Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance

Highly Sensitive Detection of Staphylococcus aureus Directly from Patient Blood

Background: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing – polymerase chain reaction (PCR) platform as a model diagnostic system. Methodology/Principal Findings: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced limit of detection (LOD) of 5 genomic copies per reaction or 10 colony forming units (CFU) per ml blood over 50 copies per reaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance of the bacteria in different components of the blood (white blood cells (WBCs), plasma or whole blood), using the above assays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in maximum PCR positives with sodA assay (90 % positive) as opposed to cell-associated bacteria (in WBCs) (71 % samples positive) or free bacterial DNA in plasma (62.5 % samples positive). Both the assays were further tested for direct detection of S. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of culture-positive patients (sensitivity 89%, 95 % CI 0.75–0.96) and 0/59 negative controls with the sodA assay (specificit

A Review of Techniques in Microbiology: a Student Handbook

<strong>Review of</strong>: <em>Techniques in Microbiology: a Student Handbook </em>; John M. Lammert; (2007) . Pearson Education, Inc., Upper Saddle River, NJ. 225 pages

Rapid Ped-2E9 Cell-Based Cytotoxicity Analysis and Genotyping of Bacillus Species

Bacillus species causing food-borne disease produce multiple toxins eliciting gastroenteritis. Toxin assays with mammalian cell cultures are reliable but may take 24 to 72 h to complete and also lack sensitivity. Here, a sensitive and rapid assay was developed using a murine hybridoma Ped-2E9 cell model. Bacillus culture supernatants containing toxins were added to a Ped-2E9 cell line and analyzed for cytotoxicity with an alkaline phosphatase release assay. Most Bacillus cereus strains produced positive cytotoxicity results within 1 h, and data were comparable to those obtained with the standard Chinese hamster ovary (CHO)-based cytotoxicity assay, which took about 72 h to complete. Moreover, the Ped-2E9 cell assay had 25- to 58-fold-higher sensitivity than the CHO assay. Enterotoxin-producing Bacillus thuringiensis also gave positive results with Ped-2E9 cells, while several other Bacillus species were negative. Eight isolates from food suspected of Bacillus contamination were also tested, and only one strain, which was later confirmed as B. cereus, gave a positive result. In comparison with two commercial diarrheal toxin assay kits (BDE-VIA and BCET-RPLA), the Ped-2E9 assay performed more reliably. Toxin fractions of >30 kDa showed the highest degree of cytotoxicity effects, and heat treatment significantly reduced the toxin activity, indicating the involvement of a heat-labile high-molecular-weight component in Ped-2E9 cytotoxicity. PCR results, in most cases, were in agreement with the cytotoxic potential of each strain. Ribotyping was used to identify cultures and indicated differences for several previously reported isolates. This Ped-2E9 cell assay could be used as a rapid (∼1-h) alternative to current methods for sensitive detection of enterotoxins from Bacillus species

Conductivity and pH Dual Detection of Growth Prfile of Healthy and Stressed Listeria monocytogenes

In this study, growth of Listeria monocytogenes in a low conductivity growth medium (LCGM) was simulaneously monitored by conductivity and pH measurements. Detection times obtained from the conductivity and pH growth curves were inversely related to the initial concentration of L. monocytogenes in the medium. Linear responses were found by plotting detection times obtained from both conductivity and pH growth curves as a function of initial cell concentration in the range of 10^2 to 10^7 cfu/mL. The detection time was approximately 12 and 2 h for 10^2 and 10^7 cfu/mL of viable L. monocytogenes, respectively, using the conductivity growth curves, whereas it was approximately 1 h less using the pH growth curves. This dual detection system was used for valuating the growth of acid-, temperature-, and salt-treated L. monocytogenes in the medium. Acid stress at pH 2 and 3 for 3 h caused approximately 12 and 4 h delay in the detection time on pH growth curves, while stress at pH 5 for 3 h did not cause a significant delay in detection time. Delay in detection times was also observed for L. monocytogenes cells exposed to 45 degrees C for more than 1 h (2 and 6 h). Exposure to 10% NaCI for 3 h did not cause visible delay in the detection time. These observations on detection times for stressed L. monocytogenes had a consistent trend with the cell number decrease determined by surface plating method

The growth profiles of DEP-treated in LCGM medium monitored by real time pH measurements, along with control samples

The DEP treatment conditions and initial cell numbers in the samples are shown in each plot. DEP voltages for all samples were at 20 Vpp. Arrows indicate the detection times on pH-growth curves.<p><b>Copyright information:</b></p><p>Taken from "Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of "</p><p>http://www.jbioleng.org/content/2/1/6</p><p>Journal of Biological Engineering 2008;2():6-6.</p><p>Published online 16 Apr 2008</p><p>PMCID:PMC2373775.</p><p></p

(a)cells exhibit negative DEP at 1 kHz and 3 Vpp; they are collected at the centers of the finger electrodes

(b) Some of the cells exhibit negative DEP, while others exhibit positive DEP, at 10 kHz and 3 Vpp; Cells are collected at both the centers of the finger electrodes and the edges of the finger electrodes. (c) As frequency increases to 50 kHz (at 3 Vpp), all the cells exhibit positive DEP and are collected at the edges of the finger electrodes.<p><b>Copyright information:</b></p><p>Taken from "Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of "</p><p>http://www.jbioleng.org/content/2/1/6</p><p>Journal of Biological Engineering 2008;2():6-6.</p><p>Published online 16 Apr 2008</p><p>PMCID:PMC2373775.</p><p></p

The ELISA results for the immuno-reactivity of DEP treated and untreated cells to PAb Lm404 (anti-InlB), PAb C639 (anti-ActA), anti-species antibody from KPL (Gathersburg, MD) and C11E9 monoclonal antibody

Cell concentrations were about 10cfu/ml. Statistically significant difference was observed at P > 0.0002 (*) and P > 0.009 (**).<p><b>Copyright information:</b></p><p>Taken from "Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of "</p><p>http://www.jbioleng.org/content/2/1/6</p><p>Journal of Biological Engineering 2008;2():6-6.</p><p>Published online 16 Apr 2008</p><p>PMCID:PMC2373775.</p><p></p