml, M. paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. paratuberculosis per ml of milk were present before heat treatment, M. paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions.     

Survival of Salmonella anatum Heated in Various Media

Survival of Salmonella anatum heated at 55 C for 35 min was determined in solutions of various chemical constituents of foods including salts, carbohydrates, amino acids, peptides, nucleic acids, gums, and stabilizers and compared with survival in 0.1 m phosphate buffer (pH 7.0). Commercially sterilized whole milk gave the most protection against heat. Trypticase Soy Broth, various peptide mixtures, and some amino acids gave substantial protection. Results with carbohydrates were variable, with mannitol, sucrose, and rhamnose providing substantial protection and glucose decreasing heat resistance. A few other substances, cysteine, glutathione, and sodium citrate, also decreased heat resistance. Pure proteins had little effect. Results show that water activity is of little significance under the test conditions. Protection from heat probably results from complexing of substances with heat-sensitive proteins in the cells. Autoclaved milk should not be considered equivalent to raw milk for studies of survival of bacteria during heating.     

Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples.

Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water. Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous. Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples. In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E. coli O157 and Salmonella typhimurium in foods and fomites. Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E. coli O157 and S. typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples. An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E. coli O157 and S. typhimurium, respectively. IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay. Some samples, especially fish, gave unexpectedly high background ECL. Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy. Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens.     

Mechanism of microwave sterilization in the dry state.

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.     

Mechanism of microwave sterilization in the dry state

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.     

Thermal and Nonthermal Effects of Discontinuous Microwave Exposure (2.45 Gigahertz) on the Cell Membrane of Escherichia coli

The aim of this study was to investigate the effects on the cell membranes of Escherichia coli of 2.45-GHz microwave (MW) treatment under various conditions with an average temperature of the cell suspension maintained at 37°C in order to examine the possible thermal versus nonthermal effects of short-duration MW exposure. To this purpose, microwave irradiation of bacteria was performed under carefully defined and controlled parameters, resulting in a discontinuous MW exposure in order to maintain the average temperature of the bacterial cell suspensions at 37°C. Escherichia coli cells were exposed to 200- to 2,000-W discontinuous microwave (DW) treatments for different periods of time. For each experiment, conventional heating (CH) in a water bath at 37°C was performed as a control. The effects of DW exposure on cell membranes was investigated using flow cytometry (FCM), after propidium iodide (PI) staining of cells, in addition to the assessment of intracellular protein release in bacterial suspensions. No effect was detected when bacteria were exposed to conventional heating or 200 W, whereas cell membrane integrity was slightly altered when cell suspensions were subjected to powers ranging from 400 to 2,000 W. Thermal characterization suggested that the temperature reached by the microwave-exposed samples for the contact time studied was not high enough to explain the measured modifications of cell membrane integrity. Because the results indicated that the cell response is power dependent, the hypothesis of a specific electromagnetic threshold effect, probably related to the temperature increase, can be advanced.     

Free amino acid concentrations in milk: Effects of microwaveversus conventional heating

Summary Microwave effects on free amino acid concentrations in milkversus a water bath heating were investigated in view of their importance for infant growth. Concentrations of few amino acids, such as aspartate, serine or lysine, are unchanged whatever the way and the temperature of heating. In contrast, tryptophan concentrations decreased similarly whatever the way of heating (110 ± 3µmol/l before heatingvs 84 ± 4µmol/l after 30°C microwave heating, p < 0.05). On the contrary, concentrations of glutamate and glycine increased more after water bath heating at 90°C (325 ± 4 and 101 ± 1µmol/l, respectively) than after microwave heating (312 ± 4 and 95 ± 1µmol/l, respectively, p < 0.05) suggesting milk proteolysis. Moreover, the accumulation of ammonia observed at 90°C with the water bath together with increase Glu levels might reflect a degradation of glutamine. An ornithine enrichment, more evident with microwave heating, was shown and could be of interest as it is a polyamine precursor. Also, considering few variations of free amino acid concentrations and the time saved, microwave heating appears to be an appropriate method to heat milk.     

Construction and operation of a multiplexed microfiltration device to facilitate rapid pathogen detection

Millions of Americans contract food poisoning or are affected by microbial pathogens each year. Rapid, sensitive detection of dilute levels of pathogens in foods, produce, water, and biomanufacturing process samples is key to consumer protection; however, current enrichment methods require as much as a full day to enrich viable bacterial pathogens to detectable levels. Our lab previously demonstrated the ability to concentrate and detect dilute levels of pathogens, within 8 hr, from various food matrices using microfiltration in our continuous cell concentration device (i.e., C3D) with one or two filter modules. This short communication describes the design, materials and construction, layout, and operational characteristics of a four filter module multiplexed system based on a four channel device. Benefits are a 2× greater sample capacity than an equivalent duplex system (achieving the same time to result of less than 8 hr from sample preparation to detection), simpler operation, and a footprint enabling operation inside a biosafety cabinet instead of requiring a BSL-2 room. Flow rate variability through four channels fit within an operational envelope of ±3%; flow rates are reproducible from one run to the next thus ensuring relatively simple, concurrent processing of samples.     

Two-Phase Slug Flow Heat Exchanger for Microbial Thermal Inactivation Research

A continuous two-phase (air-liquid), slug flow, tubular heat exchanger was developed for microbial thermal inactivation research to give exposure times and temperatures in the range of high-temperature, short-time milk pasteurization as well as heat-treated sample volumes of at least 2 ml. The use of air to compartmentalize the liquid in the capillary tubing prevented the development of laminar flow, which enabled precise identification of the residence time of the fastest flowing particles in the heating, holding, and cooling sections of the instrument. Residence time distributions were quantitated by measuring the degree of spreading of radioactive tracers for water, whole milk, chocolate milk, cream, and ice-cream mix with holding temperatures from 50 to 72 C, holding times from 2 to 60 sec, and heating and cooling times of 1.7 sec each. For a holding time of 60 sec and a fastest particle velocity of 10.2 cm/sec, the velocity ratios of the fastest moving particle to the median particle were 1.05, 1.05, 1.10, and 1.13 for whole milk, chocolate milk, cream, and ice-cream mix, respectively. With shorter holding times, these velocity ratios were even closer to unity. These velocity ratios indicated that the instrument would be an effective tool for thermal inactivation research on microorganisms suspended in homogeneous fluids with a viscosity of 15 centipoises or less at the exposure temperature.     

Membrane filtration of food suspensions.

Factors affecting the membrane filtration of food suspensions were studied for 58 foods and 13 membrane filters. Lot number within a brand, pore size (0.45 or 0.8 micrometer), and time elapsed before filtration had little effect on filterability. Brand of membrane filter, flow direction, pressure differential, age (microbiological quality) of the food, duration of the blending process, temperature, and concentration of food in the suspension had significant and often predictable effects. Preparation of suspensions by Stomacher (relative to rotary blender) addition of surfactant (particularly at elevated temperature) and prior incubation with proteases sometimes had dramatic effects of filterability. In contrast to popular opinion, foods can be membrane filtered in quantities pertinent to the maximums used in conventional plating procedures. Removal of growth inhibitors and food debris is possible by using membrane filters. Lowering of the limits of detection of microorganisms by concentration on membrane filters can be considered feasible for many foods. The data are particularly relevant to the use of hydrophobic grid-membrane filters (which are capable of enumerating up to 9 X 10(4) organisms per filter) in instrumented methods of food microbiological analysis.     

Controlled Microwave Processing Applied to the Pharmaceutical Formulation of Ibuprofen

The first successful development of controlled microwave processing for pharmaceutical formulations is presented and illustrated with a model drug (ibuprofen) and two excipients (stearic acid and polyvinylpyrrolidone). The necessary fine temperature control for formulation with microwave energy has been achieved using a uniquely modified microwave oven with direct temperature measurement and pulse-width modulation power control. In addition to comparing microwave and conventional heating, the effect of the presence of liquid (water) in aiding the mixing of the drug and excipient during formulation was also investigated. Analysis of the prepared formulations using differential scanning calorimetry and dissolution studies suggest that microwave and conventional heating produce similar products when applied to mixtures of ibuprofen and stearic acid. However, the differences were observed for the ibuprofen and polyvinylpyrrolidone formulation in terms of the dissolution kinetics. In all cases, the presence of water did not appear to influence the formulation to any appreciable degree. The application of controllable microwave heating is noteworthy as fine temperature control opens up opportunities for thermally sensitive materials for which microwave methods have not been feasible prior to this work.     

Evaluating gull diets: a comparison of conventional methods and stable isotope analysis

ABSTRACT Samples such as regurgitated pellets and food remains have traditionally been used in studies of bird diets, but these can produce biased estimates depending on the digestibility of different foods. Stable isotope analysis has been developed as a method for assessing bird diets that is not biased by digestibility. These two methods may provide complementary or conflicting information on diets of birds, but are rarely compared directly. We analyzed carbon and nitrogen stable isotope ratios of feathers of Glaucous Gull (Larus hyperboreus) chicks from eight breeding colonies in northern Alaska, and used a Bayesian mixing model to generate a probability distribution for the contribution of each food group to diets. We compared these model results with probability distributions from conventional diet samples (pellets and food remains) from the same colonies and time periods. Relative to the stable isotope estimates, conventional analysis often overestimated the contributions of birds and small mammals to gull diets and often underestimated the contributions of fish and zooplankton. Both methods gave similar estimates for the contributions of scavenged caribou, miscellaneous marine foods, and garbage to diets. Pellets and food remains therefore may be useful for assessing the importance of garbage relative to certain other foods in diets of gulls and similar birds, but are clearly inappropriate for estimating the potential impact of gulls on birds, small mammals, or fish. However, conventional samples provide more species‐level information than stable isotope analysis, so a combined approach would be most useful for diet analysis and assessing a predator's impact on particular prey groups.     

Effect of decompression drying treatment on physical properties of solid foods

This study used a decompression drying instrument to investigate the effects of a drying treatment on the physical properties of solid foods. Commercial tofu was used as a model food and was treated at different temperature and pressure conditions in a drying chamber. Overall, high temperatures resulted in better drying. Additionally, pressure in the chamber influenced the drying conditions of samples. Differences in physical properties, such as food texture, shrinkage, and color were observed among some samples, even with similar moisture content. This was caused by differences in moisture distribution in the food, which seems to have manifested as a thin, dried film on the surfaces of samples. It caused inefficient drying and changes in physical properties. Control of the drying conditions (i.e. pressure and heat supply) has relations with not only physical properties, but also the drying efficiency of solid foods.     

An optimized method for the determination of perfluorooctanoic acid, perfluorooctane sulfonate and other perfluorochemicals in different matrices using liquid chromatography/ion-trap mass spectrometry

Perfluorochemicals (PFC's) are widely spread in the environment and have been detected in blood of wildlife and humans world-wide. Recently, various toxic effects of PFC's in laboratory rats have been demonstrated, resulting in increased government concerns regarding the presence of PFC's in the environment and the implications they have on human health. In the last decade, various analytical methods have been developed for the analysis of PFC's in different matrices whereby the majority of methods have utilised liquid chromatography coupled with mass spectrometry (LC-MS). Here we describe an optimized method for the quantitation of PFC's, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), in food packaging, polytetrafluoroethylene (PTFE) sealant tape and drinking water. The method involved PFC's extraction via off-line SPE followed by separation using reversed-phase liquid chromatography on a Phenyl-Hexyl column coupled with ion-trap (IT) mass spectrometric detection. The optimized approach minimized ion-suppression effects commonly seen with conventional elution buffers, improving detection limits down to 25 pg/mL and allowed effective quantitation down to 50 pg/mL for PFOA and PFOS. The optimized LC-MS method detected PFOA and other PFC's in microwave popcorn packaging and PFOA in PTFE sealant tape in the low μg/kg. In all samples, PFOS was not detected.