Assay method aims at cleaner space exploration, cleanrooms

05/01/2003

By Mark A. DeSorbo

PASADENA, Calif.—To boldly, and cleanly, go where no man has gone before is NASA's new mission, now that a team of biotechnologists from the California Institute of Technology has discovered that assay measurement of intracellular adenosine triphophosphate (ATP) is a proven indicator of microbial contamination in cleanrooms.

"The ultimate goal is to develop quick validation methods to ensure that astronauts, microelectronics, and the spacecrafts themselves are not introducing contamination into space and to other planets," says K.J. Venkateswaran, a scientist with NASA's Jet Propulsion Laboratory (JPL) who led the Caltech research team. "We also want to prevent contaminating samples that are being brought back to earth."

At the same time, the firefly luciferase bioluminescense assay method is also being considered as the basis for developing automated environmental monitoring systems for cleanrooms.

According to the team's report, samples were taken from ordinary rooms and cleanrooms, and the air was filtered to remove particles greater than 0.5 microns. The samples were then treated with the enzyme pyrase, an ATP eliminating reagent.

"The enzyme chews up any ATP that is associated with the dead microbes," Venkateswaran says. "Then, we add a detergent to the samples, which opens the microbial cells so the ATP within viable microbial cells is available for testing. The detergent also destabilizes the enzyme that was added to remove the dead microbes."

The final phase of the 30-minute process involves adding luciferase, which within 30 seconds reflects the luminescence of the ATP. Phosphates released from the ATP light up, and the amount of ATP is directly related to the number of viable microbial cells present in the sample, Venkateswaran says.

"The luciferase is engineered to withstand a high pH, so the detergent will not affect it," he adds. "So, you will get high precision sensitivity in detection of viable microbes because the dead microbes were already eliminated."

The report says both the "cultivable and ATP-based determinations" indicated that the microbial burden was lower in cleanrooms than in ordinary rooms. "However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations," the report says.

The report, which was published in the Journal of Microbiological Methods, also indicates that cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates for comparison.

The team found that a large fraction of the samples yielded no colony formers on TSA plates, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples, while rDNA fragments were cloned and sequenced, identifying nearest neighbors— many of which are known to be non-cultivable in the media employed.

"Viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but non-cultivable organisms," the report says. "When the detection of nonviable microbes is of interest, the ATP-assay can be supplemented with DNA analysis."

"Some of the microorganisms that can pass through HEPA filters will find no nutrients and will die, but current monitoring systems fail to determine if the bacteria is dead or alive," Venkateswaran says. "This method provides a differentiation."

And that distinction is key to preventing what the Caltech team calls "forward contamination" during space exploration.

While mammals can easily perish in space, Earthling microbes live on, Venkateswaran says. In fact, common bacillus, yeast, and fungi can live for millions of years, producing spores that remain dormant until favorable conditions arise, jump-starting the replicative state.

To combat forward contamination, the Caltech team began experimenting with the firefly luciferase bioluminescense assay method, a manual test that provides differentiation between extracelluar ATP (dead cells) from intracellular ATP (viable microorganisms).

Biotechnologists looked at 27 environments—12 of which were NASA cleanrooms ranging from ISO Class 4 to Class 8—to test the method's feasibility and accuracy.