Lactose intolerance may not be life-threatening, but to those genetically incapable of breaking down ingested milk or milk products, it can be a big annoyance. After all, what is life without ice cream, milkshakes, and pizza?

Some people deal with this unpleasant condition by taking food supplements, the most popular of which contain a benign bacterium called Lactobacillus acidophilus—literally, “the sour-lover”. In the human gut, this microorganism releases enzymes that break down lactic acid. The microbial preparation can be purchased over-the-counter at most drugstores.

There’s a hitch, however. What is this acidophilus organism? Do those who buy it in food supplements know how much benefit they are obtaining? How many microorganisms in a bottle are functional, or even alive? To date, there has been no way to tell. Even the FDA does not regulate production of these items, because science has not yet produced a reliable assay that quickly and rigorously determines the percentage of viable microorganisms in a given sample.

Pushing the envelope
That appears to be changing. Dan Armstrong, a chemist at Iowa State University in Ames, has developed a brand-new focus for the established analytical techniques of capillary zone electrophoresis (CZE) and capillary isoelectric focusing. To date, these techniques have been limited to studies of molecules. Now, Armstrong’s team has shown that CZE can rapidly and efficiently separate both prokaryotic and eukaryotic cells, and even the much smaller viruses, for downstream analysis.

Extending CZE’s reach from molecules to microbes opens a new range of opportunities for the technology. One of these is the quality control of drugs, food supplements, and over-the-counter remedies containing live bacteria. Another application may create new avenues for the FDA to monitor and certify such materials. The CZE methods pioneered by Armstrong and his group are good candidates for performing these functions quickly, routinely, accurately, reproducibly, and—ultimately—even automatically.

A technical workhorse of analytical chemistry, electrophoresis involves subjecting a sample, usually containing unknown molecules in aqueous solution, to electrical potentials of fixed strength. The resultant electromotive force (EMF) acts equally on every molecule in the sample, translating it in a straight line toward the attracting pole. Electrophoresis is possible because most molecules in aqueous solution exhibit a net electrostatic charge. Thus, negative ions migrate toward the positive plate, and positive ions move toward the negative plate. In general, the lighter the molecule, the higher its acceleration, and, hence, the more distance it covers per unit time.

In this way, electrophoresis turns the molecular soup of an undifferentiated sample into a first-approximation mass spectrum, with the lightest molecules closest to the charged poles and the heaviest ones closer to the center. At this point, secondary analytical methods, such as chromatography or mass spectrometry, can further characterize each molecular band that electrophoresis has isolated in the sample.

Slab gel electrophoresis (GE) was among the first methods used by geneticists to sort tangled cellular genomes into distinct chromosomal arrays. Indeed, it is hard to imagine how cellular genetics could have made its giant strides of the past two decades without GE. Yet, for all its power, GE can be demanding on laboratory staff, because sample preparation and analysis are exacting, time-consuming, and expensive. And the EMF applicable to most GE samples is limited by what is called the Joule heat—the thermal gain imposed on the sample as the aqueous medium dissipates the electrical energy. Too high an EMF, and heat-labile compounds dissociate.

Capillary actions
CZE was first described in 1974 when Rauno Virtanen, working at Helsinki Technical University, used open-ended tubular capillaries with inner diameters of 200–500 mm. Five years later, James Jorgenson and colleagues at the University of North Carolina at Chapel Hill decided to reduce the inner diameter of the electrophoresis sample-container by orders of magnitude—in fact, to a few tens of micrometers. This, they reasoned, would reduce the threshold sample size to a few milliliters. It would also introduce a proportional gain in the ability of the sample’s aqueous matrix to dissipate the Joule heat. This, in turn, permitted much higher EMFs—on the order of several kilovolts—greatly increasing the net sorting force on a sample’s molecules. Thus, migration distance per unit time greatly increased, resulting in clearer (i.e., more striated) samples in less time. Smaller capillaries also minimized convection currents, which can remix a sample.

By the mid-1980s, CZE was commercialized, and, almost immediately, it expanded the range of analytes from light, simple inorganic compounds to the molecules of life, such as proteins and nucleic acids. CZE has derived critical data for major world research initiatives, and it has played a key role in the spectacular successes of the Human Genome Project.

One small step
The breakthrough made last year by Armstrong’s group was to extend the subject matter of high-efficiency CZE analysis from molecules to intact living cells, including yeasts and bacteria. Not only does the new method begin with an undifferentiated aqueous sample and derive a spectrum of bacteria, but the technique is also nondestructive. Thus, bacteria that are alive at the time of sample preparation, remain so. In addition, by using selective detection, the group can determine cell viability—what percentages of the cells are living or dead. Armstrong noted that a large percentage of microorganisms in many commercial preparations are in fact dead or nonviable.

Their CZE technique is so new that Armstrong’s team has yet to settle on one name, alternating between high-efficiency microbial separation (HEMS) and high-efficiency microbial analysis (HEMA).

Armstrong’s group recently turned its attention to L. acidophilus. Using HEMS, it determined that in at least one commercially available preparation, bacterial viability is only about 60%. That is, nearly half the L. acidophilus in a drugstore sample provided no health benefit.

“The principles of [our] CZE [technique] are similar to gel electrophoresis as far as charge-based migration goes,” Armstrong explains. “However, we do not use a gel. [Our] CZE is ‘miniaturized’ electrophoresis. It is much faster. [And] it uses electro-osmotic solvent flow in the separation process.”

The change in scale between HEMS and molecular CZE is impressive. Most molecules are measured in nanometers. In comparison, a yardstick for a typical bacterium is 1–10 mm—two or three orders of magnitude greater. HEMS can achieve even larger scales, isolating intact bacterial aggregates that comprise dozens of bound prokaryotes.

Figure 1. Micro mayhem. Using their new capillary zone electrophoretic technique, Dan Armstrong and colleagues separated one fungal and three bacterial microorganisms. EOF indicates the position of an internal marker compound. (Adapted from Armstrong, D. W., et al. Anal. Chem. 1999, 71, 5465–5469.)

Armstrong first got the idea for HEMS about seven years ago. “We developed a chiral stationary phase and a CZE additive for separating drug enantiomers that were based on a macrocyclic antibiotic,” he says. “[The antibiotic’s] biological action is to bind to the cell wall of Gram-positive bacteria, but not to Gram-negative bacteria. I began to wonder if we could use such compounds to separate the [bacterial species].” It turned out that he could (see Figure 1 at right).

HEMS, Armstrong says, “discriminates cells by size, shape, electrical charge, and interaction with additives.” Best of all, at least from a commercial point of view, is that the speed of HEMS—completing sample analyses in 10 min or less—admirably suits it to real-time monitoring and control of industrial processes. These processes might produce pharmaceuticals, food additives, or food supplements: anything, in fact, that contains live microorganisms and is intended for animal or human consumption. Examples include brewer’s yeast, baker’s yeast, and our old friend L. acidophilus.

When asked whether each microbial species shows a characteristic range of CZE responses analogous to an IR spectrum, Armstrong replied that the new technique’s experimental conditions could be varied to produce different selectivities. “We can arrange conditions so that all microbes of one type are focused into a single peak,” he says, in reference to a typical CZE output graph, which plots UV absorbance (at 214 nm) against time.

“Of course, if the bacteria or fungi form clumps or groups, you get a peak for each different size [of] clump,” Armstrong adds. “By pretreating the sample, we can break up the aggregate and get [the cells] to all come out together as a group of single cells.”

One giant leap

Figure 2. Medical microbe identification. Armstrong’s group distinguished and identified two bacterial species in urine samples. (Adapted from Armstrong, D. W.; Schneiderheinze, J. M. Anal. Chem. 2000, 72, 4474–4476.)

Immediate markets exist for Armstrong’s technique, and indeed commercial interest has been high. “We are working on three [medical laboratory applications] that could go commercial immediately,” he says. “The method could be invaluable to many areas of science and technology—any area affected by, or using, microbiology.” One such application is shown in Figure 2 (at right).

HEMS could, for example, readily adapt to monitoring antibiotics fermented by mycological action. It could permit real-time analysis, even process control, of products made with batch or throughput (continuous) means. Further, it could soon do so automatically. Armstrong sees a role for microchip-based devices in automating HEMS, and the group has applied for a patent on these devices.

Armstrong’s group is planning to approach the FDA about HEMS, a first step toward ultimate process certification. In the meantime, his team at Ames is “continuing to refine and decipher both theory and mechanism.” In the cards are potential applications for disease diagnosis (e.g., possible “while-you-wait” tests to diagnose throat and urinary infections), soil profiling and fertility assessment, and many types of bioenvironmental analysis. In HEMS, and even more so in the team that is developing it, Iowa State seems to have a winner.

Suggested reading

1. Armstrong, D. W., et al. Anal. Chem. 1999, 71, 5465–5469.
2. Armstrong, D. W.; Schneiderheinze, J. M. Anal. Chem. 2000, 72, 4474–4476.
3. Armstrong, D. W., et al. FEMS Microbiol. Lett. 2001, 194, 33–37.
4. Scheiderheinze, J. M., et al. FEMS Microbiol. Lett. 2000, 189, 39–44.