Detection of post-pasteurization contamination of cream by impedimetric methods

Impedimetric methods for evaluating post-pasteurization contamination and shelf-life of cream were assessed. Over 94% of the samples tested were in agreement, using selected cut-offs of 20 h for detection time measured at 21°C with creams containing inhibitors for the growth of Gram positive bacteria on standard plate count agar as growth media, and 3.2 × 10⁷ cfu/g for plate counts obtained on cream which had been pre-incubated in the presence of inhibitors for the growth of Gram positive organisms, and on cream stored at 6°C for 7 d. Agreement between the impedimetric method and plate count was not as good if either Brain Heart Infusion or Milk Agar was used in place of Plate Count Agar in the former technique. A poor correlation was obtained between plate count methods for enumerating post-pasteurization contamination and keeping quality with impedimetric measurements on cream alone. It was possible, with a reasonable degree of certainty, to determine if cream had suffered post-pasteurization contamination within 20 h of production.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni . The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO₂+ 80% N₂, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O₂+ 10% CO₂+ 85% N₂. The packaging material in the first two treatments was PA 80/PE 100–PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37°C, 20°C and 4°C for 48 h, 4 days and 25 days, respectively. At 37°C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20°C and at 4°C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37°C its numbers increased only in the optimal gas atmosphere; at 20°C the strain was not detectable after 24 to 48 h storage and at 4°C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

Starvation survival of the fish pathogen Aeromonas salmonicida in seawater

Abstract Three strains of the fish pathogenic bacterium Aeromonas salmonicida were examined with respect to their ability to survive in seawater. Five to seven days after plate counts decreased below the detection limit of 10 cells/ml, the population of respiring A. salmonicida cells comprised less than 4% of the initial total bacterial population. At this stage, samples were transferred either to sterile nutrient or to control flasks containing sterile nutrient-free medium. The addition of nutrients caused reappearance of growing bacteria, detected by plating on agar medium and an increase of the population of respiring bacteria to 85–95% of the total population. In a separate experiment it was shown that after more than six months of starvation at 15°C, a few of the starved A. salmonicida cells were able to regrow in liquid media after addition of nutrients, but not on agar media. These cells evade detection by direct microscopic respiratory measurement but appear to be reactivated after addition of nutrients.     

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.     

Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR

One of the major drawbacks of DNA-based microbial diagnostics is its inability to discriminate between live and dead bacteria. Due to the persistence of DNA in the environment after cells have lost their viability, DNA-based assays cannot assess pathogenic risk since signals can originate from both live and dead cells. Presented here is a potential application of the novel chemical propidium monoazide (PMA), which results in the selective suppression of DNA detection from dead cells. PMA can only penetrate dead cells with permeabilized cell membranes. Upon intercalation into the DNA, covalent crosslinkage of PMA to DNA is achieved through light exposure. This modification prevents the DNA from being amplified by PCR. The method, in combination with quantitative PCR as a diagnostic tool, successfully monitored the disinfection efficacy of hypochlorite, benzalkonium and heat on several model pathogens. Threshold cycle numbers increased with increasing disinfection strength after PMA treatment of samples compared to non-PMA treated samples. With some disinfectant-specific differences, monitoring viability loss with membrane integrity as an indicator seemed to be more conservative than monitoring viability loss with plate counts. Loss of viability after short UV-exposure could not be monitored with PMA as UV light affects viability by inducing DNA damage without directly affecting membrane permeability.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

Effect of starch- and lipid-based encapsulation on the culturability of two Bifidobacterium longum strains

AIMS: To assess the applicability of starch- and lipid-based encapsulation methods for improving the viability and culturability of two Bifidobacterium longum strains stored in fermented and nonfermented foods. MATERIALS AND RESULTS: Cells were encapsulated with partially hydrolysed potato starch granules combined with amylose coating, or entrapped in cocoa butter matrix. The tested B. longum strains were not adherent to the starch granules, and the culturability of the cells stored in fermented and nonfermented foods was not improved by starch-based encapsulation. Encapsulation of the cells in cocoa butter was found to increase the plate counts during storage. In addition to plate counts, viability of the cells was measured by fluorescent microscopy using LIVE/DEAD BacLight viability assay. Microscopic counts of the viable cells did not change significantly during storage, suggesting that the cells remained alive despite becoming unable to grow on nutrient agar plates. CONCLUSIONS: Encapsulation with cocoa butter increased the culturability of the cells, but encapsulation with hydrolysed potato starch had no effect. Culture-independent viability assay suggested that cells remained viable despite being unable to grow on agar plates. SIGNIFICANCE AND THE IMPACT OF THE STUDY: This study indicates that encapsulation techniques may be useful in improving the culturability of bacteria, but the plate counts may yield insufficient data on the actual viability of the cells.     

Viability of cold-adapted L cells after freezing and storage in solid CO2

Mouse L cells adapted to low temperature by repeated exposures to 4 °C for 6–8 weeks were stored in solid CO₂ at −70 °C for 7 months simultaneously with unpretreated controls. The population of the cold-adapted subline LC2 contained more living cells after thawing than did the control L cells. The increased viability of the LC2 cells was expressed by a higher number of eosin-unstained cells immediately after thawing and by a higher increase in cell number after incubation at 36 °C. The difference between the two populations was more marked after longer storage.     

Observations on the effect of raw milk quality on the keeping quality of pasteurized milk

Raw milk was stored for up to 14 d at 4°C and pasteurized on days 1, 3, 4, 7, 9 and 14. Precautions were taken to eliminate post-pasteurization contamination. The pasteurized milks were stored at 4°C and analysed at weekly intervals for standard plate counts (SPC), psychrotrophic counts (PC) and aerobic spore counts (ASC). The initial raw milk quality was very good and the keeping quality of all the pasteurized milks tested was greater than 22 d. In some cases the milk still had acceptable SPC after 42 d storage, which shows the keeping quality that can be achieved when the process is well controlled. However, the best keeping quality resulted from milk pasteurized on the third and fourth days. Even milk pasteurized on the seventh and ninth had superior keeping quality to that pasteurized on the first day. The lactoperoxidase anti-microbial system in raw milk may be most active around days 3 and 4.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4° to 25°C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 μm Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4°C > 15°C > 25°C > 37°C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R 1 drd -19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15°C or of loss of plasmid function during starvation.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Death of the Escherichia coli K-12 strain W3110 in soil and water.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.     

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.     

Starvation-Induced Thermal Tolerance as a Survival Mechanism in a Psychrophilic Marine Bacterium

Carbon-starved cultures of strain Ant-300, a psychrophilic marine vibrio isolated from the Antarctic Convergence, were compared with their nonstarved counterparts for resistance to heat. Specifically, starved and unstarved cells were exposed to 17°C, which is 4°C above the maximum growth temperature, and compared with cells maintained at the optimum temperature (5 to 7°C). Total cell counts, direct viable-cell counts, and plate counts were monitored. At a temperature of 17°C, viability (as indicated by plate counts) was lost within 40 h, with direct viable-cell counts indicating less than 5% viability at this time. However, when cells were carbon starved for 1 week prior to heat challenge, significant plateability was maintained for more than 6 days; direct viable-cell counts of starved cells maintained at 17°C indicated the presence of viable cells for at least 12 days. Because starvation is the normal physiological state of copiotrophic, heterotrophic bacteria in oligotrophic marine waters, these data suggest that starvation conditions may be a significant factor in providing heat tolerance to psychrophiles.     

Total and viable Legionella pneumophila cells in hot and natural waters as measured by immunofluorescence-based assays and solid-phase cytometry

A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter(-1), and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 10(3) viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples.     

Inducible gene expression by nonculturable bacteria in milk after pasteurization

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5 degrees C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.     

A membrane-immunofluorescent-viability staining technique for the detection of Salmonella spp. from fresh and processed meat samples

A direct staining technique was investigated for the detection of viable Salmonella in fresh and processed meats. The technique involved overnight enrichment in BPW, extraction of Salmonella cells onto a polycarbonate membrane, followed by detection of the pathogen using anti-Salmonella monoclonal antibody coupled with an antibody linked-fluorescent stain (Texas Red) and a viability stain (Sytox Green). The technique was applied to the detection of Salm. enteritidis inoculated into broth culture or minced beef and then subjected to a variety of stresses including freezing (- 20 degrees C), heating (2 or 4 min at 56.9 degrees C), low pH (5 or 3.5) or high salt (2 or 4%). The correlation between traditional plate counts and the rapid count varied widely (r2 = 0.98-0.03), depending on the type and level of stress applied to the cells. The reason for the disparity in results obtained, and the potential application of the method as a diagnostic tool, are discussed.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.