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A way to tell one spore from another
Study of humidity-induced swelling may lead to faster identification of potential bioterrorism agents such as anthrax

| 12 February 2003

Two Berkeley physicists have observed that the spores of a microbe closely related to anthrax swell with increasing humidity — a physical change that might allow quick and cheap detection of Bacillus spores like anthrax.

The swelling is a surprise to microbiologists, who have until now assumed that spores of the Bacillus bacteria, which include anthrax (Bacillus anthracis), are a dormant, resting and basically inert stage of the microbe.

The swelling, observed in spores of Bacillus thuringiensis, a bacterium now often used to kill insects that attack crops, may be diagnostic of all Bacillus spores and may allow scientists to distinguish between different types of Bacillus.

“If we are able to discriminate between spores based on size or swelling characteristics, it’s a test we could do in seconds to minutes,” says Andrew Westphal, a research physicist at Berkeley’s Space Sciences Laboratory.

Westphal and Berkeley physics professor P. Buford Price, along with microbial biologists Terrance Leighton and Katherine Wheeler of the Children’s Hospital Oakland Research Institute, reported their findings this week in the Proceedings of the National Academy of Sciences.

On Jan. 22, the federal government began to deploy environmental monitors to detect airborne bioterrorism agents, including anthrax and smallpox. The system
relies on filtering air and sending the filters to a lab, where any attached mi-crobes would be cultured and identified. Even with advanced techniques such as PCR (polymerase chain reaction) to detect microbial genes, the turnaround time would be between 12 and 24 hours. In comparison, a device to scan for Bacillus spores of a particular size and swelling time could provide an answer in about 10 minutes.

“This wouldn’t be a foolproof way of saying, ‘You’ve got anthrax spores,’” adds Price, “but it would be a flag for you to go to the next step, perhaps a PCR test to detect anthrax DNA.”

An ‘extraordinary technology’
The new technique also could mark a significant advance in biological imaging.

“The technology that Price and Westphal have developed is quite extraordinary,” says Leighton, an anthrax expert and Berkeley professor emeritus of molecular and cell biology. “There is great interest in trying to manipulate and image single microbial cells, because they are at the limit of resolution of normal optical microscopes. This technology pushes the lower limit of resolving power by perhaps a factor of 100 beyond what has been demonstrated previously.”

Price and Westphal first considered using size to discriminate between Bacillus spores, more properly called endospores, after anthrax spores began showing up in mail at spots around the East Coast shortly after the terrorist attacks of Sept. 11, 2001. In researching what is known about spore size, they found that most measurements have been very rough and have not taken humidity or other environmental factors into account. The lack of data is due partly to the spores’ size: with diameters of around two microns, they are smaller than the resolution limit of most light microscopes. More-precise electron-microscope images can only be taken of thin sections of spores in a vacuum, eliminating any possibility of gauging the effect of humidity.

About 10 years ago, the two developed a technique to extract more precision from optical microscope measurements. Using a microscope with attached CCD camera — a basic camcorder — Westphal takes hundreds to thousands of snapshots of an object. Each image is slightly offset from the others, allowing him to average the sizes to obtain a more- precise measure of the object’s size.

In practice, an automated microscope rapidly scans a surface, measuring and recording the size of every object and fitting it to the shape of an ellipse. The absolute precision of a single measurement is better than 50 nanometers. By measuring the same object multiple times, the precision can be improved by a factor of 10, to better than 5 nanometers.

“We are taking advantage of modern electronics and image processing to look at the size of individual spores with very high time resolution and rapid analysis,” says Westphal.

In their first attempts, they found that the spores of four types of Bacillus differed significantly in size — enough to let them distinguish each by size alone.

To determine whether spores change size with environmental conditions, they checked the size of B. thuringiensis spores under varying levels of humidity. To their surprise, spores swelled significantly — about 4 percent — under conditions of high humidity. The swelling took place in two stages — spores swelled about 2.9 percent in less than 50 seconds, and then increased another 0.9 percent after about eight minutes.

Price and Westphal interpret this as rapid water diffusion into the spore’s outer coat and cortex, followed by slower diffusion into the core. Because a larger, swollen spore has larger pores capable of admitting more gas, the humidity-related swelling may explain why spores are more susceptible to being killed by gases such as formaldehyde, ethylene oxide, and chlorine dioxide in conditions of high humidity.

“This paper provides one potential mechanism to explain why, with higher levels of humidity, one sees greater efficacy of spore killing,” says Leighton. “That is very important for decontamination, whether it’s medical-instrument sterilization, building de-contamination, or removing toxic mold. It’s a fundamentally important observation.”

More research needs to be done before the technique can be adapted to detection and identification of species of Bacillus spores, Price emphasizes.