Effects of Potassium Sorbate Alone and in Combination with Sodium Chloride on the Growth of Salmonella typhimurium 7136

The effects of potassium sorbate alone and in combination with 2 or 3% sodium chloride (NaCl) on the growth of Salmonella typhimurium ATCC 7136 were examined. Growth studies were performed in tryptic soy brogh (adjusted to a final pH 6.3) at 22°C for 15 days and 35°C for 48 hr without shaking. A dual plating procedure was employed to monitor growth (tryptic soy agar) and development of injury in the cell population (Levine's eosine methylene blue agar + 2% NaCl + 0.05% sodium desoxycholate). The difference between these two plate counts served as a measure of the number of injured organisms. It was found that combinations of sorbate and NaCl were more effective in the inhibition of S. typhimurium than sorbate alone under the conditions and variables studied. No injury was observed in the cell population as determined by our assay procedure. The combination of 3% NaCl + 0.3% potassium sorbate was the most effective in inhibiting growth of S. typhimurium at either storage temperature.     

Salmonella Detection in Meat and Fish by Membrane Hybridization with Chromogenic/Phosphatase/Biotin DNA Probe

The hybridization specificity of a biotin labeled 1.8 kb HindIII DNA fragment was confirmed by colony hybridization with Salmonella and non-Salmonella isolates. Culture conditions were then tested for the enrichment of salmonellae in foods with large populations of competitive flora. Different conditions of preenrichment and selective enrichment could be used for detecting low populations of salmonellae in foods. Enrichment using lactose combined tetrathionate (CTET) broth and Salmonella-Shigella (SS) agar followed by spotting the suspected isolates on a nitrocellulose membrane [CETE → SS (S)] was better. As low as 1.6 × 10⁰ salmonellae/g food could be detected.     

Comparison of selective agar media and enrichment broths for recovering heat-stressedEscherichia coliO157:H7 from ground beef

Media for detecting and enumerating healthy as well as heat-injured cells ofEscherichia coliO157:H7 in foods are highly desired. This study was conducted to evaluate the performance of eight selective and two non-selective direct plating agar media for their ability to recoverE. coliO157:H7 cells from unheated and heated ground beef, and to compare the ability of five enrichment broths to recoverE. coliO157:H7 cells from heated ground beef. Ground beef was incoulated withE. coliO157:H7 and heated at 56°C for up to 30 min. Each agar was evaluated for its ability to support colony formation byE. coliO157:H7 surviving heat treatment, and each enrichment broth was evaluated for its ability to recover low numbers of surviving cells. Of the selective media tested, modified eosin methylene blue agar (MEMB) and Rainbow^TMagar O157 supported recovery of significantly (P≤0·05) higher numbers of heat-stressed cells ofE. coliO157:H7, regardless of heating time. CHROMagar^TMO157, sorbitol MacConkey agar (SMA) supplemented with cefixime and potassium tellurite (CT-SMAC), and SMA supplemented with cefixime and rhamnose (CR-SMAC) performed less favorably, even in recovering cells ofE. coliO157:H7 that had not been subjected to heat stress. SMA and BCM^TMO157:H7 agar were similar to CT-SMAC and CR-SMAC in their ability to recoverE. coliO157:H7 from heated beef. Tryptone bile X-glucuronide (TBX) agar performed significantly better than these media, but was inferior to MEMB agar and Rainbow^TMagar O157:H7. Enrichment using tryptone soya broth with novobiocin or a procedure using brain–heart infusion and tryptone phosphate broths recovered the highest population of heat stressedE. coliO157:H7. EZ Coli^TMenrichment broth was inferior to other broths in resuscitating injured cells and supporting subsequent growth.     

EVALUATION OF PCR AND DNA HYBRIDIZATION PROTOCOLS FOR DETECTION OF VIABLE ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN IRRADIATED BEEF

The sensitivity of DNA hybridization and polymerase chain reaction (PCR), was evaluated in irradiated cooked and raw beef samples. A membrane-based colony hybridization assay and a PCR protocol, both with specificity for the enterotoxin A gene of Clostridium perfringens, were compared with viable plate counts. The results of the colony hybridization procedure were in agreement with viable plate counts for detection and enumeration of enterotoxigenic C. perfringens. The PCR procedure combined a 4 h enrichment followed by a nucleic acid extraction step and assessed the amplification of 183 and 750 base pair enterotoxin gene targets. Detection of C. perfringens by PCR did not show a reliable correlation with viable plate counts or the colony hybridization assay . C. perfringens killed by irradiation were not detected by the plate count or colony hybridization methods; however, killed cells were detected with the PCR technique. By relying on the growth of viable cells for detection and/or enumeration, the colony hybridization and plate count methods provided a direct correlation with the presence of viable bacteria .; Accepted for Publication August 6, 1997     

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.     

Comparison of Media and Influence of Petri Plate Position (Inverted or Upright) for Determining Fungal Populations in Bulk-Stored, Dry, Seed-Based Foods

Total populations of fungi and the presence of aflatoxigenic aspergilli were determined in 109 bulk-stored foods predominantly from seed origin offered for sale by retail grocers. Six media were evaluated for their suitability to support development of fungal colonies. The influence of the position of the Petri dish (inverted or upright) on colony formation during incubation was determined. Overall, dichloran-rose bengal-chloramphenicol agar, oxytetracycline-glucose-yeast extract agar, antibiotic-supplemented plate count agar and rose bengal-chlortetracycline agar were superior to acidified potato dextrose agar for recovering yeasts and molds. Considering any given medium, the position of the plate did not significantly (P ≤ 0.05) influence colony development. Aflatoxigenic strains of Aspergillus flavus or A. parasiticus were detected in 3.7% of the samples.     

Enumeration of total heterotrophic and psychrotrophic bacteria using different types of agar to evaluate the microbial quality of blue mussels (Mytilus edulis) and sea scallops (Placopecten magellanicus)

Microbial quality of Fortune Harbor, NL, cultured blue mussels stored at three temperatures (−12, 2 and 9 °C) for 10 days was evaluated using aerobic plate count (APC) and psychrotrophic plate count (PPC) on plate count agar (PCA) and marine agar (MA). The relationship between bacterial counts in Fortune Harbor mussels on PCA and MA was established using linear regression analysis. The accuracy of selected linear models to predict bacterial count on MA using bacterial counts on PCA of wild and cultured mussels and scallops, stored at 2 °C, was examined. The shelf life of stored mussels and scallops was estimated based on bacterial counts, agar type and storage temperature. Results showed that bacterial counts (APC and PPC) in Fortune Harbor mussels on MA were significantly (p < 0.05) higher than their corresponding counts on PCA agar at all storage temperatures. A strong correlation (r > 0.7, p < 0.01) was observed between bacterial counts in mussels stored at 2 and 9 °C on PCA and MA. The accuracy of the linear models to predict bacterial counts of bivalves on MA using the counts on PCA ranged from 60% to 93%. Both temperature and agar type influenced microbial shelf life estimation while the type of bacteria (APC or PPC) had a lesser effect. Results of this study strongly suggest the use of MA to evaluate the general microbial quality of bivalves instead of PCA or PCA + 1% NaCl.     

Distinctly Different Characteristics of Flocculation in Yeast1

Flocculation of cells of S. cerevisiae NCYC 1109 and S. cerevisiae NCYC 234 was studied. Both cells flocculated 48 h after inoculation although the cells grown for only 20 h were non‐flocculent. EDTA inhibited the flocculation of cells of S. cerevisiae NCYC 1109 and Ca²⁺ enhanced it. Protein‐denaturants, D‐mannose and D‐glucose depressed the flocculation of cells of NCYC 1109. Treatment with proteolytic enzymes, photo‐irradiation in the presence of methylene blue or periodate oxidation caused a loss of the floc‐forming ability of the cells of NCYC 1109. On the contrary, the flocculation of cells of NCYC 234 was not affected by EDTA and Ca²⁺. Protein‐denaturants and monosaccharides did not inhibit the flocculent ability of cells of NCYC 234. Neither proteolytic enzyme treatments, photo‐irradiation in the presence of methylene blue nor periodate oxidation, deprived the cells of NCYC 234 of the floc‐forming ability. Cycloheximide repressed induction of the floc‐forming ability of growing cells of NCYC 1109 significantly but not of growing cells of NCYC 234. These results suggest that the mechanisms of flocculation of NCYC 1109 and NCYC 234 are quite different.     

Comparison of Modified Direct-Plating Procedures for Recovery of Injured Escherichia coli

E. coli ATCC 11775 was heat injured at 59, 62, and 69°C, and recovered by a modification of the conventional coliform count on VRB. Four enrichment media, four selective overlay media, and four preincubation periods were tested to determine the optimal method for recovery of injured coliforms. Preincubation of injured E. coli for 2 hr on tryptone glucose extract (TGE) medium or plate count agar (PCA) significantly increased recovery of injured cells. Single strength violet red bile (VRB) agar gave higher recoveries than double strength VRB. No significant differences were observed between counts on boiled and sterilized VRB. We recommend the use of sterilized VRB since it is more convenient to prepare and store. Of the four enrichment and selective media examined, the recovery media of choice were TGE or PCA when used in combination with the sterilized VRB as the overlay medium. Therefore, preincubation for 2 hr with TGE or PCA overlayed with sterilized VRB is recommended for optimal recovery of injured E. coli.     

Testing matrix, inoculum size, and incubation temperature affect monolaurin activity againstListeria monocytogenes

Current literature reports conflicting minimal inhibitory concentrations (MIC) (range 10–96 g ml⁻¹) of glycerol monolaurate (monolaurin) againstListeria monocytogenes. To resolve these disagreements, the individual MIC of monolaurin against eightL. monocytogenesstrains was determined using the standard agar dilution technique on three commercial media (trypticase soy agar, plate count agar, and Oxford modified medium), and a catfish-based medium using three inocula sizes and three incubation temperatures. Mean MIC was 16 g ml⁻¹on commercial media at 25 and 35°C. Endpoints at 15°C were twofold lower (8 g ml⁻¹) than at 25 and 35°C. On catfish-based medium, MIC values were four- to eightfold higher (64–128 g ml⁻¹) compared with commercial media and depended on incubation temperature and size of inoculum tested. Poor solubility of monolaurin (85 g ml⁻¹) in aqueous solutions and its reduced activity in the presence of food components limits its application as an antimicrobial agent in foods.     

A modified methylene blue assay for accurate cell counting

Cell counting is a common technique in cellular and molecular biology research applications, such as cell culture maintenance, cell plating, cell growth and cell doubling time determinations, as well as cell proliferation and cytotoxicity measurements. Many commonly employed cell counting methods exhibit limitations that influence resulting accuracy or versatility. For example, the trypan blue method typically underestimates cell numbers in culture, and the Lowry protein assay can be influenced by cell cycle. An urgent need exists for a method of cell counting that is both accurate and versatile. This work intended to explore an adaptation of the methylene blue assay to overcome the existing limitations of the procedure, enabling application to a broader range of cell densities and various cell culture plates. This new methylene blue assay was found to be more efficient, accurate and sensitive. A linear relationship (r² > 0.99) was established between cell number and absorbance at 570 nm wavelength when the new methylene blue assay was applied to three cell lines (HepG2, Caco-2, and MCF-7) plated in a broad range of cell densities (5 × 10⁴ to 2.5 × 10⁶) in four different types of culture plates (6-, 12-, 24-, and 96-well plates). Growth curves were determined using both the trypan blue and methylene blue methods. At each time point in the HepG2 growth curve, the cell count obtained using the trypan blue assay was statistically significantly lower than that obtained using the methylene blue assay (p < 0.05). The same was true for the Caco-2 growth curve at all time points (p < 0.05) except at the 0 h. The methylene blue method proposed in this paper may serve as a direct, automated counting method for cells grown in any type of culture plates. This assay has clear advantages over traditional methods and is a powerful tool for any application requiring a versatile, efficient, and accurate method of cell counting, such as bioavailability and cytotoxicity assays, and more basic experiments such as cell growth curve or doubling time determination, especially in the research of natural products, bioactive compounds, phytochemicals, functional foods and nutraceuticals.     

PREDICTIVE MODEL FOR THE COMBINED EFFECT OF TEMPERATURE,pH, SODIUM CHLORIDE,AND SODIUM PYROPHOSPHATE ON THE HEAT RESISTANCE OF ESCHERICHIA COLI O157:H71

The effects and interactions of heating temperature (5–62.5C), pH (4 – 8), NaCl (0 – 6%, w/v), and sodium pyrophosphate (0 – 0.3%, w/v) on the heat resistance of a four strain mixture of Escherichia coli O157:H7 in beef gravy were examined. Thermal death times were determined using a submerged coil heating apparatus. The recovery medium was plate count agar supplemented with 1% sodium pyruvate. Decimal reduction times (D-values) were calculated by fitting a survival model to the data with a curve fitting program. The D-values were analyzed by second order response surface regression for temperature, pH, NaCl and sodium pyrophosphate levels. The four variables interacted to affect the inactivation of the pathogen. Thermal resistance of E. coli O157:H7 can be lowered by combining these intrinsic factors. A mathematical model describing the combined effect of temperature, pH, NaCl and sodium pyrophosphate levels on the thermal inactivation of E. coli O157:H7 was developed. The model can predict D-values for any combinations of temperature, pH, NaCl and sodium pyrophosphate that are within the range of those tested.     

新型琼脂扩散法检测食品中天然防腐剂ε-聚赖氨酸含量

为分析食品中ε-聚赖氨酸（ε-poly-L-lysine,ε-PL）含量,建立一种新型琼脂扩散法,并对分析条件进行优化,验证在检测食品中ε-PL含量时的有效性。结果表明,质量分数0.002%亚甲基蓝、0.75%琼脂、ε-PL溶液pH值为2.0、牛津杯加液量750 μL时检测结果最佳。本方法允许检测橙汁中ε-PL最低含量为2 mg/kg、糕点和猪肉火腿中最低为10 mg/kg。这种新型琼脂扩散法具有操作简便、使用成本低、专一性高等优点,可以广泛应用于ε-PL生产企业和食品工业。     

Detection of E. coli O157:H7 from Ground Beef Using Fourier Transform Infrared (FT-IR) Spectroscopy and Chemometrics

Abstract: FT-IR spectroscopy methods for detection, differentiation, and quantification of E. coli O157:H7 strains separated from ground beef were developed. Filtration and immunomagnetic separation (IMS) were used to extract live and dead E. coli O157:H7 cells from contaminated ground beef prior to spectral acquisition. Spectra were analyzed using chemometric techniques in OPUS, TQ Analyst, and WinDAS software programs. Standard plate counts were used for development and validation of spectral analyses. The detection limit based on a selectivity value using the OPUS ident test was 10⁵ CFU/g for both Filtration-FT-IR and IMS-FT-IR methods. Experiments using ground beef inoculated with fewer cells (10¹ to 10² CFU/g) reached the detection limit at 6 h incubation. Partial least squares (PLS) models with cross validation were used to establish relationships between plate counts and FT-IR spectra. Better PLS predictions were obtained for quantifying live E. coli O157:H7 strains (R²≥ 0.9955, RMSEE ≤ 0.17, RPD ≥ 14) and different ratios of live and dead E. coli O157:H7 cells (R²= 0.9945, RMSEE = 2.75, RPD = 13.43) from ground beef using Filtration-FT-IR than IMS-FT-IR methods. Discriminant analysis and canonical variate analysis (CVA) of the spectra differentiated various strains of E. coli O157:H7 from an apathogenic control strain. CVA also separated spectra of 100% dead cells separated from ground beef from spectra of 0.5% live cells in the presence of 99.5% dead cells of E. coli O157:H7. These combined separation and FT-IR methods could be useful for rapid detection and differentiation of pathogens in complex foods.     

Rapid enumeration of Bifidobacterium lactis in milk powders using impedance

An impedance method was evaluated to enumerate Bifidobacterium lactis, a probiotic, added to milk powder. Impedance changes were measured at 40 °C and recorded using the BacTrac™ 4100 microorganism growth analyser. Five different media were compared for the optimum impedance response. A raffinose-based medium (B. lactis medium) produced the fastest and most reproducible results. Good correlations were obtained between cell numbers from pure cultures of B. lactis (DR 10™) on reinforced clostridial agar plates and the impedance changes in the B. lactis medium. Enumeration of these bacteria in milk powder using the BacTrac™ 4100 impedance system showed no significant difference when compared with agar plate count results. The impedance counts estimated the cell counts of 10⁶ cells g⁻¹ within 15 h and was faster than the 3 days required to obtain a result using the agar plate count method.     

Microbial quality of domestic and imported brands of bottled water in Trinidad

A cross-sectional study was conducted to determine the microbial quality of domestic and imported brands of bottled water available in Trinidad, purchased from six geographical regions in Trinidad, and representing the whole island. A sample size of 344 bottles of water was determined by using a precision rate of 2% and a Type 1 error of 5%. The membrane filter technique was used with cultures grown on m-Endo agar and m-FC agar for total coliforms and thermotolerant coliforms, respectively. Aerobic plate count (APC) was determined on nutrient agar; Pseudomonas aeruginosa was detected on MacConkey agar, Escherichia coli was isolated on eosin methylene blue (EMB) and Salmonella spp. was assayed by using standard methods.Of the 344 water samples tested, 262 (76.2%) and 82 (23.8%) were domestic and imported brands, respectively. Eighteen (5.2%) of the 344 samples contained coliforms with a mean count of 0.88+/-6.38 coliforms per 100 ml, while 5 (1.5%) samples contained E. coli. The prevalence of total coliforms in domestic brands of bottled water was 6.9% (18 of 262) as compared with 0.0% (0 of 82) detected in imported brands. The difference was statistically significant (p=0.004). Similarly, the prevalence of aerobic bacteria in domestic brands of bottled water (33.6%) was significantly higher (p=0.001) than was found in imported brands (14.8%). Twenty-six (7.6%) of the total samples of water contained Pseudomonas species, but all were negative for thermotolerant coliforms and Salmonella spp.It was concluded that based on the recommended zero tolerance for coliforms in potable water, 5% of bottled water sold in Trinidad could be considered unfit for human consumption.     

Modified Microtiter count method for viable cell counts from pure cultures and food model samples

A modified Microtiter count method was designed to assess viable cell counts in a rapid, easy, and accurate way. A Spiral plate method was used for comparison with the modified Microtiter count method. There was a strong correlation between the two methods in microbial counts from pure cultures (Escherichia coli O157:H7, R²=0.984; Salmonella enterica serovar Enteritidis, R²=0.995; and Listeria monocytogenes, R²=0.994) and food samples (R²=0.897). This study suggests that a modified Microtiter count method can be used to determine viable bacterial cell counts.     

Survival and Recovery of Thermally Stressed Yeast in Orange Juice

Injury and survival of thermally stressed yeast in orange juice were assessed by plating on plate count agar, acidified potato dextrose agar, and orange serum agar. Thermally stressed yeast were found to have reduced plate counts on both orange serum agar and acidified potato dextrose agar in comparison to plate count agar. Severely injured populations were not able to repair injury in orange juice held at 25°C but survived storage at 25, 6, and -18°C.     

VITALITY OF CIDER YEAST GROWN MICRO-AEROBICALLY WITH ADDED ETHANOL,BUTAN-J-OL OR ISO-BUTANOL

Vitality and viability of an alcohol-tolerant wine yeast, used in cider production, were assessed after exposure to alkanol(s) during growth. Criteria employed were: methylene blue reduction, ability to form colonies on yeast extract-peptone-glucose agar medium, glucose driven proton efflux rates (“acidification power”), fermentative (CO₂ output) rates and adenylate energy charge values. We also monitored the maintenance of transmembrane electrochemical potential across the plasma membrane as measured by flow cytometry and by scanning confocal laser microscopy of oxonol dye exclusion. Growth rates were diminished by a third by 7.5% (v/v) added ethanol, 1% butan-1-ol or 1.4% iso-butanol. Exposure to 10% (v/v) ethanol gave 16% loss of “viability”, as measured by methylene blue reduction, during the first 20 h of growth. For 1% butan-1-ol, 50% loss of “viability” occurred over 40 h, whereas a similar effect of iso-butanol took 55 h. Adenylate charge values were high (<0.8) in growing cultures, remained high in early stationary phase but declined to 0.4 after 115 h. These values were hardly affected by 5 or 7.5% (v/v) ethanol whereas 10% or 15% (v/v) ethanol gave values of 0.58 and 0.16 after only 5 h exposure. 1% butan-1-ol or iso-butanol decreased adenylate charge values to a greater extent than 10% (v/v) ethanol, with the straight chain alcohol the more potent. Oxonol exclusion indicated that the vast majority of colls with greatly diminished vitality have maintained the plasma membrane potential values required to retain viability, despite extensive exposure to alkanol(s). Thus loss of ability to reduce methylene blue indicates diminished vitality but is not a reliable index of loss of viability. “Acidification power” was a more sensitive indicator of vitality than adenylate charge values. When mixtures of C₂ + C₄ alcohols were employed effects were generally additive rather than synergistic.     

Impact of Mechanical Shear on the Survival of Listeria monocytogenes on Surfaces

Abstract: The impact of mechanical surface shear on microbial viability is rarely a subject for exploration in food processing. The objective of this research was to investigate the impact of mechanical shear on the survival of Listeria monocytogenes on surfaces. Mechanical shear created by slicing a model food was explored to investigate the viability of L. monocytogenes. Cell injury/death was readily demonstrated in fluorescence images by confocal microscopy in which the live and dead cells were fluorescently stained green and red, respectively, with a viability dye kit. Images showed that a large percentage of dead cells appeared after slicing, and they were readily transferred from the slicer blade onto the surfaces of sliced agar, indicating that surface shear may cause the lethal effect on L. monocytogenes. Surface transfer results also showed that viable cell counts on agar slices (in a slicing series) followed a consistently decreasing pattern. The cell counts initially at 5 to 6.5 log CFU/slice (slices 1 to 6), decreased to 3 to 4 log CFU/slice (slices 8 to 30), then to 2 to 3 log CFU/slice (slices 31 to 40), and counts would be expected to further decrease if slicing continued. The overall cell recovery (survival) ratio was about 2% to 3% compared to the initial 8.4 log CFU/blade on a 10 cm² edge area. The impact of shear on microbial viability during slicing may contribute 99% of viable cell count reduction. This study provides clear evidence that surface shear can kill foodborne pathogens and reduce cross-contamination. The lethal effects of surface shear may further enhance food safety.