Progress 09/01/08 to 08/31/13
Outputs
Target Audience: Professionals performing field studies that involve sampling and analysis of airborne microorganisms. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Staff and students who have participated in this project had ample opportunities to learn various aspects of: instrument development; analysis and validation of bioaerosol samplers; application of various tools for bioaerosol analysis; 3D drawing and 3D printing. How have the results been disseminated to communities of interest? The results from this research have been presented at national and international conferences, published in peer-reviewed publications as well as conference abstracts and proceedings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Exposure to airborne biological agents, especially pathogenic or allergenic microorganisms, may cause a wide range of respiratory and other health disorders in occupational and general populations. Such exposures are increasingly recognized as a cause of preventable airborne infections and hypersensitivity diseases. Presence of airborne biological agents is determined by using a variety of bioaerosol samplers that employ principles of impaction, impingement and electrostatic precipitation. To improve our ability to detect airborne microorganisms, especially their low concentrations, there is an impetus to develop new and improved bioaerosol collectors as well as to investigate the performance of existing samplers. As part of our overall effort in this area, the following activities were undertaken. 1. Identification of liquid-based bioaerosol samplers and investigation of their performance. Liquid-based bioaerosol samplers are very popular, however, there is evidence that they may experience loss of already collected particles as the sampling continues. Thus, it is very important to determine the potential for the loss of particles because it may lead to underestimation of the airborne particle concentration. To analyze this potential effect, we spiked the collection fluid with a known concentration of polystyrene latex particles, bacteria (B. subtilis) and fungi (C. cladosporioides) and operated AGI-30 impinger (Ace Glass Inc., Vineland, NJ) and BioSampler (SKC Inc., Eighty Four, PA) for different time periods ranging from 15 to 60 min. The losses were studied at low and high particle concentrations. At the end of the sampling time period the concentration of remaining particles was determined and compared with the initial concentration. The range of reaerosolization rates was 0.2% to 6.9% for both samplers. When 5 ml suspension was used in the BioSampler at low particle concentrations, the internal-loss factor averaged over all particle types ranged from 22% to 36% for 15 min operating time and from 28% to 85% for 30 min sampling time. When the sampler was operated with high particle concentration, the loss factor increased by a factor 1.2 to 1.5. When the BioSampler was operated with 20 ml liquid suspension, the losses were 1% to 54% lower compared to 5ml. This is likely due to the decrease in the distance between liquid surface and the nozzles at higher liquid volume, resulting in a decreased liquid turbulence and less pronounced capture of particles by nozzle walls. Compared to BioSampler (mL), the internal-loss factors of the AGI-30 varied depending on the particle type. The obtained data show that the samplers’ performance is diminished due to internal losses. Such losses should be considered when analyzing the data obtained with these devices. 2. Development and validation of a novel electrostatic sampler. We continued our further development of a novel electrostatic collector for bioaerosols, where biological particles are electrostatically deposited onto a narrow electrode covered by a hydrophobic substance and then removed and collected by rolling water droplets (20 or 40 microliter) to achieve high sample concentration rate. As part of the collector development, it was integrated with a new electrical charger based on carbon fiber brushes. The new integrated sampler was made of static dissipative material (e.g., Delrin) and has a shape of a closed half cylinder. Its top round top part contains carbon fiber brushes, while its flat bottom holds a narrow collection electrode. We tested the sampler with various configurations of carbon brushes comprising the charger and different materials of the collection electrode. The total bioaerosol particles number collected by a water droplet was determined by microscopy and the reference concentration was monitored by a Grimm optical particle counter. Our results showed that electrode material has a great effect on collection efficiency. The collection efficiency based on the electrode material was as follows: copper (19%), brass (30%), plain steel (46%), zinc plate steel and high carbon steel (50%), alloy steel (51%), stainless steel (79%), pressed carbon fiber (87%). A combination of the charger with eight carbon brushes and carbon fiber electrode yielded virtually no particle losses and collection efficiency of 84% when sampling E. coli bacteria at 10 L/min for 10min. The efficiency was the same for 20 and 40 micro-liter collection droplets. When sampling for 60 min, the actual collection efficiency was 70% due to a reduced particle removal from the electrode. However, these results were achieved with bench scale components, such as large power supplies and use of external pumps. In the ongoing development of the sampler, it is being integrated into a field useable instrument with a battery-operated air mover, power supplies and DC-to-DC converter. The body of the integrated sampler has been 3D printed. In addition, the latest sampler configuration features two collection electrodes, thus allowing side-by-side collection of duplicate samples (droplets containing removed particles) that can be analyzed using molecular methods and/or traditional agar techniques. Laboratory tests have shown that the sampler’s average collection efficiency exceeded 80% for bacterial cells when operated at 10 L/min. In the upcoming experiments, this electrostatic collector with high concentration rate will be tested in various field environments against several commercial bioaerosol samplers.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Han, T., Fennell, D., Mainelis, G. (2013) A Field-deployable Electrostatic Collector for Bioaerosols with High Concentration Rate, Abstracts of the 32nd Annual Meeting of the American Association for Aerosol Research (Portland, OR, September 30 - October 4, 2013).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Han, T. , Cai, T., DuBois, K., and Mainelis, G. (2013) Investigation of ATP-based Bioluminescence Effectiveness for Bioaerosol Quantification, Abstracts of the 32nd Annual Meeting of the American Association for Aerosol Research (Portland, OR, September 30 - October 4, 2013).
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Exposure to airborne biological agents, especially pathogenic or allergenic microorganisms, may cause a wide range of respiratory and other health disorders in occupational and general populations. Such exposures are increasingly recognized as a cause of preventable airborne infections and hypersensitivity diseases. Presence of airborne biological agents is determined by using a variety of bioaerosol samplers that employ principles of impaction, impingement and electrostatic precipitation. To improve our ability to detect airborne microorganisms, especially their low concentrations, there is an impetus to develop new and improved bioaerosol collectors as well as to investigate the performance of existing samplers. As part of our overall effort in this area, we recently developed an electrostatic collector for bioaerosols, where biological particles are electrostatically deposited onto a 3.2 mm electrode covered by a hydrophobic substance and then removed and collected by rolling water droplets (20 or 40 microliter) to achieve high sample concentration rate. As part of the further development of the collector, it was integrated with a new electrical charger based on carbon fiber brushes. The new integrated sampler was made of static dissipative material (e.g., Delrin) and has a shape of a closed half cylinder. Its top round top part contains carbon fiber brushes, while its flat bottom holds a narrow collection electrode. The objective of this development was to optimize the charger and collection electrode for maximum performance of the sampler. We tested the sampler with various configurations of carbon brushes comprising the charger and different materials of the collection electrode. The total bioaerosol particles number collected by a water droplet was determined by microscopy and the reference concentration was monitored by a Grimm optical particle counter. PARTICIPANTS: Gediminas Mainelis, Ph.D., PI of the project. Responsible for the overall progress of the project. Supervised experiments, data collection and analysis. Taewon Han, Ph.D. performed laboratory experiments and data analysis TARGET AUDIENCES: Professionals performing field studies that involve sampling and analysis of airborne microorganisms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our results showed that electrode material has a great effect on collection efficiency. The collection efficiency based on the electrode material was as follows: copper (19%), brass (30%), plain steel (46%), zinc plate steel and high carbon steel (50%), alloy steel (51%), stainless steel (79%), pressed carbon fiber (87%). A combination of the charger with eight carbon brushes and carbon fiber electrode yielded virtually no particle losses and collection efficiency of 84% when sampling E. coli bacteria at 10 L/min for 10min. The efficiency was the same for 20 and 40 micro-liter collection droplets. When sampling for 60 min, the actual collection efficiency was 70% due to a reduced particle removal from the electrode. This bioaerosol sampler will be optimized for higher flow rates, but it already shows great promise and could be a part of various applications, including Homeland security.
Publications
- Abstract: Han, T., Fennell, D., and Mainelis, G. (2012). Investigation of an Optimized Single-Stage Electrostatic Precipitator for Bioaerosols Abstracts of the 31st Annual Meeting of the American Association for Aerosol Research (Minneapolis, MN, October 8-12, 2012).
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Exposure to airborne biological agents, especially pathogenic or allergenic microorganisms, may cause a wide range of respiratory and other health disorders in occupational and general populations. Such exposures are increasingly recognized as a cause of preventable airborne infections and hypersensitivity diseases. Presence of airborne biological agents is determined by using a variety of bioaerosol samplers that employ principles of impaction, impingement and electrostatic precipitation. To improve our ability to detect airborne microorganisms, especially their low concentrations, there is an impetus to develop new and improved bioaerosol collectors as well as to investigate the performance of existing samplers. Previous studies have analyzed collection efficiencies and reaerosolization rates of liquid-based bioaerosol collectors. However, these studies did not analyze latent internal losses in the samplers, i.e., the fraction of the particles that is aerosolized from the collection liquid, attach to the samplers' inner surfaces and do not leave the sampler via outlet. Here, we investigated the internal losses and reaerosolization rates in BioSampler (SKC Inc., Eighty Four, PA) and AGI-30 (Ace Glass Inc., Vineland, NJ) bioaerosol collectors operated with different amounts of liquid as a function of particle type (polystyrene latex particles of 0.9 and 3.2 um, B. subtilis bacteria and C. cladosporioides fungal spores), particle concentration ("low" and "high" differing by a factor of 100) and operating time (15 and 30 min). The samplers were filled with sterile deionized water containing known (reference) particle concentrations and were operated in a particle-free atmosphere for 15 or 30 minutes. The overall particle loss was determined by comparing the concentration of particles remaining in the collection liquid with the reference concentration. The reaerosolization rates were estimated by comparing the concentration of particles collected at each sampler's outlet with the reference concentration. The internal loss was determined as a fraction of particles remaining attached to the inner walls of a sampler, i.e., particles not in the collection liquid and not reaerosolized. All the investigated variables had a statistically significant effect on the overall particle loss, the reaerosolization rate, and the internal loss. Averaged for all test conditions, the internal loss for BioSampler with 5 mL liquid, BioSampler with 20 mL liquid and AGI-30 was 37.7%, 29.6%, and 22.5%, respectively. The observed reaerosolization rates were rather low and ranged from 0.2% to 6.9%. This study shows that depending on a particular set of sampling conditions a substantial fraction of already collected particles could leave the collection fluid, attach to the inner surfaces of the samplers and not be available for sample analysis thus affecting the accuracy of bioaerosol investigations. PARTICIPANTS: Gediminas Mainelis, Ph.D., PI of the project. Responsible for the overall progress of the project. Supervised experiments, data collection and analysis. Taewon Han, Ph.D., participated in experiments, data collection and analysis. TARGET AUDIENCES: Professionals performing field studies that involve sampling and analysis of airborne microorganisms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
All the investigated variables had a statistically significant effect on the overall particle loss, the reaerosolization rate, and the internal loss. Averaged for all test conditions, the internal loss for BioSampler with 5 mL liquid, BioSampler with 20 mL liquid and AGI-30 was 37.7%, 29.6%, and 22.5%, respectively. The observed reaerosolization rates were rather low and ranged from 0.2% to 6.9%. This study shows that depending on a particular set of sampling conditions a substantial fraction of already collected particles could leave the collection fluid, attach to the inner surfaces of the samplers and not be available for sample analysis thus affecting the accuracy of bioaerosol investigations. The field professionals using these sampling techniques have to be aware of their limitations and take them into account when analyzing and interpreting the data and offering recommendations based on the data.
Publications
- Han, T. and Mainelis. G. (2011) Investigation of inherent and latent internal losses in liquid-based bioaerosol samplers, Journal of Aerosol Science, 10.1016/j.jaerosci.2011.11.001.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Exposure to airborne biological agents, especially pathogenic or allergenic microorganisms, may cause a wide range of respiratory and other health disorders in occupational and general populations. Such exposures are increasingly recognized as a cause of preventable airborne infections and hypersensitivity diseases. Presence of airborne biological agents is determined by using a variety of bioaerosol samplers that employ principles of impaction, impingement and electrostatic precipitation. To improve our ability to detect airborne microorganisms, especially their low concentrations, there is an impetus to develop samplers that collect airborne microorganisms into small amounts of liquid. We recently developed an Electrostatic Precipitator with Superhydrophobic Surface (EPSS). which collects particles into a 10 to 40 microliters water droplet and achieves very high concentration rates (defined as the ratio of particle concentration in the collection liquid versus the airborne particle concentration per time unit) when sampling airborne particles. In this part of our research we analyzed the performance of this sampler when collecting three commonly found fungal spores under different operating conditions. The sampler collected the airborne test fungi and its performance was determined by comparing the amount of fungal spores that was collected with the amount of fungal spores that entered the sampler. The performance of the sampler was investigated as a function of the collecting water droplet size (10 and 40 microliters), the airborne fungal concentration (from 10^4 to 10^7 spores/m^3) and the sampling time (10, 30 and 60 min). . We also investigated the application of adenosine triphosphate (ATP)-based bioluminescence for the quantification of fungal spores in samples and adapted the methodology to analyze the collection efficiency and the concentration rates of our new sampler. In addition, we investigated conditions that resulted in the optimum performance of the ATP method for quantification of specific fungi, such as the duration of fungal growth prior to harvesting. PARTICIPANTS: Gediminas Mainelis, Ph.D., PI of the project. Responsible for the overall progress of the project. Supervised experiments, data collection and analysis. TARGET AUDIENCES: Professionals performing field studies that involve sampling and analysis of airborne microorganisms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We investigated the collection efficiency of the new sampler when collecting fungal spores and found that it ranged from 10% to 36% at a sampling flow rate of 10 L/min when the airborne fungal spore concentration was from 10^5 to 10^6 spores/m^3 resulting in concentration rates between 100,000 and 300,000 when a 10 microliter droplet was used. We also determined that the collection efficiency was inversely proportional to the airborne spore concentration, and it increased to above 60% for spore concentrations between 10,000 and 100,000 spores/m^3, which is fungi concentration commonly encountered in ambient air. Bioaerosol samplers with high concentration rates, such as the one being developed in this study, enable detection of low ambient bioaerosol concentrations in various environments, including indoor air, and would be useful for improved exposure assessment We also adapted adenosine triphosphate (ATP)-based bioluminescence for the analysis of collection efficiency and the concentration rates of bioaerosol samplers. We found that the spore concentrations determined by the ATP-based method were not statistically different from those determined by microscopy and allowed us to analyze spore concentrations that were too low to be reliably detected by microscopy. A successful adaptation of the adenosine triphosphate (ATP)-based bioluminescence assay for the quantification of fungal spores from a specific species enables fast sample analysis in laboratory investigations. This rapid assay could be especially useful when investigating performance of biological samplers as a function of multiple operational parameters.
Publications
- Han, T., Nazarenko, Y. , Lioy, P.J., and Mainelis, G. (2010) Collection Efficiencies of An Electrostatic Sampler With Superhydrophobic Surface For Fungal Bioaerosols, Indoor Air, accepted: doi: 10.1111/j.1600- 0668.2010.00685.x.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Exposure to airborne biological agents, especially pathogenic or allergenic microorganisms, may cause a wide range of respiratory and other health disorders in occupational and general populations. Such exposures are increasingly recognized as a cause of preventable airborne infections and hypersensitivity diseases. Presence of airborne biological agents is determined by using a variety of bioaerosol samplers that employ principles of impaction, impingement and electrostatic precipitation. Among the available samplers, liquid-based samplers are often employed due to their ease of use and utilization of liquid as a collection medium. Samples collected into liquid could be analyzed by a variety of techniques which is viewed as advantageous by the field users. However, there is evidence that popular liquid-based samplers may experience loss of already collected particles as the sampling continues. Thus, it is very important to determine the potential for the loss of particles because it may lead to underestimation of the airborne particle concentration. Determination of losses was the focus of this part of the project. Here, we spiked the collection fluid with a known concentration of polystyrene latex particles, bacteria (B. subtilis) and fungi (C. cladosporioides) and operated AGI-30 impinger (Ace Glass Inc., Vineland, NJ) and BioSampler (SKC Inc., Eighty Four, PA) for different time periods ranging from 15 to 60 min. The losses were studied at low and high particle concentrations. At the end of the sampling time period the concentration of remaining particles was determined and compared with the initial concentration. This allowed us to determine the potential for particle losses during samplers' operation and to analyze the factors affecting the magnitude of losses. Reaerosolization rates (fraction of already collected particles leaving the sampler during its operation) were estimated by analyzing the particle concentration in the after filter. The difference between overall loss and the reaerosolization rates represent the latent internal loss, i.e., deposition of already collected particles on internal surfaces, other than collection liquid. PARTICIPANTS: Gediminas Mainelis, Ph.D., PI of the project. Responsible for the overall progress of the project. Supervised experiments, data collection and analysis. Taewon Han, Ph.D., Performed experiments and analyzed the data. TARGET AUDIENCES: Professionals performing field studies that involve sampling and analysis of airborne microorganisms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The main outcome of this part of the project was characterization of latent internal losses and re-aerosolization rates of two popular liquid samplers, namely AGI-30 impinger (Ace Glass Inc., Vineland, NJ) and BioSampler (SKC Inc., Eighty Four, PA). The range of reaerosolization rates was 0.2% to 6.9% for both samplers. When 5 ml suspension was used in the BioSampler at low particle concentrations, the internal-loss factor averaged over all particle types ranged from 22% to 36% for 15 min operating time and from 28% to 85% for 30 min sampling time. When the sampler was operated with high particle concentration, the loss factor increased by a factor 1.2 to 1.5. When the BioSampler was operated with 20 ml liquid suspension, the losses were 1% to 54% lower compared to 5ml. This is likely due to the decrease in the distance between liquid surface and the nozzles at higher liquid volume, resulting in a decreased liquid turbulence and less pronounced capture of particles by nozzle walls. Compared to BioSampler (mL), the internal-loss factors of the AGI-30 varied depending on the particle type. The obtained data show that the samplers' performance is diminished due to their internal losses. Such losses should be considered when analyzing the data obtained with these devices. The results of this investigation are being prepared for a publication.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Exposure to airborne biological agents, especially pathogenic or allergenic microorganisms, may cause a wide range of respiratory and other health disorders in occupational and general populations. Such exposures are increasingly recognized as a cause of preventable airborne infections and hypersensitivity diseases. Presence of airborne biological agents is determined by using a variety of bioaerosol samplers that employ principles of impaction, impingement and electrostatic precipitation. As the new samplers are introduced into the market, their performance characteristics are often unknown. One of those important performance parameters is the effect of sampling time on the samplers' performance, especially for the impaction based samplers. Prolonged sampling times may cause desiccation of the collection media or the already collected organisms, which would cause under-reporting of the airborne microbial concentrations. In this part of the project we performed a study on the effect of sampling time on the performance of several portable impactors when collecting airborne bacteria and fungi from 2 to 30 minutes indoors. The performance of portable impactors was compared against the reference BioStage impactor collecting samples for 2 minutes only. The data were statistically analyzed for the effect of sampler model and sampling time on the concentration of microorganisms measured by the rest impactors relative to the reference sampler. PARTICIPANTS: Gediminas Mainelis, Ph.D., PI of the project. Responsible for the overall progress of the project. Supervised experiments, data collection and analysis. Michelle Tabayoyong. Performed experiments and analyzed the data. TARGET AUDIENCES: Professionals performing field studies that involve sampling and analysis of airborne microorganisms. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The main outcome of this part of the project were the experimental results analyzing the effect of sampling time on the performance of portable impactors when sampling bacteria and fungi indoors and outdoors. The data of the study showed that when bacteria were collected indoors, their average recovery by the test samplers relative to the reference sampler decreased from 0.45 when the sampling time was 2 minutes to 0.2, when the sampling time was increased to 30 minute. The same values for fungi being sampled for 2 and 30 min were 0.55 and 0.35, respectively. The effects of sampling time and impactor model were statistically significant for all experimental conditions. Thus, the data obtained in this study suggest that when impactors are used for the collection of airborne bacteria and fungi, sampling times should be as short as reasonably possible to minimize the under-representation of airborne microorganism concentration. These results are important for professionals performing field studies and indicate that the use of portable impactors for prolonged sampling times could substantially affect the quality of the data. Also, the statistical significance of the sampler model on the concentration of recovered airborne microorganisms suggests that only the samplers with satisfactory performance characteristics should be used in the field studies.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This research focuses on the development of an improved bioaerosol sampling system that would allow a more accurate quantification of bioaerosol presence in various environments. The system is based on particle collection by electrostatic method, where the airborne particles are electrically charged and then deposited onto a collection medium. The collected organisms can then be analyzed by traditional (culture) methods and by quantitative Real-Time PCR (QRT-PCR). Since the electrostatic collection technique subjects the microorganisms to lower levels of mechanical stress compared to traditional inertia-based techniques it is considered advantageous. In this part of our research we focused on modifying and improving the collection methodology as well as the experimental and statistical analysis of factors affecting the accuracy of QRT-PCR. Our previous electrostatic collector sampled microorganisms directly onto agar and PCR plates. While advantageous, this method was not
compatible with certain sample analysis techniques, such as microscopy. Thus, we designed and built a prototype of a new electrostatic collector that samples airborne organisms into liquid and, more importantly, small amounts of liquid (5-40uL). Once in the liquid, the collected organisms can be analyzed using a variety of techniques, including culture-based, microscopy and QRT-PCR. The design and performance of this novel collector was presented at the annual conference of the American Association for Aerosol research (AAAR). The data will be further disseminated via a publication, which is currently being prepared. In another part of this research we experimentally and statistically analyzed various factors that might affect the accuracy of QRT-PCR. The factors included bacterial species (sensitive / hardy bacteria), sample preparation methods (cultured / air sampled microorganisms), and (3) PCR preparation method (genomic DNA based PCR / whole cell PCR). The tested bacterial
species included sensitive P. fluorescens bacteria and hardy B. subtilis cells; the cells used in PCR analysis included those cultured in suspension as well as those collected from air; PCR was prepared using genomic DNA as a template as well as a whole-cell PCR. The effect of these variables was examined by building calibration curves where PCR output was correlated with the corresponding number of total cells (based on microscopy). The statistical analysis of the data was performed using two separate models. In one, the data were pooled and only the microorganisms were treated as predicting variables; in another, the data were stratified by bacterial species and effects of other variables was examined on the slopes and intercepts of the calibration curves. The testing methodology as well as the obtained results will be disseminated to the scientific community via publication (currently in preparation) and presentation at conferences.
PARTICIPANTS: Gediminas Mainelis, Ph.D., PI of the project. Responsible for the overall progress of the project. Designs, conducts and supervises experiments as well as data collection and analysis. Hey Reoun An, Ph.D. Candidate. Performed PCR-related experiments and analyzed the data.
TARGET AUDIENCES: Professionals performing sampling and analysis of airborne microorganisms.
Impacts
The new electrostatic collector was shown to have collection efficiency of 70% and higher when collecting particles of 2 um and larger. Since majority of microorganism-containing airborne particles are larger than 2um, this new collector is an effective tool for their capture. More importantly, accumulation of the captured microorganisms in a small amount of liquid allows sample analysis by multiple techniques. In addition, the collector uses low power which may lead to its wide implementation. Collection of particles on a small amount of liquid will also make this collector compatible with various laboratories on-a-chip, which would allow fast sample analysis and our improved assessment of exposure to airborne microorganisms. The analysis of factors affecting QRT-PCR output also indicated that some of the investigated factors have a statistically significant effect on the calibration curves. There was a statistical difference in calibration curves prepared with freshly
cultured bacteria and those form air samples. In addition, there was also a statistical difference among calibration curves prepared with Genomic DNA and when using whole-cell PCR. This research points to factors that play a role in application of QRT-PCR as bioaerosol sample quantification tool and, once disseminated to scientific community, will serve as guidelines for other investigators who might consider applying this method for bioaerosols analysis. The developed insights will be incorporated when applying QRT-PCR for the quantification of cells collected by a new electrostatics-based sampling method.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
This research focuses on the development of a new electrostatics-based bioaerosol collection system which would allow a more accurate determination of exposures to biological particles compared to current systems. Our previous investigations have shown that quantitative Real-Time PCR (QRT-PCR) could be applied as a promising tool for quantification of total bioaerosols (viable and non-viable) collected in air samples. The QRT-PCR relies on standard curves for the sample quantification and the parameters affecting standard curves have not yet been systematically explored. Thus, in this part of our research we generated a family of QRT-PCR standard curves by modifying various factors and analyzed the effect of these factors on the slope and intercept of the curves. The investigated factors included: (1) bacterial species (sensitive / hardy bacteria), (2) sample preparation methods (cultured / air sampled microorganisms), and (3) PCR preparation method (genomic DNA based
PCR / whole cell PCR). P. fluorescens bacteria were used as sensitive cells, while B. subtilis was used as hardy cells. Both pure cultured cells and air sampled cells of each species were tested for standard curve generation. For each sample, two different PCR preparation methods were applied: one using genomic DNA as a template in PCR reactions and the other using whole cell suspension. In addition, when determining cell counts to prepare the standard curves, we analyzed the use of culturable cell counts (based on colony forming units) and total cells (based on microscopy). The results showed that the culturability of the sensitive cells was dramatically decreased by air sampling process, while hardy bacteria were not affected. Thus, it was determined that only total cell quantity should be used as a standard when developing standard curves. When the standard curves prepared by using cultured cells and air-sampled cells were compared, there was a significant difference in standard
curves associated with sample preparation method and bacterial species used. The comparison of standard curves from two PCR preparation methods (GDNA PCR and whole-cell PCR) indicated no statistical difference for cultured cells; however there was a statistically significant difference for air-sampled cells. Overall, the obtained results indicate that factors investigated in this study can substantially affect standard curves that are used in QRT-PCR method. Therefore, for accurate bioaerosol quantification by using QRT-PCR, one should build the standard curves by using methods that mimic the actual sample collection technique.
Impacts
This research indicates that standard curve preparation method can palsy a substantial role in the accuracy of sample quantification by QRT-PCR. Thus, when applying QRT-PCR for quantification of bioaerosol samples, one should build the standard curves by using methods that mimic the actual sampling method. In addition, use of standard curves based on culturable cell quantity may lead to underestimation of bioaerosol concentration in air samples, especially when sampling sensitive organisms. The bacterial species and PCR preparation method also play an important role in accuracy of QRT-PCR. This research points to factors that might play a role in application of QRT-PCR as sample quantification tool and serve as guidelines for other investigators who might consider applying. The developed insights will be incorporated when applying QRT-PCR for the quantification of cells collected by a new electrostatics-based sampling method.
Publications
- An, H.R., Mainelis, G., and White L. (2006) Development and Calibration of Real-Time PCR for Quantification of Airborne Microorganism in Air Sample, Atmospheric Environment, 40: 7924-7939.
- An, H.R., Mainelis, G. and White, L. (2006) Development and Calibration of Whole-Cell Real-Time PCR for Quantification of Total Bacterial Number in Air Samples, Abstracts of the 7th International Aerosol Conference (St. Paul, Minnesota, September 10-15, 2006), p. 875.
- An, H.R., Mainelis, G. and White, L. (2006) Calibration of Real-Time PCR for Quantification of Bacteria in Air Samples, Abstracts of the General American Society for Microbiology Meeting, (Orlando, Florida, May 21-25, 2006), p. 180.
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Progress 01/01/05 to 12/31/05
Outputs
Numerous studies have shown that exposure to bioaerosols is strongly associated with adverse health effect and that development and validation of advanced bioaerosol quantification methods is urgently needed. In this project, we have been continuing to develop the Electrostatic Collector (ESC) capable of concurrently collecting the viable and total bioaerosol particles. Our previous experiments have shown that DNA-based methods could be used for sensitive and rapid analysis of collected total bioaerosol particles. At the same time, preliminary experiments indicated that PCR calibration method may play a crucial role in the accuracy of detection. Since one of the goals of this project is to quantitate exposures to the viable and total bioaerosols, it was necessary to explore the PCR calibration issue in detail. Thus, by using the Escherichia coli Catellani (ATCC 11775) bacteria, one of the recommended organisms when testing bioaerosol samplers, we developed a family of
standard curves by using purified genomic DNA from both pure cultured E. coli and air-sampled E. coli. The universal primer including the forward primer, 5'-TCCTACGGGAGGCAGCAGT-3' and the reverse primer, 5'-GGACTACCAGGGTATCTAATCCTGTT-3' were used in all PCR reactions. For all conditions, a family of standard curves was generated based on the linear relationship between the threshold cycle value and corresponding cell number determined by both culturable cell counts and total cell counts. The developed family of standard curves was used to quantify airborne E. coli cells collected by air sampling. Our results indicate that the Real-Time PCR output substantially depends on the standard curve preparation method. Standard curves built using a traditional approach, where genomic DNA is extracted from pure culture bacteria, diluted in series, and then amplified by the Real-Time PCR yielded underestimation of sample quantities compared to airborne microorganism concentration as measured by
optical particle counter. The underestimation was especially pronounced (by a factor of 20) when the standard curves generated by using colony forming units were used in quantification. In contrast, the estimate of cell concentration in an air sample by Real-Time PCR was much more accurate (60% compared to more established methods) when the standard curve was built using air samples in various concentration. The accuracy improved even further (100%) when air samples used to built the standard curves were diluted first, DNA extracted from each dilution and then amplified by the Real-Time PCR - to mimic the handling of air samples with unknown and possibly low concentration. Thus, by developing and comparing various standard curves we were able to accurately quantify the amounts of microorganisms collected in air samples. Overall, our results demonstrate that to improve accuracy of the Real-Time PCR assay, standard curves should be prepared using the same environmental matrix and
procedures as handling of the environmental sample in question. Reliance only on pure cultured bacterial suspension may lead to substantial underestimation of microorganism quantities in environmental samples.
Impacts
The development of new collection method allowing to concurrently determining viable and total airborne microorganisms will improve our exposure assessment capabilities. The use of such collector will allow extracting more information from one sample and will eliminate the need for samples separately analyzed for total and viable microorganisms. As part of this project, we investigated the calibration of Real-Time Polymerase Chain Reaction (PCR) assay. Our results demonstrate that to improve the accuracy of the Real-Time PCR assay, calibration curves should be prepared using the same environmental matrix and procedures as handling of the environmental sample in question. Reliance only on pure cultured bacterial suspension (classical approach) may lead to substantial underestimation of microorganism quantities in environmental samples. Overall, our data provides important insights into the application of quantitative air sample analysis using Real-Time PCR.
Publications
- An, H.R., Mainelis, G., and White L. (2005) Development and Calibration of Real-Time PCR for Quantification of Airborne Microorganism in Air Sample, Applied and Environmental Microbiology, submitted.
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Progress 01/01/04 to 12/31/04
Outputs
In our continuing development of Electrostatic Collector (ESC) for viable and total bioaerosol particles, we explored application of DNA-based methods for sensitive and rapid air sample analysis. In this part of our study, to quantify the total airborne bacterial number collected by the ESC, we used Real-time PCR assay along with both Colony Forming Units (CFUs) and Microscopy method. Common laboratory test microorganisms, including Escherichia coli, were used in the experiments. Escherichia coli Catellani (ATCC 11775) was one of the bacteria used to obtain standard curves in this study. Standard curves were generated using both pure cultured E. coli suspensions (whole cells) and purified E. coli genomic DNA as the templates in PCR reaction. The universal primer including the forward primer, 5'-TCCTACGGGAGGCAGCAGT-3' and the reverse primer, 5'-GGACTACCAGGGTATCTAATCCTGTT-3' were used in PCR reaction and produced 466bp amplicon. Standard graphs were obtained when the CT
value for each amount of DNA was plotted against the log of starting quantity. The linear relationship between the threshold cycle (CT) and viable bacteria (CFU /mL); CT values and total bacteria (bacterial number/mL as determined by microscope) were determined and respective standard curves were generated. The Melt curves were run immediately after completion of PCR amplification. A negative first derivative plot was presented as the rate of change in fluorescence over temperature and generated graph represented the amplicon as distinct melting peaks with specified melting temperature (Tm). Using our standard curves based on purified E. coli genomic DNA we consistently detected between 10 cells (corresponding to 49.6 fg DNA) and 1xE7 cells (corresponding to 50 ug DNA). When pure cultured E. coli suspensions (whole cells) were used as the template in PCR amplification, the acquired standard curves indicated that we can detect between 10 cells of E. coli (corresponding to 49.6 fg DNA)
and 1xE6 cells of E. coli (corresponding to 5 ug DNA). The specificity of universal primer in PCR reaction was confirmed by melting curve graph that showed specified melting temperature of amplicon. In conclusion, the development of standard curves using Real-time PCR assay with universal primers (16S rDNA from the E. coli and other bacteria) as a rapid and sensitive detection method to determine unknown microbial quantities in air samples was effectively investigate. The sensitivity and specificity of the universal primers were confirmed by melting curve graphs. The obtained standard curves will be used to quantify total airborne bacterial load in indoor and outdoor environmental samples taken by the new Electrostatic Collector (ESC). Concurrent sample analysis by traditional culturing techniques and RT-PCR will provide more comprehensive exposure assessment.
Impacts
The development of new method allowing to concurrently determining viable and total airborne microorganisms will improve our exposure assessment capabilities. The use of concurrent determination will allow extracting more information from one sample and will eliminate the need for samples separately analyzed for total and viable microorganisms.
Publications
- Mainelis G., An., H.R., Yao, M. (2004) Use of Electrostatic Method for Collecting Culturable and Total Airborne Microorganisms, Abstracts of the 2nd Joint Conference on Point Detection for Chemical and Biological Defense, (Williamsburg, Virginia, March 1-5, 2004), p.81.
- Mainelis G., An., H.R., Yao, M., and Lioy, P. (2004) A Low Power Collector for Concurrent Measurement of Viable and Total Bioaerosols, Abstracts of the 14th annual conference of International Society for Exposure Analysis (Philadelphia, Pennsylvania, October 17-21, 2004), p. 152.
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Progress 01/01/03 to 12/31/03
Outputs
This research investigates a hypothesis that electrostatic collection method could be applied in determining not only the viable, but also total airborne microorganisms. In this part of research, we built an Electrostatic Collector (ESC), which simultaneously collects particles onto two square Petri dishes as well as into a 96-well ELISA microtiter plate. The plates are positioned along the airflow flow axis. Electrostatic field created by connecting voltage to copper plates positioned below and above collection plates deposits the incoming microorganisms onto agar and ELISA surface. In the first set of experiments we investigated the efficiency of the collector when sampling airborne microorganisms. The physical performance was tested with Pseudomonas fluorescens and Bacillus subtilis var niger bacteria and the ESC was operated at precipitation voltages ranging from 0 to -6,000V and at collection flow rates of 4, 8, and 16 L/min. These tests were performed to
determine optimal operational parameters (collection voltage and flow rate) resulting in the best overall collection efficiency of the device. In the next set of experiments, we investigated the microorganism deposition onto the agar and into the ELISA plates inside the collector. For this purpose, the ELISA plate was filled with agar and along with agar plates was placed inside the collector. The collection of airborne microorganisms was performed at a collection voltage of -1,500V and the collection flow rate of 8 L/min. After completing the sampling, the bacterial colonies were grown and counted. The obtained counts were compared to concentrations of bacteria entering the collector and the biological efficiency of the collector was determined. The experiments showed that the physical collection efficiency of the ESC reached 50% at collection voltage of -500V, when sampled at 4 and 8 L/min. The collection efficiency reached more than 80%, when collection voltage was increased to
-4,000V. The biological efficiency of the electrostatic aerosol collector was about 15-25% for both test microorganisms. From 40 to 50% of the enumerated microorganisms landed on ELISA plate. The concentration of the airborne microorganisms that have landed in the ELISA could be enumerated using ELISA procedure, which will be a topic of our further experiments. The difference between the physical and biological efficiencies of the new collector when collecting B. subtilis var. niger vegetative cells and P. fluorescens vegetative cells were not statistically significant for different bacteria (p>0.05). From the performed experiments we concluded that the electrostatic collector built for determining both viable and total airborne microorganisms showed a satisfactory physical collection efficiency. The experiments demonstrated that electrostatic method is capable of depositing airborne microorganisms on both agar and ELISA plates and could be applied in determining both viable and total
airborne microorganisms. Further research will further investigate the efficiency of concurrent analysis of electrostatically collected culturable and total microorganisms by using ELISA and agar methods.
Impacts
The development of new method allowing to concurrently determining viable and total airborne microorganisms will improve our exposure assessment capabilities. The use of concurrent determination will allow extracting more information from one sample and will eliminate the need for samples separately analyzed for total and viable microorganisms.
Publications
- Mainelis, G., An, H.-R., Yao, M., Lioy, P., and Lioy, M. J. (2003) Concurrent Determination of Culturable and Total Airborne Microorganisms Using Electrostatic Collection Method, abstracts of the Annual Meeting of the American Association for Aerosol Research (Anaheim, California, October 20-24, 2003), p. 107.
- Mainelis, G., Lioy, P., Lioy, M.J., and An, H.R. (2003) Application of Electrostatic Precipitation for Simultaneous Determination of Culturable and Total Airborne Microorganisms, abstracts of the General American Society for Microbiology Meeting (May 18-22, 2003, Washington D.C.), Q-219.
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