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1.  Mass and Density Measurements of Live and Dead Gram-Negative and Gram-Positive Bacterial Populations 
Applied and Environmental Microbiology  2014;80(12):3622-3631.
Monitoring cell growth and measuring physical features of food-borne pathogenic bacteria are important for better understanding the conditions under which these organisms survive and proliferate. To address this challenge, buoyant masses of live and dead Escherichia coli O157:H7 and Listeria innocua were measured using Archimedes, a commercially available suspended microchannel resonator (SMR). Cell growth was monitored with Archimedes by observing increased cell concentration and buoyant mass values of live growing bacteria. These growth data were compared to optical density measurements obtained with a Bioscreen system. We observed buoyant mass measurements with Archimedes at cell concentrations between 105 and 108 cells/ml, while growth was not observed with optical density measurements until the concentration was 107 cells/ml. Buoyant mass measurements of live and dead cells with and without exposure to hydrogen peroxide stress were also compared; live cells generally had a larger buoyant mass than dead cells. Additionally, buoyant mass measurements were used to determine cell density and total mass for both live and dead cells. Dead E. coli cells were found to have a larger density and smaller total mass than live E. coli cells. In contrast, density was the same for both live and dead L. innocua cells, while the total mass was greater for live than for dead cells. These results contribute to the ongoing challenge to further develop existing technologies used to observe cell populations at low concentrations and to measure unique physical features of cells that may be useful for developing future diagnostics.
doi:10.1128/AEM.00117-14
PMCID: PMC4054131  PMID: 24705320
2.  The Effect of 3-Thiopheneacetic Acid in the Polymerization of a Conductive Electrotextile for Use in Biosensor Development 
Biosensors  2013;3(3):286-296.
Investigations were conducted to develop an electrotextile using a nonwoven polypropylene fiber platform conformally coated in a conductive, functionalized copolymer of polypyrrole and 3-thiopheneacetic acid (3TAA). The objectives of this study were to determine: (1) if the inclusion of 3TAA in the polymerization process would have an effect on the availability of binding sites in the high-surface area electrotextile for biorecognition elements and (2) how the increase in the concentration of 3TAA would affect the physical characteristics of the coating, resistivity of the sample and availability of binding sites. It was found that the addition of 3TAA to the polymerization process resulted in an increase in the size of the polypyrrole coating, as well as the material resistivity and available binding sites for biorecognition elements. These factors were used to determine which of the tested concentrations was best for biosensor development. A polymer coated membrane sample containing a concentration within the range of 10–50 mg/mL of 3TAA was selected as the best for future biosensor work.
doi:10.3390/bios3030286
PMCID: PMC4263581  PMID: 25586259
3-thiopheneacetic acid; electrotextile; biosensor; polypyrrole; antibody attachment
3.  Synthesis of a Functionalized Polypyrrole Coated Electrotextile for Use in Biosensors 
Biosensors  2012;2(4):465-478.
An electrotextile with a biosensing focus composed of conductive polymer coated microfibers that contain functional attachment sites for biorecognition elements was developed. Experiments were conducted to select a compound with a pendant functional group for inclusion in the polymer, a fiber platform, and polymerization solvent. The effects of dopant inclusion and post-polymerization wash steps were also analyzed. Finally, the successful attachment of avidin, which was then used to capture biotin, to the electrotextile was achieved. The initial results show a nonwoven fiber matrix can be successfully coated in a conductive, functionalized polymer while still maintaining surface area and fiber durability. A polypropylene fiber platform with a conductive polypyrrole coating using iron (III) chloride as an oxidant, water as a solvent, and 5-sulfosalicylic acid as a dopant exhibited the best coating consistency, material durability, and lowest resistance. Biological attachment of avidin was achieved on the fibers through the inclusion of a carboxyl functional group via 3-thiopheneacetic acid in the monomer. The immobilized avidin was then successfully used to capture biotin. This was confirmed through the use of fluorescent quantum dots and confocal microscopy. A preliminary electrochemical experiment using avidin for biotin detection was conducted. This technology will be extremely useful in the formation of electrotextiles for use in biosensor systems.
doi:10.3390/bios2040465
PMCID: PMC4263555  PMID: 25586036
electrotextile; biosensor; polypyrrole; immobilization
4.  Bacteriophage cocktail significantly reduces Escherichia coli O157 
Bacteriophage  2012;2(3):178-185.
Foods contaminated with Escherichia coli O157:H7 cause more than 63,000 foodborne illnesses in the United States every year, resulting in a significant economic impact on medical costs and product liabilities. Efforts to reduce contamination with E. coli O157:H7 have largely focused on washing, application of various antibacterial chemicals, and gamma-irradiation, each of which has practical and environmental drawbacks. A relatively recent, environmentally-friendly approach proposed for eliminating or significantly reducing E. coli O157:H7 contamination of foods is the use of lytic bacteriophages as biocontrol agents. We found that EcoShield™, a commercially available preparation composed of three lytic bacteriophages specific for E. coli O157:H7, significantly (p < 0.05) reduced the levels of the bacterium in experimentally contaminated beef by ≥ 94% and in lettuce by 87% after a five minute contact time. The reduced levels of bacteria were maintained for at least one week at refrigerated temperatures. However, the one-time application of EcoShield™ did not protect the foods from recontamination with E. coli O157:H7. Our results demonstrate that EcoShield™ is effective in significantly reducing contamination of beef and lettuce with E. coli O157:H7, but does not protect against potential later contamination due to, for example, unsanitary handling of the foods post processing.
doi:10.4161/bact.22825
PMCID: PMC3530527  PMID: 23275869
EcoShield™; Escherichia coli O157:H7; bacteriophage; beef; food safety; genomics; ground beef; lettuce; phage; phylogeny
5.  Application of a biotin functionalized QD assay for determining available binding sites on electrospun nanofiber membrane 
Background
The quantification of surface groups attached to non-woven fibers is an important step in developing nanofiber biosensing detection technologies. A method utilizing biotin functionalized quantum dots (QDs) 655 for quantitative analysis of available biotin binding sites within avidin immobilized on electrospun nanofiber membranes was developed.
Results
A method for quantifying nanofiber bound avidin using biotin functionalized QDs is presented. Avidin was covalently bound to electrospun fibrous polyvinyl chloride (PVC 1.8% COOH w/w containing 10% w/w carbon black) membranes using primary amine reactive EDC-Sulfo NHS linkage chemistry. After a 12 h exposure of the avidin coated membranes to the biotin-QD complex, fluorescence intensity was measured and the total amount of attached QDs was determined from a standard curve of QD in solution (total fluorescence vs. femtomole of QD 655). Additionally, fluorescence confocal microscopy verified the labeling of avidin coated nanofibers with QDs. The developed method was tested against 2.4, 5.2, 7.3 and 13.7 mg spray weights of electrospun nanofiber mats. Of the spray weight samples tested, maximum fluorescence was measured for a weight of 7.3 mg, not at the highest weight of 13.7 mg. The data of total fluorescence from QDs bound to immobilized avidin on increasing weights of nanofiber membrane was best fit with a second order polynomial equation (R2 = .9973) while the standard curve of total fluorescence vs. femtomole QDs in solution had a linear response (R2 = .999).
Conclusion
A QD assay was developed in this study that provides a direct method for quantifying ligand attachment sites of avidin covalently bound to surfaces. The strong fluorescence signal that is a fundamental characteristic of QDs allows for the measurement of small changes in the amount of these particles in solution or attached to surfaces.
doi:10.1186/1477-3155-9-48
PMCID: PMC3216855  PMID: 22024374
6.  Enumeration of bacteriophage particles 
Bacteriophage  2011;1(2):86-93.
Bacteriophages are increasingly being utilized and considered for various practical applications, ranging from decontaminating foods and inanimate surfaces to human therapy; therefore, it is important to determine their concentrations quickly and reliably. Traditional plaque assay (PA) is the current “gold standard” for quantitating phage titers. However, it requires at least 18 h before results are obtained, and they may be significantly influenced by various factors. Therefore, two alternative assays based on the quantitative real-time polymerase chain reaction (QPCR) and NanoSight Limited (NS) technologies were recently proposed for enumerating phage particles. The present study compared the three approaches' abilities to quantitate Listeria monocytogenes-, Escherichia coli O157:H7- and Yersinia pestis-specific lytic phages quickly and reproducibly. The average coefficient of variation (CVS) of the PA method including all three phages was 0.15. The reproducibility of the PA method decreased dramatically when multiple investigators performed the assays, and mean differences of as much as 0.33 log were observed. The QPC R method required costly equipment and the synthesis of phage-specific oligonucleotide primers, but it determined phage concentrations faster (within about 4 h) and more precisely than did PA (CVS = 0.13). NS technology required costly equipment, was less precise (CVS = 0.28) than the PA and QPCR methods, and only worked when the phages were suspended in clear medium. However, it provided results within 5 min. After the overall correlation is established with the PA method, either of the two assays may be useful for quickly and reproducibly determining phage concentrations.
doi:10.4161/bact.1.2.15456
PMCID: PMC3278645  PMID: 22334864
bacteriophage; phage; plaque assays; phage titer
7.  Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef by Escherichia coli O157:H7▿  
Applied and Environmental Microbiology  2008;74(20):6230-6238.
A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (1010, 109, and 108 PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 109 PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.
doi:10.1128/AEM.01465-08
PMCID: PMC2570303  PMID: 18723643

Results 1-7 (7)