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Electrophoretic Method Adapted for Separating Microbes

Faced with an expanding array of products such as probiotics that contain live bacteria, it behooves those who handle them and may be responsible for certifying their content and quality to have quick and accurate tests to determine species, strain, and percentage of viable microorganisms in a particular product. Recently, Dan Armstrong and his team of chemists at Iowa State University in Ames adapted capillary zone electrophoresis (ZE) and isoelectric focusing (IF) methods to this purpose—providing a new, rapid, and efficient analytic means for separating a wide range of microorganisms, including bacteria, yeasts, and viruses.

Although used extensively for separating and analyzing biological macromolecules such as DNA and proteins, capillary ZE and IF methods were not previously applied to microbes, which typically are at least 100- to 1,000-fold larger than such molecules. These methods separate particles such as cells or molecules on the basis of size, shape, electrical charge, and their interaction with various additives that may be supplied to the analytic medium. Conditions, including pH and buffer type, are tinkered with to encourage particular microbes to migrate as single peaks during an analytic run.

According to Armstrong’s high-efficiency microbial separation (HEMS) method, capillary tubes with internal diameters of 50 to 100 microns can replace the traditional slab gel used for many kinds of electrophoresis-based analyses—short-circuiting the time-consuming and thus costly step needed to prepare this workhorse of many analytical laboratories. The alternative capillary-based "miniaturized electrophoresis" procedure uses samples consisting of a mere few microliters, and bacteria or other microorganisms typically remain viable throughout the analytic process, according to Armstrong. Because a routine analysis may last only 10 to 15 minutes, HEMS lends itself to real-time monitoring and high-throughput screening.

Armstrong stumbled on the idea for adapting capillary ZE and IF analytic methods to separating microorganisms about eight years ago while he was exploring ways to separate gram-positive and gram-negative bacteria. Exploiting the capacity of the antibiotic vancomycin to bind gram-positive bacteria, he initially used this antibiotic-binding property to develop a capillary zone electrophoresis method for separating these two broad classes of bacteria. "That led to this more general approach," he says.

For example, early this year, Armstrong and his collaborators published a report describing how they used HEMS to separate, identify, and quantitatively measure Lactobacillus acidophilus, which is advertised as the active component of a widely used dietary supplement for individuals with lactose intolerance (FEMS Microbiology Letters, January 1, 2001). As an adjunct to the HEMS procedure, they also determine cell viability by pretreating cells with a mixture of green and red fluorescent stains that cause viable cells to glow green while dead cells appear red under a dual- channel laser. Their analysis of the dietary supplement indicated that only about 60% of L. acidophilus cells in a popular commercial brand are viable.

In addition to verifying the content of probiotics, HEMS also holds promise for quickly identifying pathogens involved in causing infections, such as those of the urinary tract, according to Armstrong. In a few microliters of urine and without pretreating specimens, "we can check for all likely bacteria simultaneously," he says. The method is faster and cheaper than waiting days for bacterial cultures to grow, or using PCR-based probes, each of which is designed to identify only one type of bacteria.

Armstrong also predicts that HEMS will find wide use in the food and biotechnology industries to monitor fermentation processes for producing pharmaceuticals, food additives, or other products that contain live microorganisms. "There’s no way to look at microbes directly" he says. Instead, fermentation processes typically are followed by monitoring the amount of product generated or the decline of starting materials. HEMS can also detect contaminants and microbes containing mutations. "In the next five to ten years, we’ll see hundreds of people leaping into this useful technology," he predicts. Faced with keen interest from companies and venture capitalists who may help in commercializing the HEMS procedure, he adds, "we’re trying to decide the best way to go."

Carol Potera
Carol Potera is a freelance writer based in Great Falls, Mont.