"Our results suggest that commonly used sampling methods detect only a small fraction of what is actually in the air," said Timothy Buckley, the study's senior author and an associate professor of public health at Ohio State.

"And what they detect is often so damaged - due to the collection method - that the pathogens no longer possess the same infectious potential as they did while in the air."

Such damage can make it nearly impossible for public health workers to determine if a pathogen is viable - that is, whether or not it has the potential to infect.

Buckley and his colleagues found that a relatively new device called the BioSampler caused the least amount of damage to the non-infectious microorganisms used in this study. The BioSampler was developed in the late 1990s by a team of researchers from the University of Cincinnati. Although it's not yet a commonly used method for detecting airborne pathogens, it gave Buckley and his team the most accurate reading of the degree of the microorganism's viability, its ability to grow in the human body.

The results currently appear online at the website of the journal Environmental Science and Technology. Ana Rule, a postdoctoral researcher at the Johns Hopkins University Bloomberg School of Public Health, led the study.

In a series of experiments, the researchers tested the BioSampler along with two traditional methods used to sample air - a simple membrane filter, which traps microorganisms on a tightly-woven mesh screen, and the AGI-30 (All-Glass Impinger-30), which collects organisms in a fluid-filled glass chamber. While the BioSampler also collects microorganisms in a glass chamber, its design is slightly different. That difference may result in less damage as the organism is trapped, Buckley said.

The researchers used Pantoea agglomerans, a non-pathogenic bacterium that is a distant relative to E. coli and to Yersenia pestis, the bacterium that causes plague. Y. pestis is among the pathogens listed by the Centers for Disease Control and Prevention as a Category A bioterrorism agent, meaning that it is easily transmitted from person to person and may cause high mortality rates. E. coli can contaminate food and water and also pose an air hazard, Buckley said.

The researchers loaded a sample of P. agglomerans onto each device to determine how efficient the device was in retaining the microorganism in its original state and which sampling method damaged P. agglomerans the least. They collected the bacterial samples from each device after a predetermined amount of time, and then tested the microorganisms' viability - that is, whether or not the organism's cellular membrane was intact. They also tried to grow each sample in a laboratory dish, a trait the researchers call the pathogen's "culturability," which reflects whether or not the organism can replicate and grow.

"From a health standpoint, the current gold standard is to determine if a pathogen can be grown in the lab," Rule said. "In reality, its viability may be a more accurate assessment of its potential threat to human health.

"Even when damaged by the sampling process, pathogens have repair mechanisms that with time in the right medium - for example, the human body - would allow them to replicate and grow," she continued. "Growing the cells in a medium immediately after sampling may not truly represent what happens biologically."

The researchers used a technique called flow cytometry to assess the viability of the P. agglomerans organisms collected by each sampling method. In flow cytometry, cells are stained with a fluorescent dye and passed through a beam of laser light. The resulting color, which scientists see as the organisms pass through this light, tells them whether the cells are live (viable) or dead.

"Just because we can't grow something in medium in the lab doesn't mean it won't grow in a human," Buckley said.

Flow cytometry also tells researchers how many total cells there are in a given sample, which allows them to determine if any cells were lost by the sampling method (they had determined the exact counts in each bacteria sample prior to using each method.)

For each sampling method, the researchers evaluated the number of total, viable and culturable bacteria.

They found that three to six times more cells were viable than culturable after using the filtering method and the AGI-30, suggesting that most of the P. agglomerans had been damaged in the collection process. But P. agglomerans samples taken from the BioSampler showed extremely close agreement between the numbers of viable and culturable bacteria. It was also the sampler with the fewest cell losses.

"Based on these results, it's fair to conclude that what conventional analysis methods measure may not always represent the actual presence and composition of a microorganism," Buckley said. "Understanding what effects a sampling method has on a pathogen is important for designing better sampling strategies."

Buckley and Rule conducted the work with Kellogg Schwab, an associate professor with the Johns Hopkins School of Public Health, and Jana Kesavan, who is with the Aerosol Sciences Team, RDECOM, at the Edgewood Chemical and Biological Center in Edgewood, Md.

Funding for the work was provided by the U.S. Army Edgewood Chemical and Biological Center through a Scientific Services Agreement with Battelle.

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