Real-time determination of the efficacy of residual disinfection to limit wastewater contamination in a water distribution system using filtration-based luminescence.

Water distribution systems can be vulnerable to microbial contamination through cross-connections, wastewater backflow, the intrusion of soiled water after a loss of pressure resulting from an electricity blackout, natural disaster, or intentional contamination of the system in a bioterrrorism event. The most urgent matter a water treatment utility would face in this situation is detecting the presence and extent of a contamination event in real-time, so that immediate action can be taken to mitigate the problem. The current approved microbiological detection methods are culture-based plate count methods, which require incubation time (1 to 7 days). This long period of time would not be useful for the protection of public health. This study was designed to simulate wastewater intrusion in a water distribution system. The objectives were 2-fold: (1) real-time detection of water contamination, and (2) investigation of the sustainability of drinking water systems to suppress the contamination with secondary disinfectant residuals (chlorine and chloramine). The events of drinking water contamination resulting from a wastewater addition were determined by filtration-based luminescence assay. The water contamination was detected by luminescence method within 5 minutes. The signal amplification attributed to wastewater contamination was clear-102-fold signal increase. After 1 hour, chlorinated water could inactivate 98.8% of the bacterial contaminant, while chloraminated water reduced 77.2%.

Rapid detection of enterococci in marine beach water by immunomagnetic capture and bioluminescence and its comparison with conventional methods.

A rapid method has been developed to determine enterococci using immunomagnetic separation and bioluminescence. Small paramagnetic beads are coated with antibodies having a specific affinity to enterococci. The captured enterococci are quantified by bioluminescence. The entire procedure takes 30 minutes without a pre-enrichment step. After developing the method, field tests were performed in California. Eight beach samples were provided for determining enterococci using the rapid method. At the same time, the same samples were tested with traditional methods performed by 26 local laboratories. The results show a significant agreement between the two methods. In six out of the eight locations, the predictions based upon the rapid method agreed well with the average values by the traditional methods in determining the quality of swimmable water. Among these, the four predictions were very close to the average colony count. The results also showed the sensitivity of the rapid method (<104 CFUs/100 ml).

Quantification, distribution, and possible source of bacterial biofilm in mouse automated watering systems.

The use of automated watering systems for providing drinking water to rodents has become commonplace in the research setting. Little is known regarding bacterial biofilm growth within the water piping attached to the racks (manifolds). The purposes of this project were to determine whether the mouse oral flora contributed to the aerobic bacterial component of the rack biofilm, quantify bacterial growth in rack manifolds over 6 mo, assess our rack sanitation practices, and quantify bacterial biofilm development within sections of the manifold. By using standard methods of bacterial identification, the aerobic oral flora of 8 strains and stocks of mice were determined on their arrival at our animal facility. Ten rack manifolds were sampled before, during, and after sanitation and monthly for 6 mo. Manifolds were evaluated for aerobic bacterial growth by culture on R2A and trypticase soy agar, in addition to bacterial ATP quantification by bioluminescence. In addition, 6 racks were sampled at 32 accessible sites for evaluation of biofilm distribution within the watering manifold. The identified aerobic bacteria in the oral flora were inconsistent with the bacteria from the manifold, suggesting that the mice do not contribute to the biofilm bacteria. Bacterial growth in manifolds increased while they were in service, with exponential growth of the biofilm from months 3 to 6 and a significant decrease after sanitization. Bacterial biofilm distribution was not significantly different across location quartiles of the rack manifold, but bacterial levels differed between the shelf pipe and connecting elbow pipes.

Simple and rapid method for detection of bacterial spores in powder useful for first responders.

The need for a rapid method by which first responders can screen for the presence of spores in powder samples has been increased since the anthrax attack of 2001. The majority of powders that were sampled in the context of that attack were hoaxes and did not contain Bacillus anthracis. The large number of samples overwhelmed the analysis capacity of public health laboratories. A rapid screening method for determining the presence of viable spores would eliminate much laboratory work and expedite procedures for identification if spores were detected. In the study reported here, Bacillus thuringiensis was used as a surrogate for B. anthracis to investigate if heat shock followed by luminescence analysis would allow rapid quantification of viable spores. The aim of the study was to investigate the best and fastest heat shock conditions that would trigger the breakdown of endospore dormancy of Bacillus species and give a higher luminescence signal. Heat shock and luminescence do not identify the type of spores in a powder sample, but they can detect the presence of viable spores in near real time. Different types of germinant were tested at different conditions (germination duration, temperature, concentration). The rapid method was compared with the traditional plate count method. The results showed that the rapid method can be used as a rapid (< 15 min), sensitive (< 100 spores), and inexpensive detection technique. The rapid method can be applied on site when suspicious powder is found and enables decision making for further identification.

Rapid assessment of microbial hazards in metalworking fluids.

Industries that use metalworking fluids require a test method that can rapidly estimate the total number of bacteria. Such a test method would improve the ability to manage the metal working fluid by allowing near real-time decision making. The ability to manage the fluid more consistently and make critical decisions as they arise would reduce occupational exposures to contaminated metalworking fluid and likely result in fewer worker complaints and/or work stoppages. In this study, a filtration-based rapid adenosine triphosphate bioluminescence assay, which takes less than 10 min to perform, was evaluated as a test method for estimating total bacteria populations in metalworking fluid. This evaluation used two types of metalworking fluid (soluble and semisynthetic) that were inoculated using Pseudomonas aeruginosa and spoiled metalworking fluid. Daily parallel testing was completed on metalworking fluid samples using the rapid adenosine triphosphate assay and the standard plate count methods. Daily test results were evaluated by calculating r2 values using statistical correlation and regression procedures for each fluid type. Study results indicate the rapid adenosine triphosphate assay is strongly correlated to the standard plate count method for soluble and semisynthetic fluids, producing results of r2 = 0.74 and r2 = 0.89, respectively.

A rapid screening method for the detection of viable spores in powder using bioluminescence.

A rapid diagnosis of a biological threat in a powder sample is important for fi rst responders who have to make decisions on-site. The present culture-based method does not provide timely results, which is a critical barrier for a quick response when a suspicious powder sample is found. The ATP bioluminescence method, combined with a heat shock, was investigated to determine the presence of spores in powder. The results show that only spore-containing powder samples provided a dramatic increase in the bioluminescence signal after the heat shock, which induces germination of the spores. Various conditions were tested to fi nd the most effective and rapid germination procedure. Elevated temperatures (37 degrees C and 50 degrees C) were more effective in germination than room temperature. At 50 degrees C, a double-strength germinant was more effective in germination than the regular strength. The 37 degrees C/15 min procedure induced the germination of spores most effectively, while a 50 degrees C/2 min procedure provided reasonably high signals, so it could make the entire procedure even faster (< 5 min). The detection limit of the bioluminescence method is < 100 spores.

Detection of E. coli in beach water within 1 hour using immunomagnetic separation and ATP bioluminescence.

The contamination of beach waters occurs from the discharge of storm water and sanitary sewer overflows containing faecal material. Additional faecal material derives from discharge of animals and waterfowl. In order to protect public from exposure to faecal-contaminated water, it is required to test enteric indicators in beach water. The problem is that the traditional culture-based methods cannot meet this goal because it takes too long (>24 h), so the results are not available until a day later. A rapid method for testing beach water for Escherichia coli within 1 h has been developed. Immunomagnetic separation (IMS) and ATP bioluminescence were used for selective capture and quantification, respectively. This rapid method was compared to the current method (m-TEC) using beach water samples. The beach samples were prefiltered with a 20 microm pore size filter in order to remove algae, plant debris and large particles. The results showed that the prefiltration step did not trap the bacteria which were present in the original water samples. The prefiltered water was then passed through a 0.45 microm pore size filter for concentration. The deposited bacteria were resuspended and then mixed with superparamagnetic polystyrene beads (diameter of 0.6 microm) that were coated with E. coli antibodies. After IMS, the quantification of the E. coli was done by ATP bioluminescence. The results obtained with IMS-ATP bioluminescence correlated well with the plate count method (Rsq = 0.93). The detection limit of the assay was about 20 CFU/100 mL, which is well below the US EPA limits for recreational water. The entire procedure can be completed in less than 1 hour. The necessary equipment is portable and was tested on-site.