SAN LUIS OBISPO- In what may be the ultimate Rip Van Winkle experiment, researchers claim to have revived bacterial spores found in the belly of a bee frozen in amber some 25 million years ago.

Researchers at the California Polytechnic State University cracked open a piece of Dominican amber and extracted material from the abdomen of a bee encased therein. The amber is dated at 25 and 40 million years based on the microscopic fossils found in the geographic strata in which the amber was found.

The extracted material was placed in Petri dishes containing a nutrient solution (trypticase soy broth). Within two weeks, bacteria subsequently identified as Bacillus sphericus grew in the culture. A comparison of the DNA of ancient and modern
bacteria appeared to confirm that the bacteria had indeed grown from the ancient spores. Morphological, enzymatic and biochemical identification methods were used to make a specific identification.

Bacillus is an ancient genus of bacteria that is known to form endospores. The spores are protected from the effects of heat, radiation, pressure and environmental contamination by a thick, protective protein coat. Bacteria can remain in this dehydrated cryptobiotic state for millions of years, said Dr. Raul Cano, a microbiologist at Cal Poly.

What was the bacteria doing in the bee's stomach? Bacillus species form symbiotic relationships with numerous bee species. While the bees provide a home for the bacteria, the bacteria aid metabolic processes within the bees. When facing starvation, the bacteria undergo transformation to spore form, he explained.

Critics were quick to claim that the bacteria was not ancient at all, but represented some form of modern-day contamination of the sample. Dr. Cano anticipated these objections and included a number of safeguards in his experiments. First of all, rigorous decontamination procedures were used to sterilize the surface of the amber before it was cracked. Second, the entire procedure was conducted under a decontaminated, class II laminar flow hood that had never been used for any other bacterial extraction processes.

The researchers also instituted a number of control measures during the procedure to monitor for external contamination. For example, pieces of the broken amber were incubated with the solutions used in the sterilization process for two weeks, with no evidence of bacteria. Petri dishes containing soy agar were also placed under the hood throughout the tissue removal process. These control dishes were incubated for two weeks with no evidence of bacterial contamination.

"We have discovered a brand new source of organisms that could produce life-saving pharmaceuticals or be used in valuable industrial processes. There is almost no downside. These bacteria are different enough to give us new substances, but not different enough that we can't recognize them. There's no more danger with these bacteria than there is with any newly discovered modern microorganisms," he said.

Cano actually first succeeded in reviving the bacteria in 1991 but waited to publish in order to repeat and validate the initial results. In 1992, Cano announced the extraction and cloning of insect DNA from the Jurassic period, coinciding with the opening of the film "Jurassic Park". Since that time he and colleagues have isolated bacteria as old as 135 million years.

However, the current research does not mean scientists are one step closer to reviving dinosaurs, he noted: "It has been known for some time that because of their size, structure and composition, some bacteria can survive as spores for long periods, much as seeds outlive a plant. That is not true of more complex organisms."

Dr. Cano is now working with a company to try and develop useful diagnostic and therapeutic applications for ancient bacteria. The discovery of such ancient organisms could also yield significant new information on the evolution of bacteria.

For more information on this study please refer to: Science, v.268, 5/19/95, pp 1060-1064. Cano et al.