Investigating the effect of electric field on growth of bacterial

ABSTRACT

High-intensity electric field and magnetic field have shown to decrease the growth rate of bacteria. Some pathogenic and non-pathogenic have even shown to decrease the growth rate to zero. This property along with some physical laws (such as high potential, properties of the conductor, coronary discharge, LMP [lipid membrane potential] polarization) can be used to develop an anti-infective instrument. Such auto-sterilized instruments can be of great use in the medical field leading to fewer casualties. Many of the casualties due to medical surgery are caused due to infection and growth of bacteria on medical instruments. On the other hand, in some specific cases, genetically modified bacteria are needed to grow faster for their biotechnological applications. The results reported here in the presence of low electric field and high electric field are found to be different. The growth is more prominent in case of low applied altering voltage while it is slower at higher applied voltage.

INTRODUCTION:

Because of the functionality of ion pumps and ion channels in the cellular membrane, the membrane gets electrically repolarized and depolarized following regular cycle. These membranes are characterized by a resting potential. Any change in this potential can bring adverse effect on the membrane functionality. It has been reported in various research articles that application of high electric and magnetic fields can change the membrane potential which ultimately can lead to cell death [1, 2].

The aims of the present project were to:

(1) design prototype to conduct an experiment.
(2) grow and culture bacteria.
(3) record growth of bacteria data as a function of applied electric field.
(4) predict optimum condition for the bactericidal effect.
(5) finding a relation between the electric field applied and the growth rate.

The growth rate of bacteria is affected by many factors and is a complicated phenomenon to study. Techniques such as UV-Visible spectrophotometry, spot assay and colony forming unit (CFU) were used to quantify the growth of bacteria under the electric field.

Instead of a high electric field, the effect of low electric field was studied and reported here in the present study. The growth rate of bacteria with varying intensity of electric field was monitored with alternating voltage source. Surprisingly, the experimental result is observed to deviate from the result reported for the high electric field.

MATERIALS:

Escherichia coli (E. coli: DH5α) bacteria, Agar broth, and Agar media were the main ingredients for the experiments. De-ionized Millipore water (DI water) (resistivity ~ 18 MΩ cm) with pH ~ 7.0 was used to prepare the aqueous solution of these samples. 25 gm sigma LB agar was mixed in 1 liter of DI water.

Other important lab instruments and consumables were petri dish, UV-Visible spectrophotometer, incubator, 70 % ethanol, weighing balance, indium tin oxide (ITO) coated glass slide, normal glass slide, glass cutter, silver paste, conducting wire with non-conducting coating and function generator for applying the voltage.

EXPERIMENTAL METHODS:

(A) UV-Visible spectroscopy: It is a spectroscopy technique that uses UV-visible spectrum of light in order to check the optical density (O.D.) of a liquid sample. For the experiment, a fixed OD of 600 nm was used. Glass cuvette with 3.5 ml of sample was used for the measurement. (DI water) (resistivity ~ 18 MΩ cm) with pH ~ 7.0 was used to prepare the LB broth that was used as blank for the experiment.

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Figure 1: The experimental setup to monitor the effect of low-intensity electric field on the growth of bacteria. The function generator was used to apply the voltage across two parallel ITO coated glass slides with the E. coli solution in between the plates.

(B) Electric field set up: For the application of perpendicular electric field on bacteria, ITO (Indium Tin Oxide) coated glass slides were kept parallel to each other. The solution of bacteria was dropped on the lower slide and the other slide was kept on top of it, maintaining a gap of 0.5 cm. A schematic diagram of the experimental set up is shown in figuree1. The solution of silver nano-particle was used to make electrical contact between the function generator (Figure 2) and the ITO coated glasses. A distance of ~ 0.5 cm was maintained between the two plates.

The primary culture was made from the stock E. coli by overnight incubation and secondary culture was then made. Multiple O.D (optical density) readings were taken, and an experiment was started when the O.D reached a minimum value. Then, the bacterial culture was taken and added dropwise to three substrates with altered conditions. 50 µl of culture was added to (a) glass slide (b) IT0 coated glass slide and (c) ITO coated glass slide with the applied electric field. All the three substrates were then incubated at 37°C. After overnight incubation (~18 hrs.), the bacterial culture was extracted using 0.9% saline solution. From this, 50 µl of 0.9% saline solution was added dropwise to a plate for mixing completely and then dilution series was made (till 104 dilutions). An agar plate was then marked into four different quadrants, each denoting a dilution series. Then 10 µl of each dilution series was added to each quadrant and streaked. Results were recorded after 24 - 32 hrs. of incubation. The incubation conditions maintained was 37 °C (optimum for E. coli).

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Figure 2 : Left: Function generator to produce alternating voltage to figure out the effect of applied electric field on bacterial growth. Right: Function generator attached to the incubator shaker with a fixed temperature of 37oC.

(C) Dilution Series: This is the succession of step dilutions, each with the same dilution factor, where the diluted material of the previous step is used to make the subsequent dilution. The ratio followed was 50 microliters of the bacterial sample (after treatment) was added to 450 ml of .9% saline solution to create a dilution series of order 10.Subsequently, the other dilution series was also made.

(D) CFU Count: CFU stands for colony forming unit that is used to measure the number of viable cells in a bacterial colony. The viability is defined as the ability to multiply under a set of given conditions and parameters. Counting the colony forming units requires the culturing of the bacteria of interest and counts the number of living cells, in contrast to the traditional microscopic counting which takes into account all the cells irrespective of them being dead or alive. As explained above, three samples were taken for the CFU measurement in each experiment; (1) bacteria grown on glass substrate, (2) bacteria grown on ITO coated glass substrate, and (3) bacteria grown on ITO coated glass substrate under applied electric field of various intensity.

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Figure 3: Serial Dilution used to measure CFU, plated on AGAR plate.

The bacteria grown on various substrates were cultured on LB agar plates. Dilution series till a range of 104 were made and then grown on the plate. The formula used for the CFU measurement is:

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where, N is the number of colonies, D.F the dilution factor and V the volume plated in ml. For the experiment. Each LB agar plate was divided into four quadrants named Ⅰ, Ⅱ, Ⅲ, Ⅳ with respect to the dilution series as shown in figure 3.

RESULTS AND DISCUSSIONS:

To understand the effect of electric field on the growth of E-coli bacteria, numerous set of experiments were performed. In many cases, experiments were repeated but the experimental parameters were kept constant as to figure out a statistically averaged results. The parameters are enlisted below in table 1. Indium tin oxide (ITO) coated surface was used for the growth experiment.

Table 1: Experimental parameters related to the AC applied voltage

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In all experiments, spot assay was followed. A representative data set is shown in figure 3. For getting more quantitative analysis, the CFU calculation was done. As is evident from table 2 of the first experiment, an applied voltage of 4 volts leads to an increase in the growth of bacteria on ITO surface. These results are obtained at dilution series 1 (101). Interestingly, in case of 25 volts, the growth decreases considerably. Note that other dilution series have not been shown here. Other series have been shown from other experiments in the consecutive sections.

Table 2: CFU calculated from the spot assay of bacteria grown on indium tin oxide (ITO) coated surface with and without applied electric voltage (first dilution).

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Figure 4: Bar diagram representing the CFU of bacteria grown on ITO coated substrate at different applied electric field (experiment 1 and in first dilution).

This is to be noted that before growing bacteria on ITO coated glass slide, only glass was used as the substrate to figure out the effect of ITO on the growth. Interestingly, all experiments have shown that the bacteria are more viable on the glass surface which was observed through more CFU counts from the colony grown on glass compared to ITO coated surface. As voltage cannot be applied across the pure glass substrate, the result was not compared with that of ITO coated glass with applied voltage. Hence, the ITO coated surface is toxic to the bacterial cell.

Table 3: Comparing the CFU of bacteria grown on glass and ITO coated surface (No electric voltage applied)

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Table 4 CFU calculated from the spot assay of bacteria grown on indium tin oxide (ITO) coated surface with and without applied electric voltage (second dilution).

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Figure 5: Bar diagram representing the CFU of bacteria grown on ITO coated substrate at different applied electric field (experiment 2 and second dilution).

The result of the second dilution series of the second experiment is shown in table 4 and in figure 5. It is seen again statistically that at low applied voltage, the growth is higher compared to that of high voltage. Similar observations have been noted in table 5 and figure 6 for higher dilution. For each experiment, the data of only one dilution series has been shown in this report as to avoid the repeats of similar observations.

Table 5. CFU calculated from the spot assay of bacteria grown on indium tin oxide (ITO) coated surface with and without applied electric voltage (third dilution).

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Figure 6: Bar diagram representing the CFU of bacteria grown on ITO coated substrate at the different applied voltage (experiment 3 and third dilution).

CONCLUSION:

The effect of alternating voltage on the growth of bacteria has been reported in the literature to be toxic which slows down the growth rate of bacteria and hence the lower number of colony formed in a given span of time. However, the results reported in the literature were observed at higher applied voltages (≥ 25 volts, peak to peak). In this present study, it has been observed that lower applied voltage helps in growing bacteria. It is to be noted that the results of this report are verified using the ITO-coated surface only. It would be interesting to check if the results hold good for other surfaces such as zinc, copper, and silver as these materials are well known to have anti-bacterial activities. Further investigations are needed to answer this question and to understand the molecular mechanism of such an effect.

The method reported here may be helpful in many ways such as more and faster growth of genetically modified bacteria which are used for the production of various biotechnologically important bacteria. The higher yields could be beneficial economically.

ACKNOWLEDGMENT:

I am thankful to Shiv Nadar University (SNU) for providing me the financial and academic support for this ‘OUR’ project.

I would like to thank my mentor, Dr. Sajal Kumar Ghosh, Department of Physics for giving me an opportunity to work under him and for guiding and helping me in conducting different experiments. He also helped me in understanding the results reported here.

I would like to thank Ms. Ritika Gupta for her generous help in every step of performing the experiment and making me understand the results. I enjoyed the scientific discussions with all the lab members of Soft Matter Physics Laboratory.

REFERENCES:

[1] Snezana BRKOVIC, Srdjan POSTIC, Dragan ILIC “Influence of the magnetic field on microorganisms in the oral cavity “2015;23(2):179-86

[2] Shilpee Jain, Ashutosh Sharma, Bikramjit Basu “Vertical electric field induced bacterial growth inactivation on amorphous carbon electrodes “CARBON 81 (2015) 193 – 202