To really understand what’s going on in the soil, scientists must learn about the microorganisms that live there. Which kinds? How many? What are they doing?

Above: Mary Ann Bruns uses the techniques of molecular biology to study soil bacteria. Right: Bacterial “bar codes” are read from top to bottom in a fingerprint gel illuminated with ultraviolet light. The bright horizontal bands are DNA fragments of different sizes. The outside lanes contain “marker” DNA bands of known sizes, while the inner lanes contain DNA fingerprints of 16 different bacteria isolated from soil. Bacteria of the same genetic makeup produce identical fingerprints.

Microbiologists have always studied soil microorganisms by growing them in laboratory cultures. They add nutrient mixtures to soil and incubate these to get microbes to grow.

But when scientists examine soils under the microscope, they find huge discrepancies between the numbers of living cells they see and the numbers of microbes that grow in cultures. Scientists readily admit that they have studied only a tiny fraction of the microbes living in soil.

In the mid-1980s, new techniques using molecular biology and computers allowed microbiologists to begin “reading” the genetic material (DNA) of uncultured soil microbes. It was as if a light turned on to reveal whole new realms of microbial diversity.

“We now know that 99 percent of the soil bacteria haven’t been cultured,” says food microbiologist Steve Knabel, who teaches a course in microbial diversity. “It’s causing a renaissance in microbiology. It’s also raising an awareness that we can’t keep carelessly mucking around with the soil, or we’re going to lose some of this tremendous diversity.”

Soil microbial ecologist Mary Ann Bruns is exploring the microbial “Who’s Who” of central Pennsylvania soils. When she joined Penn State in 1999, she set up a molecular biology lab to study soil bacteria and fungi.

“In a molecular biology lab, tests are run on a much smaller scale than in a traditional soil microbiology lab,” she explains. “The techniques are sensitive enough to analyze a few billionths of a gram of DNA. Instead of test tubes, we use tiny plastic vials that hold microdroplets, and pipettes that measure microliters.”

When you extract DNA from soil, you get a mix of everything that’s there—from worm to germ to virus. A key piece of equipment in any molecular lab is a polymerase chain reaction (PCR) machine, which makes millions of copies of specific genes from DNA mixtures. “This allows us to copy genes from soil microorganisms that have never been studied before,” Bruns says.

After PCR, gene mixtures are added to a gel, which is then subjected to an electric field. The “electrophoresis” process in the gel causes gene fragments of different sizes to separate into bands, thus making a unique DNA “fingerprint.”

“To see the fingerprint, we put the gel in an ultraviolet light box and use a dye that makes DNA glow pink under ultraviolet light,” Bruns says. “Then we photograph the glowing bands for our records. When we detect bands from unknown organisms, we cut out the bands to analyze their DNA sequences and identify these microbes.”

To prevent sample contamination, Bruns’ molecular biology lab is located in a “clean room” to minimize dust. “We also wear gloves when handling our samples to protect them,” she says. “We have enzymes on our fingers that can break down DNA.”