The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% ؎ 2%) and FCM (4% ؎ 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD PFGE ] ‫؍‬ 3% ؎ 2% and RSD FCM ‫؍‬ 1.2% ؎ 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional bandbased methods.DNA fragment sizing is arguably the most widely used analytical method in molecular biology, biochemistry, and microbiology. Specific applications of DNA fragment sizing include microbe identification and discrimination, genotyping, and sequencing. Traditionally, DNA fragments are characterized via size-dependent separation methods such as gel electrophoresis.Conventional gel electrophoresis, using a solid polymer (e.g., agarose or polyacrylamide) and a static electric field, is ubiquitous in today's molecular biology and biochemistry labs. Such methods are routinely used to separate and size DNA fragments of Յ20 kb (4).Pulsed-field gel electrophoresis (PFGE), developed in 1984 by Schwartz et al. (44), extended the fragment sizing range to over 1 Mb (4,6,9,19,44). For PFGE, large DNA fragments (i.e., Ͼ10 kb) are separated in an agarose gel by a pulsed electric field. A popular application of PFGE has been strainlevel bacterial fingerprinting through the sizing of DNA fragments resulting from the digestion of whole genomic DNAs with rare-cutting restriction endonucleases (5,11,28,35,47,49). Macrorestriction-based bacterial fingerprinting has found applications primarily in the public health and food safety industries. For example, the Centers for Disease Control and Prevention (CDC) have formed PulseNet, the National Molecular Subtyping Network for Foodborne Disease Surveillance (46; http://www.cdc.gov/pulsenet). PulseNet utilizes macrorestriction fi...

Virus detection and enumeration has become increasingly important in fields ranging from medicine and biotechnology to environmental science. Although there are a wide variety of techniques that can be used to count viruses, there is demand for a rapid and more accurate means for virus enumeration. In this work, the performance of a flow cytometer that was designed and custom-built specifically for rapid detection of single viruses was evaluated. The instrument, designated a single nanometric particle enumerator (SNaPE), was characterized and calibrated using fluorescent polystyrene nanospheres. The reliability of the instrument with respect to virus enumeration was demonstrated for three medically relevant viruses, adenovirus-5, respiratory syncytial virus, and influenza A, treated with a fluorescent nucleotide stain. In each case, the SNaPE yielded a virus particle concentration consistent with, but slightly lower than, transmission electron microscopy (TEM) results, as expected. In addition, on the basis of calibration of signal intensity, the average peak height for a given virus was correlated with genome size, as expected. In contrast to time-consuming analyses such as TEM and plaque titers, SNaPE analysis of pure virus samples (including sample handling, data collection, and data processing) can be completed within 1 h.

The utility of a new instrument for rapid virus quantitation, the Virus Counter, was evaluated in a blind study conducted at three sites. This instrument is a substantially improved version of the original academic research instrument described previously by Stoffel et al. (2005a). The addition of hydrodynamic focusing, a self-contained fluidics system and customized software for system control and data analysis has resulted in a commercially viable and available design.

Surface-enhanced Raman spectroscopy (SERS) has been used to study the mechanism of “photooxidation” of decanethiol self-assembled on roughened Ag. Direct exposure of self-assembled decanethiol to light from a low-pressure Hg lamp under ambient conditions results in adsorbed oxidized sulfur species. Absorbance spectroscopy was used to quantify ozone levels (∼ 20 ppm) generated by the Hg lamp. When decanethiol samples were exposed to the same amount of 254 nm light from the lamp but were environmentally isolated, so that oxygen was present but ozone generated by the lamp could not diffuse to the surface, no oxidation was observed. In addition, when decanethiol samples were exposed to an equivalent amount of electrically generated ozone in the absence of light, the SERS oxidation product spectra were identical to those generated by the lamp under ambient conditions. Quantitative investigation of the kinetics of oxidation yielded second-order rate constants of 0.0029 ± 0.0005 ppm−1 s−1 and 0.0024 ± 0.0006 ppm−1 s−1, for electrically generated ozone and the lamp, respectively. The two rate constants are the same within error, indicating that under the conditions of our experiment the mechanism of oxidation is dominated by ozone and not light.

Background:The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N Ͼ 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters. Methods: Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of singlemolecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM). Results: Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five