Cisplatin 2. Control Experiments for the Effect of Electric Fields on E. coli

With the result that application of an electric field apparently halted bacterial cell division and led to filamentous growth, Barnett Rosenberg’s group sought to understand better the cause of this phenomenon. (Recall that filamentous growth occurs when cell division—but not cell growth—is inhibited.) For this reason, the researchers conducted a variety of control experiments in which they varied one experimental parameter at a time. They knew that the filamentous growth they had observed in E. coli bacterial cells could be caused by several known physical and chemical agents such as the following:

• dyes such as methylene blue and penicillin,
• transfer to an unaccustomed medium,
• osmotic pressure changes,
• near ultraviolet irradiation,
• and magnesium deficiency or excess.

Based on the experimental conditions, Rosenberg and his colleagues were able to rule out the first three possibilities. They then conducted a variety of tests and ruled out not only the last two possibilities but also several others:

• ultraviolet light,
• temperature,
• pH.

They also found that adaptive mechanisms and mutation effects in the bacteria did not play a role in filamentous growth.¹ The researchers then considered the possibility that the application of an electric field to the bacterial medium might have led to an electrolysis reaction and that the chemical products of this reaction might have affected bacterial cell division. For this reason, they conducted more control experiments. In one experiment, they decided to separate the electrodes from the E. coli bacteria. Their new apparatus had two chambers: an electrolysis chamber containing the electrodes but no bacteria, and a bacterial chamber containing E. coli cells but no electrodes. In the experiment, the nutrient was pumped into the electrolysis chamber and a voltage was applied to the electrodes. The nutrient was then pumped from the electrolysis chamber into the bacterial chamber. The idea was that if a new chemical species was produced in the electrolysis chamber, and if it was relatively stable (meaning long-lived), then it would be pumped into the bacterial chamber along with the nutrient. Such a new chemical species might be the cause of the observed filamentous growth (also called elongation) of the bacterial cells. The researchers found that under this new set of experimental conditions, they again observed that the bacterial cells formed long filaments.

From the results of this experiment, they concluded that the application of the electric current (voltage) was not itself responsible for the observed effects on bacterial growth, but rather that the electric current led to the formation of a new chemical species that affected bacterial elongation. Furthermore, they again observed that oxygen had to be present in the electrolysis chamber for bacterial elongation to occur.1 This result led them to consider the possibility that electrolysis was generating an oxidizing agent, which might be contributing to the observed effects on bacterial growth. They then conducted another control experiment to look for the presence of an oxidizing agent. One test commonly used to detect the presence of an oxidizing agent is the potassium iodide-starch test. For example, if the putative oxidizing agent is a metal ion with a +2 charge (represented here as \(M^{2+}\)), the metal ion gains two electrons to form the neutral, elemental metal (M0) and is therefore reduced. The two electrons come from two iodide ions (\(I^-\)), each of which gives up one electron, producing a molecule of elemental iodine (\(I_2\)). When elemental iodine exists in the presence of excess iodide ion, an acid-base reaction occurs in which elemental iodine acts as a Lewis acid and iodide ion acts as a Lewis base to produce a triiodide ion, \(I_3^-\). Triiodide ion is a linear species and forms a blue complex with starch. Therefore, the potassium iodide-starch test is able to detect the presence of an oxidizing agent, here \(M^{2+}\). For more information on oxidations and reductions (i.e., redox chemistry), go to the redox chemistry and electrochemistry modules.

\[M^{2+} + 2 I^- → M^0 + I_2 \tag{redox reaction} \]

\[I^- + I_2 →I_3^- \tag{acid-base reaction} \]

+ _____________________________

\[M^{2+} + 3 I^- → M^0 + I_3^- \tag{net reaction} \]

The researchers used the potassium iodide-starch test to see whether any oxidizing agents were present in the medium they were using to grow bacterial cells. They found that the ordinary medium gave no reaction with potassium iodide and starch but that the electrolyzed medium gave a positive test, in which a blue color developed after 5 minutes. From this result, they concluded that an oxidizing agent was indeed present in the medium. Furthermore, they found that the appearance of the blue color indicating the presence of oxidizing agent coincided with the elongation of the bacterial cells. And finally, they observed that the intensity of the blue color corresponded to the frequency of the applied voltage. When the frequency was 500 c/s, the blue color was most intense, indicating that the concentration of the oxidizing agent was highest at this frequency; when the frequency was 6,000 c/s, the blue color was not detectable (recall that in the electric fields module, we learned that when the applied voltage was 500 c/s, filamentous growth was at a maximum, whereas at 6,000 c/s, no filaments were observed). These last two results strongly suggested that a new chemical species, an oxidizing agent, was causing the changes in bacterial cell growth.1 The next step for the researchers was to determine the identity of this mystery oxidizing agent. To do this, they ran another set of control experiments. They deduced that several possible oxidizing agents could be created from the medium during electrolysis:

• hypochlorite ion (\(ClO^-\)),
• chlorite ion (\(ClO_2^-\)),
• chlorate ion (\(ClO_3^-\)),
• perchlorate ion (\(ClO_4^-\)),
• hydrogen peroxide (\(H_2O_2\)),
• hydroxylamine (\(NH_2OH\)),
• and persulfate ion (\(S_2O_8^{2-}\)).

Sensitive qualitative tests showed that none of these ions existed in the medium. In a separate set of control experiments, each component of the medium was made up in the appropriate concentration and electrolyzed for 6 amp-h. Following electrolysis, each solution was subjected to the potassium iodide-starch test. A mixture of negative and positive results were obtained. Negative tests resulted in the following cases, meaning that no oxidizing agents were found in these electrolyzed solutions: phosphate ion (PO₄^3-); sulfate ion (SO₄^2-); phosphate ion + glucose; phosphate ion + sulfate ion + glucose; sodium sulfate (Na₂SO₄); and sodium carbonate (Na₂CO₃). However, oxidizing agents were found in the following cases, as evidenced by positive tests: ammonium sulfate ((NH₄)₂SO₄); ammonium carbonate ((NH₄)₂CO₃); ammonium chloride (NH₄Cl); and other chlorides. Sodium chloride (NaCl) gave a faint positive response.1 To learn the conclusions that Rosenberg and his coworkers reached as a result of this set of control experiments, go to the module on the role of platinum electrodes.

References

1. Rosenberg, B., Camp, L. V., Krigas, T. Nature ,1965, 205, pp. 698-699.