The Center for Food Safety Engineering - Purdue University...IFT Myron Solberg Award Dr. Richard Linton was the 2006 recipient of Myron Solberg Award presented at the Institute of

The Center for Food Safety Engineering2005 - 2006 Research Report

“Collaborating to make our food safer”

The mission of the Center for Food Safety Engineering is to develop new knowledge, technologies and systems for detection and prevention of chemical and microbial contamination of foods.

Through CFSE, Purdue University positions itself as a national leader in multi-disciplinary food safety research. Our multi-disciplinary approach, including a strong engineering component, makes Purdue University truly unique.

Visit us @ www.cfse.purdue.edu

2005-2006 Research Report 2 Welcome from the Director • Message from Richard Linton, Center Director

2 Message from USDA • Message from our Partnership with USDA-ARS

3 Researchers developing food pathogen biosensors garner Agriculture Team Award

3 Center Director captures IFT Myron Solberg Award

4 Engineering of biosystems for the detection of Listeria Monocytogenes in foods • Michael R. Ladisch, Rashid Bashir, Arun Bhunia, J. Paul Robinson

6 Multiplexed detection of pathogens using fl uorescence resonance energy transfer in spatial detection format

• Bruce Applegate

7 Optical forward scattering for bacterial colony differentiation and identifi cation • Arun K. Bhunia, E. Dan Hirleman, J. Paul Robinson, Bartek Rajwa, Padmapriya Banda

8 Multi-pathogen screening and/or confi rmation via microarray detection • Arun K. Bhunia, Mark Morgan, B.K. Hahm, Viswaprakash Nanduri, Shu-I Tu

9 Multipathogen screening using immunomicroarray • Arun K. Bhunia, Viswaprakash Nanduri, Andrew Gehring

10 Infrared sensors for rapid detection of select microbial foodborne contaminants • Lisa Mauer, Maribeth Cousin, Jay Gore, Jean Guard-Petter, Brad Reuhs, Sivakumar Santhanakrishnan

11 Infrared sensors for rapid identifi cation of living vs. dead select microbial foodborne contaminants

• Lisa Mauer, Maribeth Cousin, Jay Gore, Brad Reuhs

12 Development of bioreporter-based chemical biosensor technology for the detection of chemical threat agents

• David Nivens, Michael Franklin, Bruce Applegate, Carlos Corvalan

13 Scientifi c publications and presentations

16 Center staff

Welcome from the Director

The Center for Food Safety Engineering (CFSE) at Purdue University, celebrates its 6th anniversary. It has been a very successful and exciting year for our interdisciplinary group. One of the key highlights was our biosensor team receiving the Purdue University College of Agriculture Team Award. This award is given each year to an interdisciplinary team that has demonstrated the most signifi cant research impact. Our partnership with USDA-ARS Eastern Regional Research Laboratory continues to breed success, leading to 19 peer-reviewed research publications, 21 presentations at national meetings, graduation of 5 Masters and Ph.D. students, and granting of 3 patents. This past summer, we were invited again to conduct a half-day program at the Annual Rapid Methods Workshop held at Kansas State University.

Our research teams continue to work on emerging technologies to improve microbial and chemical detection. Detection systems are being developed for bacterial foodborne pathogens including Listeria monocytogenes, E. coli O157:H7, Campylobacter spp., Salmonella spp., and a wide variety of chemical toxins. We are researching many detection technologies such as enzyme-linked immunosorbant assays, polymerase chain reactions, impedance-based microbiology, infrared spectroscopy, scanning microscopy, bioluminescense, and DNA/RNA probes. Some of the research breakthroughs include detection of live versus dead cells using infrared sensing devices, use of light scattering technology to produce images to distinguish foodborne pathogens from an agar plate, further development of a biochip for detection of Listeria monocytogenes, and signifi cant improvement of techniques to separate microorganisms from complex food systems.

As our center grows, I continue to be amazed with the capabilities and talents of our scientists. To learn more about our center, please visit our web site at www.cfse.purdue.edu or feel free to contact me directly.

Dr. Richard H. Linton

Director of the Center forFood Safety Engineering

Message from USDA

As the collaboration between the Center for Food Safety Engineering (CFSE) at Purdue University and the Eastern Regional Research Center (ERRC) of the Agricultural Research Service (ARS) is entering its 7th year, I am grateful to witness the growth and maturation of this Congressional initiative. I am proud that the biosensor team of our Purdue colleagues was honored by winning the 2006 Purdue University College of Agriculture Team Award. Together, our Purdue-ERRC team has earned the reputation as a major contributor to the technology advancement of pathogen detection in food, as evident by the invitation from the International Workshop on Rapid Methods and Automation in Microbiology, to conduct a half-day symposium on molecular methodologies in August 2006. Furthermore, our team will have a dedicated issue of the Journal of Rapid Methods and Automation in Microbiology to report the progress of our collaboration team.

In 2006, ERRC has continued to train visiting postdoctorates and graduate students from CFSE. Currently there are two visiting scientists from CFSE working at ERRC to develop phage-display antibodies, protein microarrays, and Salmonella detection methods using fi ber optics and time- resolved fl uorescence. We at ERRC valued the opportunity to be directly involved in and contribute to the development of future scientists in the food safety area. This year, the ERRC research team has started a 5-year research project to continue its efforts in biosensor research. This development assures the commitment by ARS to continue and strengthen the collaboration with CFSE for many more years.

Dr. Shu-I Tu

Supervisory ResearchChemist USDA-ARS,

Eastern RegionalResearch Center

2Center for Food Safety Engineering

Researchers developing food pathogen biosensors garner Agriculture Team AwardAn interdisciplinary team of scientists, who are inventing new ways to protect our food supply from potentially deadly food pathogens, has garnered the 2006 Purdue Agriculture Team Award.

The Biosensor Detection Team’s research focuses on rapidly determining whether such microbes as Listeria monocytogenes or E. coli exist in food, particularly meat and milk products. The technologies the team has developed include an innovative biochip that analyzes very small amounts of food and does it faster and less expensively than current methods.

The researchers also have found a way to take large samples of foods and concentrate the microorganisms into small volumes to inject onto the chip.

“We have a number of different platforms in the team’s research because we have brought together many scientifi c disciplines,” said Arun Bhunia, a microbiologist in the Department of Food Science. “The interdisciplinary work not only has enabled us to produce these new technologies, but it also has helped us better teach and train undergraduate and graduate students in biology, microbiology and engineering. This award is a highlight and a result of the teamwork. We could not have achieved the same results if just one research specialty had been involved.”

The biochip sensor, about the size of a fi ngernail, can determine if pathogen cells are present and whether they are dead or alive. Live cells cause disease, and it requires fewer than 10 cells to cause illness. The diagnostic method that includes the biosensor and the cell concentration technology has decreased detection time from two to three days to a few hours.

“The technologies that the Biosensor Detection Team has developed are a major step in protecting food throughout the supply system from accidental or purposeful contamination by potentially deadly pathogens,” said Randy Woodson, Glenn W. Sample Dean of Agriculture. “This technology eventually will be available to many segments of the food-producing industry and will save time and money.”

Approximately 76 millions cases of food-borne illness occur annually in the United States, according to the Centers for Disease Control and Prevention. This results in 325,000 people being hospitalized and 5,500 deaths, and costs the health-care industry and individuals as much as $23 billion.

Consumption of pathogen-contaminated food can cause meningitis, encephalitis (swelling of the brain), liver abscess, headache, fever and gastrointestinal problems. Listeria monocytogenes has a 20 percent fatality rate for those affected. The very young, people 60 and over, pregnant women and those with weakened immune systems are most at risk of being sickened by these pathogens.

Center Director captures IFT Myron Solberg AwardDr. Richard Linton was the 2006 recipient of Myron Solberg Award presented at the Institute of Food Technologists (IFT) Annual Meeting in Orlando, Florida. The award honors an IFT member for providing world-class excellence in food science leadership in the establishment and successful development and continuation of industry, government, and academia cooperation. Linton was recognized for his outstanding contributions for a) detection and control of foodborne pathogens, b) development of education and training programs for food manufacturing and retail food establishments, and c) exceptional service to science/industry boards, committees, and task forces.


Project RationalePathogenic bacteria cause 90% of reported foodborne illnesses. Listeria monocytogenes has emerged as one of the most important food pathogens, having a “zero tolerance” in ready-to eat processed (lunch) meats and dairy foods. This bacterium not only causes serious illness but also is lethal in infants, people over 60, and immune-compromised individuals.

Current methods to detect this bacterium rely upon enrichment to increase the number of bacteria present in a sample. The food or food extract is incubated in special growth media for 12 to 24 hours and the resulting culture is tested for L. monocytogenesusing procedures that require an additional 3 to 24 hours. The food industry includes many small food processors and producers that do not have in-house microbiological laboratories for the purpose of testing for food pathogens. Therefore, many companies send out samples for analysis. This adds up to another 24 hours to the time that elapses between when the food is sampled and the bacterium, if present, is detected. An overall time of 2 to 3 days typically elapses from when the food is sampled and the test results are available. The elapsed time, referred to as “time to result” or TTR, is problematic since some foods are consumed before test results would be available.

Rapid and affordable technologies to detect low numbers of L. monocytogenes cells directly from food, and which distinguish living from dead cells, are needed. This multi-disciplinary, multi-departmental research project is addressing the fundamental engineering and science required for development of microchip, bio-based assays that are transportable to the fi eld, useable in a manufacturing plant environment, and capable of rapidly detecting L. monocytogenes at the point of use. This research has the goals of (a) microscale detection of Listeria monocytogenes on a real-time or near real-time basis with a time-to-result of 4 hours, and, (b) reducing the time of culture steps with rapid cell concentration and recovery based on membrane technology.

Our multidisciplinary research team is addressing the development, engineering and validation of such a microchip system that combines bioseparations technology for rapid concentration with recovery of microbial cells and bionanotechnology to construct systems capable of interrogating fl uids for pathogens. Our approach is resulting in a technology platform capable of detecting other types of foodborne and medically relevant pathogens, even the focus of the research is on rapid detection of Listeria monocytogenes by a combination of technologies that will ultimately give a time to result of hours.

Project ObjectivesBiochips are needed that are affordable, capable of rapid detection of food pathogens, and easy to use by small food processors as well as major food companies. The goals and associated milestones of our research are:

• Rapid concentration and recovery of microorganisms from food samples for subsequent interrogation of pathogens.

• Sampling and conditioning fl uids containing the cells while maintaining their information content (i.e., the molecules or cells that represent possible targets of the chip).

• Transporting sample fl uids on and/or off the chip so that target microbes are captured and retained so that they can be probed for presence of pathogens.

• Interfacing biological molecules (i.e., biomolecules) with electronic components.

• Electronically detecting and amplifying biomolecular interactions between target and the biomolecules that form the biorecognition components of the chip.

• Achieving in vitro biospecifi city for the target molecule.• Interfacing biochip systems with electronic reading devices.

“Biochips are needed that are affordable, capable of rapid detection of food pathogens, and easy to use by small food processors as well as major food companies.”

Engineering of biosystems for the detection of Listeria Monocytogenes in foodsInvestigators: Michael R. Ladisch (Principal), Rashid Bashir, Arun Bhunia, J. Paul Robinson (College of Engineering) (College of Agriculture)

Ladisch, Bashir, Bhunia and Robinson


Project HighlightsThe integration of cell concentration and recovery, sample introduction to the biochip, parallel detection of pH and conductivity, and improvements in specifi city and sensitivity have a common basis in rapid detection and quantitation of small differences in conductivity or changes in pH. This difference is maximized using a low conductivity buffer that will support cell viability and growth. The low conductivity growth medium (LCGM) that includes compounds such as tryptone, yeast extract, glucose, BSA and other constituents to yield a low conductivity of 1.2 mS. Proteins expressed by LCGM were identifi ed to include superoxide dismentase, thiol peroxidase, and unknown lipoproteins. These over-expressed proteins could be used as target proteins for development of new antibodies, and for direct or indirect measurement on-chip, thereby enhancing sensitivity. Overall, the validation of LCGM over the last year resulted in the practical impact of increasing sensitivity of on-chip detection of L. monocytogenes.

Our interdisciplinary research approach has enabled us to learn about the biology of interactions between microorganisms and nutrient-rich foods; mechanisms of their transport and capture in microfl uidic systems; expression of biomarkers under environmental stress (i.e., conditions during food handling and/or sampling); and characteristics that impact their viability under environmental stress. The stress that the microorganisms may experience is related to changes in micro-environments during the course of sampling and rapid detection protocols.

Initial runs with milk and vegetables have been carried out to begin examining rapid recovery of microorganisms internal to the biological tissue as found in various types of foods. Ultimately, the fundamental knowledge gained will apply to a broad range of samples (water, air, packages, animals, and people) for which fl uids are probed to rapidly assess the level of risk by interrogating these samples for biomarkers that

indicate biological or chemical toxins. Such biomarkers may include proteins, peptides, low molecular weight metabolites, chemical or bio-toxins, and lipids, as well as nucleic acids. This is being studied in the context of on-chip pathogen detection and integration of preprocessing steps. The rapid concentration and recovery of the microorganisms has advanced with the use of hollow-fi ber membranes that are able to process extracts from foods that contain the microorganisms, and are amenable to use in a hands-off system that ultimately will lead to an automated instrument. A team approach is required by the complex interactions of the various components – both biological and electrical – of the detection system. These include dielectrophoresis and antibody-mediated selective capture of microorganisms in microfl uidic biochips. The multidisciplinary cooperation among the team has enabled signifi cant progress to be made in the integration of the various subsystems.

Ladisch, Bashir, Bhunia and Robinson

“Listeria monocytogenes has emerged as one of the most important food pathogens, having a “zero tolerance” in ready-to eat processed (lunch) meats and dairy foods.”


Project RationaleInadvertent contamination of foods with harmful microorganisms can result in multiple problems including loss in productivity, expenses related to healthcare, investigation, litigation, destruction of vast quantities of agricultural products, and loss of human life. To streamline efforts in the circumvention of food poisoning-related incidents, FSIS requires a fully automated testing system for the rapid throughput analysis of foods for contaminant pathogens (E. coli O157:H7, Salmonella, Listeriaspp., etc.). Ideally, the testing platform will be a technician-operated instrument that can simultaneously screen food samples for the presumptive presence of multiple bacteria and, if desired, confi rm their presence and characterize the pathogens through identifi cation of virulence-related or other genes. This approach would ultimately eliminate the need for the time-consuming conventional cell culture/isolation/confi rmation procedures currently used. The specifi c objective of this project is the development of a nucleic acid microarray for the detection of multiple PCR products for the identifi cation of foodborne pathogens E. coli O157:H7, Salmonella spp. and L. monocytogenes, based on molecular beacon technology. This project leverages capabilities in DNA microarray technology to develop a gene-specifi c assay that does not require costly labeling and purifi cation methods to detect the presence of the target gene.

Project Objectives

• Construct an initial prototype gene array consisting of four marker genes for E. coli O157:H7 utilizing molecular beacon probes immobilized on a glass slide. (2005)

• Determine hybridization conditions and evaluate the prototype beacon array utilizing amplifi ed target genes.(2005)

• Contruct microarrays containing four targets from Salmonella spp. and L. monocytogenes from previously constructed target probe and amplicon sequences along

with microarrays including all three sets of probes for the simultaneous detection of E. coli O157:H7, Salmonella spp. and L. monocytogenes. (2006)

• Design and develop probe and amplicons for Campylobacter, Clostridium, and S. aureus. (2006)

• Develop and integrate a hybridization and PCR control into the array to provide quality assurance. (2006)

• Construct arrays for multiple organisms and evaluate for hybridization specifi city. (2006)

• Evaluate previously developed multiplex PCR reactions for simultaneous amplifi cation of multiple pathogen targets utilizing the developed microarrays. (2006)

Project HighlightsThis project focuses on the integration of the microarray work with the previously developed spatial array format. A DNA hybridization-based optical biosensor for the detection of foodborne pathogens was developed with virtually zero probability of a false negative signal. This portable, low-cost and real-time assaying biosensor utilizes the color-changing molecular beacon as a probe for the optical detection of the target sequence. The computer-controlled biosensor exploits the target hybridization-induced change of fl uorescence color due to the Förster (fl uorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fl uorophores conjugated to the opposite ends of a beacon. Unlike the traditional fl uorophore-quencher beacon design, the presence of two fl uorescence molecules allows one to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection that is characteristic of the fl uorophore-quencher beacon for which bleaching of the fl uorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. The two-color design allows us to quantify the concentration of the target DNA in a sample down to ≤0.5 ng/μl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format.

Multiplexed detection of pathogens using fl uorescence resonance energy transfer in spatial detection formatInvestigator: Bruce Applegate (Principal) (College of Agriculture)

Applegate

“Inadvertent contamination of foods with harmful microorganisms can result in multiple problems including loss in productivity, expenses related to healthcare, investigation,

litigation, destruction of vast quantities of agricultural products, and loss of human life.”6Center for Food Safety Engineering

Optical forward scattering for bacterial colony diff erentiation and identifi cationInvestigators: Arun K. Bhunia (Principal), E. Dan Hirleman, J. Paul Robinson, Bartek Rajwa, Padmapriya Banda (College of Agriculture, College of Engineering)

Project RationaleListeria monocytogenes and Escherichia coli are the major foodborne pathogens of concern in the United States. For the detection and evaluation of foods contaminated with Listeria monocytogenes or E. coli, USDA/FSIS recommends initial enrichment and subsequent plating on a selective agar media, which is often followed by further identifi cation procedures. These procedures are often time consuming and lengthy, taking more than 2-3 days. The present industrial demand is to increase the rapidity of the detection assays leading to strategies for decreasing bacterial contamination and thus reducing the economical loss. Our main objective was to reduce the time of identifi cation of these pathogens after plating, by developing a simple light scattering sensory method. The method has now been improved to differentiate among the different strains of Listeria, based on the varying patterns.

There have been increasing foodborne illnesses, multiple outbreaks, product recalls, and loss of lives, resulting from pathogens in processed, ready-to-eat food products. Bacterial contamination in products not only puts the public at risk, it is costly to companies due to loss of production time, product recalls and liability.

Project Objectives

• To improve the BARDOT (Bacteria Rapid Detection using Optical Scattering Technology) design, including supporting physics-based models, for more repeatability and maximum discrimination of forward scattering signatures of colonies.

• To acquire scatter images of colonies of select foodborne bacterial colonies including pathogens.

• To analyze the bacterial colonies of different foodborne bacteria on non-selective and selective agar media.

• To validate the technology by using naturally or deliberately contaminated food samples.

• To analyze cellular composition, cell arrangement, refractive index and colony contents using electron microscopy, FT-IR or GC-MS.

• To analyze the scatter signal images using ‘Standard feature extraction’ and ‘Moments of shape analysis’ methods.

Project HighlightsThe design and development of the BARDOT system was the major accomplishment during the year 2005-2006. This was a signifi cant accomplishment since it provided a platform for using a simple laser beam to scatter the bacterial colonies growing on an agar plate and simultaneously capturing and storing the images using a CCD camera attached to the instrument. The computer-stored images were used for image analysis. This system was able to differentiate genus Listeria, Salmonella and Vibrio with 89-98% accuracy. Furthermore, species within genus Listeria can be differentiated with 92-94% accuracy, Salmonella with 95-98% accuracy, and Vibrio with 84-90% accuracy.

“the BARDOT system… provided a platform for using a simple laser beam toscatter the bacterial colonies growing on an agar plate and simultaneously

capturing and storing the images…”

Bhunia, Hirleman, Robinson, Rajwa and Banda


Project RationaleFoodborne disease is one of the most common causes of morbidity and mortality in the world, and more than 200 known diseases are transmitted through food. In the United States there are about 76 million cases each year, of which 325,000 require hospitalization and 5,000 die. The foodborne pathogens of concern are E. coli O157:H7, Salmonella, Listeria monocytogenes, Toxoplasma and Campylobacter. Therefore, detection of these pathogenic bacteria during food processing and storage is crucial for the microbiological safety and prevention of possible outbreaks.

Antibody-based detection methods are regarded as rapid and effi cient and are widely used in conventional ELISA and dipstick methods. In recent years, antibodies have been successfully used in biosensor tools for rapid detection. Therefore, specifi city and avidity of a given antibody for the target bacteria is extremely important, specifi cally those originating from stressful environments of food. We have also learned that stress can affect antigen expression on bacterial cells thus affecting antibody-based detection.

The goals of this project were to: (a) develop specifi c antibodies for Listeria monocytogenes, Salmonella enterica and E. coli O157:H7, (b) analyze effect of environmental and physiological stresses on antigen expression and antibody based detection, and (c) to develop antibody-based microarray system for simultaneous detection of multi-pathogens.

Once completed, this would allow development of a detection kit for multiple pathogens, thus saving time and money for product testing and also helping regulatory agencies for evaluation of a product for key food pathogens.

Project Objectives

• Develop antibody specifi c for L. monocytogenes, Salmonella enterica and E. coli O157:H7.

• Determine the effect of environmental and physiological stresses on antigen expression.

• Develop sandwich ELISA for each pathogen.• Determine the selective enrichment media affecting the

antibody-based detection of stress-exposed Listeria monocytogenes.

• Development of pathogen enrichment and detection device (PEDD).

Project HighlightsTwo polyclonal antibodies, PAb Lm404 and LmC369, were demonstrated to be specifi c for Internalin B (InlB) and actin polymerization protein (ActA) of L. monocytogenes, respectively. These antibodies could be potentially used for specifi c detection of this bacterium. These antibodies showed differential expression of antigens in different enrichment broths: selective media (like BLEB, UVM and FB) suppressed PAb Lm 404 reactive InlB expression whereas only FB suppressed Lm C369 reactive ActA expression. PAb Lm404 could be used only when bacteria are cultured in non selective media while PAb-Lm C369 could be used in an immunoassay with bacteria directly taken from selective enrichment broth. Surface localization of these two epitopes was confi rmed by immuno-electron microscopy.

Multi-pathogen screening and/or confi rmation via microarray detectionInvestigators: Arun K. Bhunia (Principal), Mark Morgan, B.K. Hahm, Viswaprakash Nanduri, Shu-I Tu (College of Agriculture) (USDA-ARS, ERRC)

Bhunia, Morgan, Hahm, Nanduri and Tu

“Foodborne disease is one of the most common causes of morbidity and mortality in the world…”


Multipathogen screening using immunomicroarrayInvestigators: Arun K. Bhunia (Principal), Viswaprakash Nanduri, Andrew Gehring (College of Agriculture)

Project RationaleAntibody-based methods are widely used for the detection of most pathogenic bacteria, and are regarded as rapid and effi cient. Their application to a conventional ELISA assay and further adaptation in modern biosensor tools shows promise in rapid detection. Most assays are developed for detection of a single target pathogen or toxin. Therefore, it is very expensive to test for multiple pathogens from a single product because separate assay methods need to be used, thus adding cost and labor per test. Also, large laboratory space is required to perform separate tests for each target pathogen because separate enrichment reagents and procedures need to be applied for different pathogens. If a single test is available that can detect multiple pathogens enriched in a single enrichment broth, this will not only reduce cost but also will be convenient and provide results in a short period of time. This would also benefi t the regulatory agencies when evaluating food products for key food pathogens.

In the last decade, several rapid detection methods, such as antibody-based, nucleic acid-based, and biochemical-based, were developed. Even though these methods have shortened the analysis time compared to the conventional detection method, still we have to allow time for selective enrichment of samples prior to employing rapid detection methods. An antibody-based method such as ELISA requires at least 106 CFU/ml for detection. Thus, to achieve that level of cells, it is important to use proper enrichment media for detection of foodborne pathogens. Furthermore, cell injury or stress encountered during food processing may affect cell numbers.

Additionally, selective agents in media can delay growth and recovery of stressed or injured cells. Thus, currently used selective media may not be suitable for use with modern detection methods. Furthermore, current trends emphasize the detection of multiple targets with a single device. To achieve that, a medium that can allow selective enrichment of multiple pathogens is desirable.

Project ObjectivesThe overall objective of this project is to develop immunomicroarray for concurrent detection of viable cells of three pathogens; L. monocytogenes, E. coli O157:H7 and Salmonella enterica.

The specifi c objectives are:

• Development of microarray assay in 96-well plate and glass slide using sandwich immunoassay for three pathogens.

• Optimizing growth and enrichment of three pathogens (healthy or stressed) spiked in model food samples in a selective enrichment broth for use with microarray.

Project HighlightsOur group has developed a selective enrichment medium for simultaneous growth and detection of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella. The media, designated SEL (Salmonella, E. coli, Listeria) was formulated using Buffered Listeria Enrichment Broth (BLEB) base and various combinations of antibiotics: acrifl avine, cycloheximide, fosfomycin and nalidixic acid. Initial testing indicated that this medium supports the growth of E. coli O157:H7, L. monocytogenes and S. enteritidis well, and the growth rate of each is comparable to the respective selective enrichment broth such as modifi ed EC medium for E. coli, RV for Salmonella and FB for Listeria.

“If a single test is available that can detect multiple pathogens enriched in a single enrichment broth, this will not only reduce cost but also will be convenient

and provide results in a short period of time.”

Bhunia, Hirleman, Robinson, Banda, Rajwa and Bayraktar


Project RationaleConventional detection methods take at least 24 to 48 hours to differentiate and identify microorganisms; therefore, measures taken to counteract food contamination must wait at least that long. To facilitate timely intervention measures, the food industry needs more rapid detection methods and a sensor able to accurately and rapidly identify low levels of microbial foodborne contaminants within food systems or culture media. We are investigating the effi cacy of infrared (IR) technology as a means for rapid detection of select bacterial pathogens. To accomplish this goal, we: (a) Created a library of Fourier-transform infrared (FT-IR) spectra of bacterial cell wall components and whole cells needed for pathogen identifi cation and differentiation. (b) Developed FT-IR methods for identifi cation and quantifi cation of these pathogens from water, cultural media, and select foods. This included standardizing sampling procedures, quantifi cation methods, and spectral analysis procedures, as well as developing an overall chemometric approach for the analysis of FT-IR data. (c) Designed an IR sensor based on the most promising few-wavelength algorithms developed using FT-IR data generated from research activities in the fi rst two objectives.

Project Objectives

• Create a library of FT-IR spectra of bacterial cell wall components and whole cells (from Salmonella, Campylobacter jejuni, and Escherichia coli O157:H7) needed for cell identifi cation and differentiation.

• Develop FT-IR methods for cell identifi cation and quantifi cation in water, cultural media, and foods.

• Develop a limited wavelength approach for cell identifi cation.

• Build and validate an IR sensor based on the most promising few-wavelength algorithm developed using FT-IR techniques selected in the fi rst two milestones.

Project HighlightsWe successfully completed the development of sample preparation techniques for FTIR spectral collection and data analysis that are able to both quantify and identify Salmonellaand E. coli O157:H7 from mixtures of bacteria in culture media and select food items. To develop this approach, we evaluated a variety of sample preparation methods, FTIR data collection methods, and analytical approaches for raw spectra. Further development of this approach enabled the design of a sensor that can be used in a production or retail facility to characterize a food sample as contaminated or free of select pathogenic bacteria in less time than current methods for detection.

Infrared sensors for rapid detection of select microbial foodborne contaminantsInvestigators: Lisa Mauer (Principal), Maribeth Cousin, Jay Gore, Jean Guard-Petter, Brad Reuhs, Sivakumar Santhanakrishnan

(College of Agriculture, College of Engineering)

Mauer, Cousin, Gore, Guard-Petter, Reuhs and Santhanakrishnan

“To facilitate timely intervention measures, the food industryneeds more rapid detection methods…”


Infrared sensors for rapid identifi cation of living vs. dead select microbial foodborne contaminantsInvestigators: Lisa Mauer (Principal), Maribeth Cousin, Jay Gore, Brad Reuhs (College of Agriculture, College of Engineering)

Project RationaleTo keep the food supply safe, food production, processing, and retail establishments must be able to identify microbial foodborne pathogens, such as Salmonella, Campylobacter jejuni, and Escherichia coli O157:H7. The CDC estimates that annual foodborne-related outbreaks result in 76 million cases of illness, 325,000 hospitalizations, and 5,000 deaths.

To facilitate timely intervention measures, the food industry needs rapid detection methods. Our group has developed such a system, using an infrared (IR) sensor, that is able to accurately identify low levels of microbial foodborne contaminants. In this phase of the study we have tested the fi eld instrument, and we have applied the technology to a second problem: distinguishing live bacterial cells from dead cells. To accomplish this goal, we developed FT-IR methods and analytical approaches to differentiate between living and dead cells of Salmonella spp. strains and E. coli O157:H7 in water, culture media, and select foods. This is important in both the determination of contamination potential in fresh products and the effi cacy of decontamination efforts. We also validated a miniature IR sensor for detecting the live bacteria. In this part of the project we addressed sample handling procedures, the time needed for detection, detection limits, and wavelength bands and algorithms appropriate for the sensor design.

Project Objectives

• Use an existing library of FT-IR spectra of bacterial cell-wall components and whole cells of E. coli O157:H7 and E. coli K12, as well as Salmonella, for cell identifi cation and differentiation of live versus dead cells in water, cultural media, and foods. This milestone also involves the application and improvement of capture (fi ltration, etc), concentration, and enrichment techniques.

• Apply a limited wavelength technique that has already been developed for cell identifi cation in the validation of a fi eld-size IR using the most promising few-wavelength algorithm developed during FT-IR techniques evaluations in the previous project.

Project HighlightsIn the studies using variations on the selective and non-selective capture of live versus dead cells of E. coli O157:H7 and E. coli K12, and the subsequent spectral analyses, we showed that live cells could easily be distinguished from dead cells, especially when the cells were killed by the harsh techniques commonly used in the food industry (e.g., heat, salt, UV, etc). In contrast, the use of antibiotics that disrupt metabolism with out physical damage to the cell resulted in less distinct spectral separation of dead cells, but differentiation was still possible. Importantly, a relatively quick multi-step procedure allowed for a quantitative analysis of the relative abundance of dead versus live cells of E. coli O157:H7.

“Our group has developed such a system, using an infrared (IR) sensor, that is able to accurately identify low levels of microbial foodborne contaminants.”

Mauer, Cousin, Gore and Reuhs


Project RationaleOur nation must not only protect against environmental sources of pollution that can contaminate the food supply but also guard against deliberate acts of terrorism intended to degrade human health and/or weaken our economic base. The overall goal for our interdisciplinary research is the development of a core bioreporter-based chemical biosensor (BCB) platform for the eventual production of inexpensive biosensors to detect chemical agents that threaten our environment and our food supply. The BCB platform consists of an enclosure/microenvironment system that contains minimal nutrients, genetically-modifi ed bioreporters, and in some applications an analytical transducer. The technology exploits the abilities of living microorganisms: (i) to sense and react to chemical stimuli; (ii) to be genetically manipulated to contain reporter genes; and (iii) to form biofi lms that promote survival. Enclosures/microenvironment systems will be developed to facilitate resuscitation of microorganisms from storage and sustain them within biofi lms in an optimal sensing state for the detection of chemical agents. Our research involves the construction of bioreporters that are used in a novel biosensor confi guration to detect an organic pesticide (paraquat), a toxic metal (arsenic), and aromatic compounds (solvents). This research is expected to enable the development of BCB technology for rapid, selective and sensitive detection of many chemical threat agents. After core technology is developed, inexpensive application-specifi c devices will be designed for potential use by farmers and/or scientists in a testing laboratory to identify environmental contamination or product tampering in both point-of-use and long-term monitoring applications.

Project Objectives

• Develop prototype enclosure(s) that contains a micro-environment that supports bio-reporting biofi lms and in some applications contains a transducer(s) to facilitate rapid detection and long-term monitoring of biological responses to toxic compounds.

• Develop biofi lm bioreporters for use in the enclosure/micro-environment for the detection of arsenic, paraquat, and aromatic solvents.

• Combine constructed bioreporters with the prototype enclosure/micro-environments and obtain concentration- dependent bioreporter response data for detection of a chemical threat agent in food.

• Use empirical data to develop models for understanding the nature of bacterial responses for improving the analytical performance of the biosensors.

Project HighlightsWe have nearly completed the infrastructure for the core bioreporter-based sensor technology that will allow the development of application-specifi c sensors: (1) selection and testing of host strains, (2) constructing genetic systems that can be effi ciently and rapidly inserted into the genome of the host strain, (3) development of test systems to understand natural and confi ned biofi lms, (4) selection of appropriate transducers, and (5) development of mathematical models for testing.

Development of bioreporter-based chemical biosensor technology for the detection of chemical threat agentsInvestigators: David Nivens (Principal), Michael Franklin, Bruce Applegate, Carlos Corvalan (College of Agriculture)

Nivens, Franklin, Applegate and Corvalan

“Our research involves the construction of bioreporters that are used in anovel biosensor confi guration to detect an organic pesticide (paraquat), a toxic

metal (arsenic), and aromatic compounds (solvents).”12Center for Food Safety Engineering

Scientifi c Publications and Presentations

Peer Reviewed Journal Publications (2005-2006)

• Bayraktar, B., Banada, P.P., Hirleman, E.D., Bhunia, A.K., Robinson, J.P., Rajwa. B. Bacterial phenotype identifi cation using Zernike moment invariants. Proceedings of the Society for PhotoOptical Instrumentation Engineers. v. 6080. p. 155-162 (2006).

• Burgula, Y., Khali, D., Krishnan, S.S., Cousin, M.A., Gore, J.P., Reuhs, B. L., Mauer, L. J. Detection of E. coli O157:H7 and Salmonella Typhimurium Using Filtration followed by FT-IR Spectroscopy. Journal of Food Protection. 69:8. p. 1777-1784 (2006).

• Chen, W-T., Hendrickson, R. L., Huang, C-P., Sherman, D., Geng, T., Bhunia, A. K., Ladisch, M. R. “Mechanistic Study of Membrane Concentration and Recovery of Listeria monocytogenes,” Biotechnol. Bioeng., 89, 263-273 (2005).

• Chen, W-T., Geng, T., Bhunia, A. K., Ladisch, M. R. “Membrane for Selective Capture of the Microbial Pathogen Listeria monocytogenes,” AIChE J., 51(12), 3305-3308 (2005).

• Hahm, B. K., Bhunia, A. K. Effect of environmental stresses on antibody-based detection of Escherichia coli O157:H7, Salmonella enterica subsp. Enteritidis and Listeria monocytogenes. Journal of Applied Microbiology. v. 100. p. 1017-1027 (2006).

• Huang, T. T., Taylor, D. G., Sedlak, M., Mosier, N. S., Ladisch, M. R. “Microfi ber-directed Boundary Flow in Press-fi t Microdevices Fabricated from Self-adhesive Hydrophobic Surfaces,” Analytical Chemistry, 77, 3671-3675 (2005).

• Jedlica, S.S., McKenzie, J.L., Leavesley, S.J., Little, K.M., Webster, T.J., Robinson, J.P., Nivens, D.E., Rickus, J.L. Surface features of sol-gel derived silica infl uence protein conformation and neuronal differentiation. Journal of Materials Chemistry. 16:3221-3230 (2006).

• Kim, H., Kane, M. , Kim, S., Dominguez, W., Applegate, B. Savikhin, S. A molecular beacon DNA microarray system for rapid detection of Escherichia coli O157:H7 that eliminates the risk of a false negative signal. Biosensors and Bioelectronics. In press (2006).

• Kim, K.-P., Jagadeesan, B., Burkholder, K., Jaradat, Z.W., Wampler, J.L., Lathrop, A.A., Morgan, M.T., Bhunia, A.K. Adhesion characteristics of Listeria adhesion protein (LAP) – expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor. FEMS Microbiology Letters. v. 256. p. 324-332 (2006).

• Kim, S. S., Huang, T. T., Fisher, T. S., Ladisch, M. R., “Effects of Carbon Nanotube Structure on Protein Adsorption,” IMECE 2005-2008 1395, Proceedings of IMECE 2005, 2005 ASME International Mechanical Engineering Congress and Exposition, Orlando, FL, November 5-100 (2005).

• Kim, S., Kim, H., Reuhs, B.L., Mauer, L.J. Differentiation of outer membrane proteins from Salmonella enterica serotypes using Fourier transform infrared spectroscopy and chemometrics. Letters in Applied Microbiology. v. 42. p. 229-234 (2006).

• Kim, S., Burgula, Y., Ojanen-Reuhs, T., Cousin, M.A., Reuhs, B.L., Mauer, L.J. Differentiation of crude lipopolysaccharides from Escherichia coli strains using Fourier transform infrared spectroscopy and chemometrics. Journal of Food Science. v. 71. p. 57-61 (2006).

• Lim, K. S., Chang, W.J., Koo, Y.M., Bashir, R., “Reliable Fabrication Method of Transferable Micron Scale Metal Pattern for Poly(dimethylsiloxane) Metallization”, Lab. Chip. v. 1. 578 – 580 (2006).

• Yang, L., Banada, P.P., Liu, Y.S., Bhunia, A.K., Bashir, R. Conductivity and pH dual detection of growth profi le of healthy and stressed Listeria monocytogenes. Biotechnology and Bioengineering. v. 92. p. 685-693 (2005).

• Yang, L., Banada, P., Chatni, M. R., Lim, K, Ladisch, M., Bhunia, A., Bashir, R. “A MultiFunctional Micro-Fluidic System for Dielectrophoretic Concentration Coupled with Immuno-Capture of Low Number of Listeria monocytogenes”, Lab on a chip, appeared online. DOI: 10.1039/b607061m (2006).

“Our partnership with USDA-ARS Eastern Regional Research Laboratory continues to breed success, leading to 19 peer-reviewed research publications, 21 presentations at national

meetings, graduation of 5 Masters and Ph.D. students, and granting of 3 patents.”13

Center for Food Safety Engineering

“At the Center for Food Safety Engineering, we direct our

Scientifi c Publications and Presentations

Abstracts for Major Papers/Posters Presented (2005-2006)

• Bashir, B. “BioMEMS and Bionanotechnology and Applications to Diagnostics”, Plenary Session Monday Oct 31st, Topical Conference “Biomedical Applications of Nanotechnology (Bionanotechnology)”, AIChE Annual Meeting. Cincinnati, OH. 2005 (invited).

• Bashir, R. BioMEMS and Bionanotechnology and Applications to Diagnostics, in N.A. Peppas and J.Z. Hilt, eds., “Advances in Bionanotechnology”, pp. 1-5, AIChE, New York, NY. 2005 (invited).

• Bayraktar, B., Banada, P. P., Hirleman, E. D., Bhunia, A. K., Robinson, J. P., Rajwa, B. Feature Extraction, Identifi cation and Classifi cation of Listeria colonies from light scatter patterns. 14th Annual GLIIFCA Meeting. 2005.

• Bayraktar, B., Banada, P.P., Hirleman, E D, Bhunia, A.K., Robinson, J.P., Rajwa, B. Feature extraction from light-scatter patterns of Listeria colonies for identifi cation and classifi cation. Journal of Biomedical Optics. 2006. v. 11 paper no. 034006.

• Burgula, Y., Cousin, M.A., Reuhs, B.L., Mauer, L.J. Differentiation of Live vs. Dead cells of E. coli O157:H7 based upon incubation and immunomagnetic separation. Institute of Food Technologists’ Annual Meeting and Food Expo. Orlando, FL. 2006.

• Burgula, Y., Cousin, M.A., Applegate, B., Linton, R., Reuhs, B.L., Mauer, L.J. Effects of processing treatments on FT-IR based classifi cation of dead E. coli K12 cells in comparison to live cells. Institute of Food Technologists’ Annual Meeting and Food Expo. Orlando, FL. 2006.

• Burkholder, K.M., Bhunia, A.K. Interaction of Salmonella with cultured intestinal cell lines during heat stress. American Society for Microbiology General Meeting. 2005. p. 445. Abstract No. P-041.

• Gomez, R., Bashir, R. “Microscale Impedance-Based Detection of Bacterial Metabolism”, in Encyclopedia of Rapid Microbiological Methods, Volume 3, Series Editor, Michael J. Miller, Davis Healthcare International Publishing (DHI). 2006. pp. 333-362.

• Huff , K., Banada, P.P., Bayraktar, B, Bae, E, Rajwa, B, Robinson, J.P., Hirleman, E.D. Bhunia, A.K. Detection and Identifi cation of Foodborne Pathogens in Genus and Species Levels Using a Non-invasive Modifi ed Light Scatterometer-BARDOT. American Society for Microbiology Annual Meeting. 2006. Abstract no. P-075.

• Jagadeesan, B., Burkholder, K., Wampler, J.L., Bhunia, A.K. Interaction of Listeria adhesion protein (LAP) with human Hsp60 on the surface of stressed epithelial cells. American Society for Microbiology General Meeting. 2006. p. 48. Abstract No. B-103.

• Kwan S.L., Chang, W.J., Koo, Y.M., Bashir, R. “Embedding Microscale Metal Patterns In Polydimethylsiloxane Substrate” Ninth International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS), Boston Marriott Copley Place, Boston, Massachusetts, USA October 9th - 13th, 2005.

• Lathrop, A.A., Bhunia, A.K. Expression of Listeria monocytogenes InlB and ActA surface proteins under different growth media. American Society for Microbiology General Meeting. 2005. p. 446, Abstract No. P-046.

• Paarlberg, K., Burgula, Y., Cousin, M.A., Reuhs, B.L., Mauer, L.J. Development of a FT-IR spectral library for select microorganisms. Institute of Food Technologists’ Annual Meeting and Food Expo. Orlando, FL. 2006. Abstract.

• Shen, X., Nivens, D.E., Corvalan, C.M. Predicting analytical response in bioreporter-based sensors for food and agriculture systems. Institute for Food Technologists, Orlando, FL. 2006. Session number 020B.


efforts toward detecting problems and protecting consumers.”

Thesis/Dissertations (2005-2006)

• Burgula, Y., “Detection of select foodborne pathogens using FT-IR spectroscopy,” Ph.D. Dissertation. 2006. Purdue University. 294p.

• Chen, W., Biomedical Engineering, “Engineering and Analysis of Immobilized Enzyme System in Microfl uidic Device,” Ph.D. Dissertation. 2006. Purdue University. 135 p.

• Davis, K.D. “Assessment of molecular virulence gene profi ling and antibodies for rapid detection of pathogenic Escherichia coli isolates.” Ph.D. Dissertation. 2005. Purdue University. 138 p.

• Lathrop, A.A. “Development of Listeria monocytogenes specifi c antibodies using a proteomic/genomics approach and expression of antibody-specifi c antigens InlB and ActA under different environments.” Ph.D. Dissertation. 2005. Purdue University. 142 p.

• Nagel, A.C. “Development and analysis of bioreporters in biofi lms,” Masters Thesis. 2006. Purdue University. (In Progress)

Books and Book Chapters (2005-2006)

• Amass, S.F., Bhunia, A.K., Chaturvedi, A.R., Dolk, D.R., Peeta, S., Atallah, M.J. editors. Purdue University Press, West Lafayette, IN. Advances in Homeland Security Vol. 1. The Science of Homeland Security, 2006. p. 109-149.

• Bhunia, A.K. Detection of signifi cant bacterial pathogens and toxins of interest in homeland security.

Invited Lectures and Seminars (2005-2006)

• Bhunia, A.K. Bacterial Pathogen Detection: Micro/Nano-Technology Approaches. Indian Institute of Chemical Biology, Calcutta, West Bengal, India (Dec 21, 2005).

• Bhunia, A.K. Listeria monocytogenes Pathogenesis: Intestinal Phase of Infection. Indian Institute of Technology, Guwahati, Assam, India (Dec 5, 2005).

• Bhunia, A.K. Microbiology Meets Nanotechnology. Indian Institute of Technology, Guwahati, Assam, India (Dec 5, 2005).

• Bhunia, A.K. Optical immunosensors and cell-based detection for Listeria monocytogenes. International Rapid Methods Workshop, Kansas State University, Manhattan, KS (July, 2006).

• Bhunia, A.K. Pathogenesis of Listeria monocytogenes and its detection strategies using micro/nano sensors. Department of Biology, Ball State University, Muncie, Indiana (Nov 4, 2005).

• Ladisch, M. R., “Microfl uidic Characterization of Press Fit Microdevices,” University of Akron, Akron, OH (October 21, 2005).

• Ladisch, M. R., “Rapid Prototyping of Microfl uidic Separation Platforms,” North Carolina State University (February 3, 2006).

Patents Granted (2005-2006)

• Bhunia, A.K., Hahm, B.K., Morgan, M.T. Pathogen enrichment detection device and methods of use. Ref. No. 64303.P1US (2005).

• Hirleman, E.D., Guo, S., Bhunia, A.K., Bae, E. System and method for rapid detection and characterization of bacterial colonies using forward light scattering. Ref. No. 64142.00.US (2005).

• Ladisch et al, Cell Concentration and Pathogen Recovery, 11/081 378 (2005).


Center Staff

Dr. Richard H. LintonDirector

[email protected]

Staff

Kevin T. Hamstra, Multimedia Technical Specialist• 765.496.3833 • [email protected]

Kiya A. Smith, Center Coordinator• 765.496.3827 • [email protected]

Dr. W. R. (Randy) Woodson, Dean of Agriculture• 765.494.8391 • [email protected]

Dr. Shu-I TuSupervisory Research Chemist

[email protected]

USDA-ARS

Dr. Jeff rey Brewster • 215.233.6447 • [email protected]. John P. Cherry • 215.233.6595 • [email protected]. Pina Fratamico • 215.233.6525 • [email protected]. Andrew Gehring • 215.233.6491 • [email protected]. Peter Irwin • 215.233.6420 • [email protected]. James A. Lindsay • 301.504.4674 • [email protected]. John B. Luchansky • 215.233.6620 • [email protected]. George Paoli • 215.233.6671 • [email protected]. Gary Richards • 302.857.6419 • [email protected]. Christopher Sommers • 215.836.3754 • [email protected]. Howard Zhang • 215.233.6583 • [email protected]

Dr. Arun K. BhuniaPI

[email protected]

Co-PIs

Padmapriya Banada • 765.496.3826 • [email protected] Bayraktar • 765.494.0757 • [email protected] Gehring • [email protected]. K. Hahm • 765.496.7356 • [email protected]. Dan Hirleman • 765.494.5688 • [email protected] Morgan • 765.494.1180 • [email protected] Nanduri • [email protected] Rajwa • 765.494.0757 • rajwa@fl owcyt.cyto.purdue.eduJ. Paul Robinson • 765.494.6449 • jpr@fl owcyt.cyto.purdue.eduShu-I Tu • 215.233.6466 • [email protected]

Staff / Graduate Students

EuiWon Bae • 765.494.4762 • [email protected] Banerjee • 765.496.7356 • [email protected] Huff • [email protected] Kim • 765.496.7354 • [email protected]

Dr. Bruce ApplegatePI

[email protected]

Co-PIs

Dr. Michael Kane • [email protected]. Lynda Perry • [email protected]


Preciaus Heard • [email protected]


Dr. Michael LadischPI

[email protected]

Co-PIs

Padmapriya Banada • 765.496.3826 • [email protected] Bashir • [email protected] Bhunia • 765.494.5443 • [email protected] Liu • 765.494.7052 • [email protected] Mosier • 765.494.7025 • [email protected]. Paul Robinson • 765.494.6449 • jpr@fl owcyt.cyto.purdue.eduMiroslav Sedlak • 765.494.3699 • [email protected]


Ben Baker • [email protected] Burton • [email protected] Bwatwa • [email protected] Gregory • [email protected] Jegadeeshan • [email protected] Kim • [email protected] Liu • [email protected] Manier • [email protected] (Heyjin) Park • 765.494.7031 • [email protected] Serafi n • ajserafi @purdu.eduHunter Vibbert • [email protected] Valadez • 765.496.3824 • [email protected]

Dr. David E. NivensPI

[email protected]

Co-PIs

Bruce Applegate • 765.496.7920 • [email protected] Corvalan • 765.494.8262 • [email protected] Franklin • 406.994.5658 • [email protected]


Bonnie Co • 765.496.1805 • [email protected] Ionita • 765.496.7354 • [email protected] Aaron Nagel • 765.496.3832 • [email protected] Schroeder • 765.496.1805 • [email protected] Shen • 765.496.3825 • [email protected]

Dr. Lisa MauerPI

[email protected]


Yashodar Burgula • 765.496.3805 • [email protected]


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The Center for Food Safety Engineering - Purdue University...IFT Myron Solberg Award Dr. Richard Linton was the 2006 recipient of Myron Solberg Award presented at the Institute of

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