1. Study of the Effect of Electric Fields on E. coli
|Our story begins in 1961 when a young physics professor named Barnett Rosenberg left his position at New York University to help found a new department of biophysics at Michigan State University. As a physicist, he had long been intrigued by microphotographs depicting the process of cell division, called mitosis. These photographs reminded him of a natural physical phenomenon: the magnetic dipole field created when one places iron filings over a bar magnet. The field lines created by magnets have the same shape as those created by electricity between two equal and opposite point charges.|
His observation of the similarities between the shape of cells as they are undergoing mitosis and the shape of electrical and magnetic field lines presented him with a perfect avenue for shifting his research focus more toward biology. He and others hypothesized that this similarity in shape was perhaps not an accident of re, but of nature that such a dipole might be involved in cell division. His idea was that if the mitotic dipole was exposed to electromagnetic radiation of a resonant frequency, the dipole might absorb some energy. His goal, then, was to see how the cell was affected by the absorption of this energy.3 As is often the case in scientific research, particularly in interdisciplinary fields,Figure 1. Representations of mitosis: (a) a schematic diagram identifying the three classes of microtubules in a mitotic spindle (not to scale); (b) a confocal image of a mitotic spindle at metaphase from a Drosophila embryo, with the microtubules fluorescently labeled. (Courtesy of William Theurkauf); (c) an experiment in which tubulin molecules (which form microtubules) labeled with two different fluorescent dyes (appearing green and orange) were injected into the living cell; the cell is shown here at metaphase. (Courtesy of G. Borisy) Reprinted with permission.1
(b) (c)Figure 2. Magnetic field lines: (a) magnetic field patterns surrounding a bar magnet as displayed with iron filings; (b) magnetic field patterns between unlike poles of two bar magnets. (Courtesy of Henry Leap and Jim Lehman) Reprinted with permission.2
Figure 3. Electric field lines: (a) the electric field lines for two equal and opposite point charges (an electric dipole); note that the number of lines leaving the positive charge equals the number terminating at the negative charge; (b) the photograph was taken using small pieces of thread suspended in oil; the pieces of thread align with the electric field. (Courtesy of Harold M. Waage) Reprinted with permission.2
Rosenberg decided that it would be a good idea to have a collaborator who had been trained in biology. Therefore, he hired a biologist and together they set up a specially designed continuous culture apparatus for the cells they were planning to study. Their plan was to use mammalian cells, which are known to undergo mitosis. They decided to test the apparatus first, however, by using Escherichia coli bacterial cells, which (along with other prokaryotes) do not form the mitotic figures in cell division, as shown above. For their experiments, they chose a medium commonly used for studying the growth of E. coli bacteria; this medium was enriched with glucose and magnesium chloride. The culture chamber was kept at a constant temperature of 37° C, and compressed air was bubbled into the chamber through a glass frit bubbler. Two strains of E. coli were used to inoculate the chamber: E. coli B and E. coli K-12. After about 24 hours, the bacterial population reached a steady state, as determined by turbidity measurements. These measurements showed that the steady state was maintained for 10 to 14 days longer. Therefore, all experiments were performed within 10 days of inoculation.4 To study the effect of electric fields on growth processes in bacteria, an audio oscillator was used to supply sinusoidal voltages of 50 to 100,000 cycles/second (c/s). These signals were amplified by a conventional audio power amplifier and then sent to a pair of platinum electrodes in the chamber. (Platinum was chosen as the material for the electrodes because of its well-known chemical inertness.) The impedance of the chamber was approximately 6 ohms and was perfectly matched to the output impedance of the amplifier, allowing for the most efficient transfer of power. In the first experiment, the electric field was turned on to 1,000 c/s, 2 amp (peak to peak) through the chamber. After 1 hour, the turbidity began to decrease, meaning that cell growth was slowing down. After 2 hours, the bacterial population was almost completely washed out, and the voltage was turned off. After 8 more hours, the population density of the bacteria returned to its previous value. This process could be repeated.
Rosenberg and his colleague found a surprising and unexpected phenomenon at this point. The researchers observed that the bacterial cells stopped dividing 1 to 2 hours after the voltage had been applied and that the cells started to elongate in that time, forming long filaments. For 1 to 2 hours after the voltage was removed, the cells continued to increase in length, but after 2 hours had elapsed, cell division started to occur again.4 These effects were tested over a range of applied voltages. When the frequency of the applied voltage was between 500 to 6,000 c/s, bacterial growth was affected and filamentous growth of the bacteria occurred. Filamentous growth occurs when cell division is inhibited, but cell growth is not. During filamentous growth, E. coli are still able to grow, but since they cannot divide, they form long filaments, as shown in Figure 4, part (b). Under normal conditions, E. coli form short rods, as shown in Figure 4, part (a). The extent of filamentous growth varied over the 500 to 6,000 c/s frequency range: At 500 c/s, filamentous growth was at a maximum, whereas at 6,000 c/s, no filaments could be seen. Moreover, at frequencies above 6,000 c/s (i.e., from 6,000 to 100,000 c/s), no effects were observed in 24-hour trials.4
Along with variations in frequency, the presence of oxygen was an important factor in causing filamentous growth in E. coli cells. Filamentous growth occurred at the frequencies mentioned above only when oxygen was present. If another gas, such as nitrogen or helium, were bubbled through the cell, no effect was observed. Having observed the unexpected result that electric fields indeed seemed to affect bacterial cell division, Rosenberg and his coworkers conducted more experiments to try to determine the exact cause of this effect. In their subsequent experiments, they varied one experimental parameter at a time in an effort to determine exactly why filamentous growth of bacterial cells occurred under their experimental conditions. Such control experiments are essential in scientific research. As we will see, negative control experiments can often give as much information as positive ones.
Figure 4. Normal and elongated E. coli: (a) scanning electron microphotograph of normal E. coli (Gram-negative rods); (b) scanning electron microphotograph of E. coli grown in medium containing a few parts per million of cis-diamminedichloroplatinum(II). Same magnification in both pictures. The platinum drug has inhibited cell division, but not growth, leading to long filaments. (Courtesy of D. Beck) Reprinted with permission.
(1) Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J. D. Molecular Biology of the Cell, 3rd ed. Garland Publishing, Inc.: New York, 1994, pp. 919, 925. (2) Serway, R. A. Principles of Physics, 2nd ed. Harcourt Brace & Company: Fort Worth, 1998, p. 639. (3) Rosenberg, B. In Nucleic Acid-Metal Ion Interactions, T. G. Spiro, Ed., John Wiley & Sons, Inc.: New York, 1980, Vol. 1, pp. 1-29. (4) Rosenberg, B., VanCamp, L., Krigas, T. Nature, 1965, 205, pp. 698-699.
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