Showing papers by "Arun K. Bhunia published in 2002"

Efficacy of Chlorine Dioxide, Ozone, and Thyme Essential Oil or a Sequential Washing in Killing Escherichia coli O157:H7 on Lettuce and Baby Carrots

Abstract: Chlorine dioxide (ClO2), ozone, and thyme essential oil has been found to be effective in reducing pathogens, including Escherichia coli O157:H7, on selected produce. The efficacy of these sanitizers was evaluated, alone or through their sequential washing to achieve a 3 or more log reduction of mixed strains of E. coli O157:H7 on shredded lettuce and baby carrots. Samples sprinkle inoculated with mixed strains of E. coli O157:H7 were air-dried for 1 h at 22±2°C in a biosafety cabinet, stored at 4°C for 24 h, and then treated with different concentrations of disinfectants and exposure time. Sterile deionized water washing resulted in approximately 1log reduction ofE. coli O157:H7 after 10 min washing of lettuce and baby carrots. Gaseous treatments resulted in higher log reductions in comparison to aqueous washing. However, decolorization of lettuce leaves was observed during long exposure time. A logarithmic reduction of 1.48–1.97log10 cfu/g was obtained using aqueous ClO2 (10.0 mg/L for 10 min) ozonated water (9.7 mg/L for 10 min) or thyme oil suspension (1.0 mL/L for 5 min) on lettuce and baby carrots. Of the three sequential washing treatments used in this study, thyme oil followed by aqueous ClO2/ozonated water, or ozonated water/aqueous ClO2 were significantly (P<0.05) more effective in reducing E. coli O157:H7 (3.75 and 3.99log, and 3.83 and 4.34 log reduction) on lettuce and baby carrots, respectively. The results obtained from this study indicate that sequential washing treatments could achieve 3–4log reduction of E. coli O157:H7 on shredded lettuce and baby carrots.

Microscale electronic detection of bacterial metabolism

Abstract: In this paper, we present a microscale impedance-based technique for detecting the metabolic activity of a few live bacterial cells. Impedance-based detection relies on measuring changes in the ac impedance of two electrodes in contact with a liquid where the bacteria are cultured, caused by the release of ionic species by metabolizing cells. Rapid detection of a few live cells (1–10) is, in theory, possible if the cells are confined into a volume on the order of nanoliters. A microfluidic biochip prototype has been fabricated to explore this technique, consisting of a network of channels and chambers etched in a crystalline silicon substrate. The complex impedance of bacterial suspensions is measured with interdigitated platinum electrodes in a 5.27 nl chamber in the biochip at frequencies between 100 Hz and 1 MHz. After 2 h of off-chip incubation, the minimum number of live cells suspended in a low conductivity buffer that could be easily distinguished from the same number of heat-killed cells was on the order of 100 Listeria innocua , 200 L. monocytogenes , and 40 Escherichia coli cells, confined into the 5.27 nl chamber. A number on the order of 100 live L. innocua cells suspended in Luria–Bertani (LB) broth produced a significantly higher signal than the same number of heat-killed cells, and a difference is evident even down to ∼5–20 cells. To the best of our knowledge, this is the first demonstration of microscale impedance-based detection of bacterial metabolism.

Glucose and Nutrient Concentrations Affect the Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

Abstract: Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K(2)HPO(4) reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.

Electrical characterization of micro-organisms using microfabricated devices

Abstract: This article describes the electrical characterization of live and heat-inactivated micro-organisms within microstructures. Electrophoretic movement of live and heat-inactivated Listeria innocua on interdigitated fingered electrodes confirmed the presence of negative charges on the live micro-organisms and demonstrated positive or neutral charges on the heat-inactivated micro-organisms. A micropore was fabricated in an oxide coated silicon diaphragm, which was placed between two chambers containing ionic buffer solutions. When the micro-organism electrophoretically traversed the pore, the background ionic current was blocked and a decrease in the current was observed. As an initial test case, negatively charged polystyrene beads, which were 2.38 μm in diameter, were electrophoretically driven across the pore. Then, Listeria innocua, suspended in tris-glycine buffer, were also moved through the pore and their electrophoretic mobility was determined. In addition, the values of mobility of the micro-organism...

Microfabricated Device for Impedance-Based Detection of Bacterial Metabolism

Abstract: We present the use of a microfabricated device for impedance-based detection of a few live bacterial cells. Impedance-based detection relies on measuring changes in the AC impedance of two electrodes immersed in a liquid were the bacteria are cultured, caused by the release of ionic species by metabolizing bacterial cells. Rapid detection of a few cells (1 to 10) is possible if the cells are confined into a volume on the order of nanoliters. A microfluidic biochip prototype has been fabricated to test this miniaturized assay. The conductance of the bacterial suspensions is extracted from measuring their complex impedance in a 5.27 nl chamber in the biochip, at several frequencies between 100 Hz and 1 MHz. Measurements on suspensions of the bacteria Listeria innocua, Listeria monocytogenes, and Escherichia coli in a low conductivity buffer demonstrate that, under the current experimental conditions, the minimum detection level is between 50 and 200 live cells, after two hours of off-chip incubation. Work is in progress to develop techniques for selective capture of bacteria inside the chip, and to minimize background changes in impedance during on-chip incubation.