1 23

The Environmentalist ISSN 0251-1088 EnvironmentalistDOI 10.1007/s10669-011-9365-2

The frequency-dependent effect ofextremely low-frequency electromagneticfield and mechanical vibration atinfrasound frequency on the growth,division and motility of Escherichia coliK-12Varsik Martirosyan, Levon Markosyan,Hrachik Hovhanesyan, KarlenHovnanyan & Sinerik Ayrapetyan

1 23

Your article is protected by copyright and

all rights are held exclusively by Springer

Science+Business Media, LLC. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you

wish to self-archive your work, please use the

accepted author’s version for posting to your

own website or your institution’s repository.

You may further deposit the accepted author’s

version on a funder’s repository at a funder’s

request, provided it is not made publicly

available until 12 months after publication.

The frequency-dependent effect of extremely low-frequencyelectromagnetic field and mechanical vibration at infrasoundfrequency on the growth, division and motility of Escherichia coliK-12

Varsik Martirosyan • Levon Markosyan •

Hrachik Hovhanesyan • Karlen Hovnanyan •

Sinerik Ayrapetyan

� Springer Science+Business Media, LLC 2011

Abstract The aim of this work was to investigate the

frequency-dependent effects of extremely low-frequency

electromagnetic field (ELF-EMF) and mechanical vibra-

tion at infrasound frequency (MV at IS frequency or MV)

on growth and development of Escherichia coli K-12, by

using classical microbiological (counting colony forming

units), isotopic, spectrophotometric and electronmicro-

scopic methods. The frequency-dependent effects of MV

and ELF-EMF were shown that they could either stimulate

or inhibit the growth and the division of microbes

depending on the periods following exposure. However,

the mechanism through which the MV and ELF-EMF

effects affect the bacteria cell is not clear yet. It was sug-

gested that the aqua medium could serve a target through

which the biological effect of MV and ELF-EMF on

microbes could be realized. To check this hypothesis, the

frequency-dependent effects (2, 4, 6, 8, 10 Hz) of both MV

and ELF-EMF on the bacterial growth, division and their

motility in cases of exposure, the preliminary treated

microbes-free medium and microbes containing medium

were studied. Both MV and ELF-EMF effect on microbes

have frequency and post-exposure period duration-depen-

dent characters. The [3H]-thymidine involving experiments

shown that EMF at 4 Hz exposure has pronounced

stimulation effect on cell proliferation while 4 Hz MV has

inhibition effect. But at 8–10 Hz, the both EMF and MV

have inhibitory effects on cell proliferation. It is suggested

that 4 and 8 Hz EMF have different biological effects on

microbes.

Keywords MV at IS frequency � ELF-EMF � Escherichia

coli K-12 � Cell division � Motility

1 Introduction

There is vast literature devoted to the effect of physical and

chemical factors on the activity of various organisms,

whereas information on the effect of MV and ELF-EMF on

living organisms is limited. Depending on the frequency,

MV and ELF-EMF have stimulating or depressing effect

on the growth and division of microorganisms. However,

the mechanism by which MV and ELF-EMF affect the

bacteria cell is not clear yet.

Numerous hypotheses on the mechanisms of the bio-

logical effects of non-ionizing radiation (NIR) on cells and

organisms have been proposed. One of the most popular

hypotheses is the so-called ‘‘Water Hypothesis.’’ Since

water is a dominant component of environmental medium

and biological systems, and its molecule ionization is

extremely sensitive to different environmental factors, it

was suggested as a sensor for NIR, through which its

biological effects are realized (Klassen 1982; Ayrapetyan

and Markov 2006; Gudkova et al. 2005; Voikov 2006;

Chaplin 2011).

Previously it was shown that MV and ELF-EMF have

frequency-dependent effect on physicochemical properties

of water and water solutions. At frequency of 4 Hz the

effects were more pronounced (Stepanyan et al. 1999). It is

V. Martirosyan � S. Ayrapetyan (&)

UNESCO Chair in Life Sciences International Postgraduate

Educational Center, 31 Acharyan St., 0040 Yerevan, Armenia

e-mail: info@biophys.am

L. Markosyan � H. Hovhanesyan

Institutes of Microbiology of SPC Armbiotechnology,

NAS of Armenia, Yerevan, Armenia

K. Hovnanyan

Institute of Molecular Biology, Yerevan, Armenia

123

Environmentalist

DOI 10.1007/s10669-011-9365-2

Author's personal copy

known that such treatments of water lead to changes of the

CO2 solubility in water and at the presence of oxygen it

promotes the formation of reactive oxygen species (ROS),

such as singlet oxygen (1O2), super oxide radical (O2-),

peroxide anion (O2-2), hydroxyl radical (OH�) and hydro-

gen peroxide (H2O2), through which the modulation of cell

metabolic activity takes place (Klassen 1982; Chaplin

2011). To check this hypothesis, the effect of low fre-

quency of MV and EMF (2, 4, 6, 8, 10 Hz frequencies,

30 dB intensity and B = 0.4 mT intensity correspondingly,

for 30 min) on the bacterial growth and division was

studied. As the non-ionizing radiation leads to formation of

H2O2 and decreases the CO2 solubility in aqua medium, we

have decided to check the morphological changes of bac-

terial cells under the action of these biocides and visual-

izations of the ultra structural mechanisms actions of these

substances on E. coli K-12.

The biological effects of electromagnetic fields have

been studied, particularly on cells (mammalian, T-lym-

phocytes, tissue, tumors), biomolecules, chemical reactions

(influencing the electronic spin states of reaction interme-

diates) and microorganisms, as well as on other animal and

human cells (Polk and Postow 1996) and more recently, the

interaction with cell membranes (Baureus et al. 2003). It

has been reported that the stimulation or inhibition of the

growth of bacteria species and yeasts was dependent on the

field strength, frequency and bacterium kind (Moore 1979;

Ceon et al. 1987; Chacon et al. 1996; Ivanova et al. 1996;

Rao et al. 1997; Fojt et al. 2004).

On the other hand, the living cells are under continuous

effects of internal and external micro-mechanical factors

(such as gravity, tension, pressure and shear), intensities of

most of which are changed within the range of IS fre-

quency. At the cellular level, IS vibration (or MV at IS

frequency) leads to the changes of intracellular ion

homeostasis (Pei et al. 2006; Wei et al. 2007), enzyme

activities (Dang et al. 2007; Steplewski et al. 1964),

cytoskeleton organization, cell shape (Zhu et al. 2006),

growth (Wang et al. 2002), division (Naruse 2002), pro-

liferation (Wang et al. 2002), locomotion and survival.

So, the study of the action of MV and ELF-EMF on the

activity of microorganisms is of particular interest, since

microorganisms are very sensitive to various physical and

chemical factors.

2 Materials and methods

2.1 A special setup for the treatment of the bacterial

culture by ELF-EMF and MV

A glass test tube (1) by 7 cm in diameter and 50 ml volume

was fixed in the holder of the vibrator (2) as MV at IS

frequency source, and placed in the coil as ELF-EMF

source. The vibrator (IS) and the coil were controlled by

the generator (4) and the signal went to the double pole

switch (6): in the 1st position the generator functions as

EMF, while in the 2nd position as MV source (Fig. 1). To

obtain IS waves, the vibrating device was used for gener-

ating vertical vibrations by set frequency and intensity. To

keep the vibration intensity constant (30 dB) at different

frequencies, a coil with a feedback amplifier system (3)

was used. EMF was generated by the controlled generator

and low-noise amplifier on the coil (5). The coil consists of

Helmholtz rings generating the homogeneous magnetic

field. Helmholtz rings are formed by two equal ring coils,

located coaxially, and parallels. The magnetic field created

by these rings had high homogeneity.

To study the affect of MV and EMF on bacterial cells,

two types of experiments have been setup: in the first setup,

1 ml bacteria culture (1010 cells) with NEB (nutrient-

enriched broth-media, 30 ml) have been treated together,

and in second setup, NEB (30 ml) has been treated during

30 min in advance and then 1 ml bacterial culture (1010

cells) was added to the medium. In both setups, the con-

centration of cells in suspensions was evaluated by plating

them on complete agar medium at certain time intervals.

The cell proliferation was investigated by radioactive

thymidine assay.

The motility of cells was measured using two commu-

nicating cuvettes (10 9 10 9 40 mm), which are com-

monly employed for measuring optical densities (Fig. 2).

One of the cuvettes contained nutrient medium with the

MV and EMF-pretreated cells (in the control experiment,

Fig. 1 The schematic diagram of a device for investigation of MV

and ELF-EMF treatments. 1 Glass test tube by diameter of 7 cm and

volume of 50 ml. 2 Vibrator with the coil of feedback coupling. 3 The

low-noise amplifier. 4 The generator of a harmonic field. 5 The coil:

Rings of Helmholtz. 6 The switch (has 2 positions: I and II, where

I- EMF and II- MV)

Environmentalist

123

Author's personal copy

cells were untreated). The other cuvette contained the same

nutrient medium but without cells; changes in the optical

density of this medium were recorded at 600 nm for

60 min. The media in the cuvettes were linked through a

polyethylene tubing 2 mm in diameter filled with the

nutrient medium. Cells from the first cuvette could pass to

the nutrient medium of the second cuvette, thereby

increasing its optical density. The small sizes of the tubing

and cuvettes served to minimize hydrodynamic flows in

them. The reproduction of the cells that arrived to the

second cuvette could not significantly influence the result

of the experiment, since the conditions of cell reproduction

in the both cuvettes were the same.

2.2 Experimental setup and apparatus for CO2

and H2O2 treatments

The processing apparatus used in this work contained 50

liter CO2 vacuum tank, the airline tubing with regulator

valve and the CellStar CO2 incubator (USA, Model:

SWJ500TV BA), where the samples (100 ml) were located

for CO2 exposure. The experimental tubes containing

bacterial culture were placed in the CellStar incubator at

the beginning of each experiment, and the temperature was

kept constant (20�C) in this incubator. Then, carbon

dioxide (air liquid, 99.7% pure) was injected into the

incubator at the selected pressure (atmospheric pressure).

All the samples, which were incubated in CellStar incu-

bator, were treated by CO2 for 10 min at 20�C. The

appropriated control tube was also placed in the same CO2

incubator. After CO2 treatment the samples were closed

and removed to thermostat at 37�C, 4 h for the future

processing.

Since CO2 dissolves in aqueous solutions, to form an

acid we need to keep constant the level of pH in order to

avoid the effects of latter on bacterial killing processes. We

kept the level of pH constant by adding NaOH (0.1 mol/l)

during CO2 treatment. It should be mentioned that we kept

the value of pH constant (pH = 6.0), because in case of

this value the growth of E. coli bacterial culture was

maximal (Presser et al. 1997).

For hydrogen peroxide challenge, a fresh hydrogen

peroxide solution was prepared immediately before starting

the experiments and added to the bacterial culture bringing

to the corresponding concentration (5.2 mM) of H2O2 in

aqua media. Samples were incubated for at 20�C, 4 h for

the future processing.

2.3 Bacterial strain, culture media

All experiments were performed on the wild type of E. coli

K-12, which was obtained from the Armenia Collection of

Microorganisms (Institute of Microbiology of the Center of

Microbiology and Microbial Depository of Armenian

NAS). Prior to each test, the bacterial cultures (previously

stored at 4�C in nutrient-enriched agar slants (NEA, con-

taining: NEB ? 1.5% agar (Difco), final pH 7.1 ± 0.2 at

37�C) were grown in nutrient-enriched broth (NEB, con-

taining: peptone 15 g/l, sodium chloride 6.0 g/l, yeast

extracts 3.0 g/l, final pH 7.5 ± 0.2 at 37�C), at 37�C in

thermostat for overnight (18 h), which were used for the

future treatments.

2.4 Enumeration of living microorganisms

The viability of E. coli K-12 was determined by counting

the number of colony forming units (CFUs) per milliliter.

Measuring techniques involved indirect viable cell counts,

also called plate counts, which was performed by plating

out (spreading) a sample of a culture on a nutrient agar

surface by triplicate plating. Each treated sample (5 ll)

was diluted with 495 ll of an aqueous NEB solution before

plating. The contents of the tubes were then shaken well by

magnetic stirring for two min. By shaking the tubes the

microorganisms dispersed in the solution. The dispersion

obtained was then diluted repeatedly, and 100 ll of the

appropriate dilution was plated on triplicate petri dishes

containing a suitable growth medium. After plated on a

suitable medium and after incubation in thermostat (37�C),

each viable unit grew and formed a colony and the number

of CFUs was related to the viable number of bacteria in the

sample. Microbial cells in the control samples were

Fig. 2 Communicating cuvettes for the estimation of cell motility:

1 Cuvette with the culture tested for cell motility. 2 Cuvette with the

nutrient medium whose optical density, measured at 600 nm,

increased as motile cells passed from cuvette 1. 3 Tubing with the

nutrient medium linking the cuvettes

Environmentalist

123

Author's personal copy

counted by the same procedures described for treated

samples.

2.5 Electronmicroscopy assay

The methods of negative contrasting were done by 2%

phosphoric-tungsten acid solution at pH 6.8–7.0. For pre-

paring the electronic microscopic preparations of bacteria

suspensions after centrifugation at 3,000 rpm (CLR-1)

during 10 min bacterial sediment was fixed by 2.5%

solution of glutaraldehyde in 0.1 M cacodylate buffer

under pH 7.4 during 2 h under room temperature. After

three-phase washing in 0.1 M cacodylate buffer was fixed

under room temperature by 1% of solution tetraoxide

osmium in 0.1 M cacodylate buffer under pH 7.4 during

not less than 1 h. After washing in the same buffer,

dehydration of sample was conducted in the ethanol

increasing concentrations for 30, 50, 70, 96 and 100% or

acetone increasing concentrations and potting by mixture

of Araldite on Lufft. The bacterial sediment after poly-

merization in thermostat at 37� and 59�C, with further

dehydration, saturating was polymerized in Araldite prep-

arations. Ultrathin sections preparation was conducted by

using the ultra cut ‘‘Reichert Ultracut-Young.’’ Ultrathin

sections were stained in the uranyl acetate and lead citrate

(Reynolds-Veinable). These were observed with electronic

microscopes Tesla BS-500 (Czechoslovakia). The photo-

micrographies were scanned in permit of 900 pixels for

inch and processed on computer programme Corel Draw

versions 11 and Photoshop versions 8. Computer mor-

phometric and stereometric analysis of electronic micro-

scopic imaging was performed according to the program

‘‘Video Test Structure-5, nanotechnology.’’

2.6 Radioactive labeled [3H]-thymidine-based cell

proliferation assay (liquid scintillation counter)

For the measurement of the cell proliferation the bacteria

cultures were grown in 100 ml of NEB, overnight 18 h at

37�C. Then, 1 ml of overnight culture (1010 cells) was

added to the 30 ml NEB, in which content is 40 ll [3H]-

thymidine (1.3 lCi/ml) ([6-3H]-thymidine, PerkinElmer,

Boston, MA, 14.4 Ci/mmol specific activity). [3H]-thymi-

dine containing samples were treated by MV and ELF-

EMF at 30 min and then incubated in thermostat at 37�C,

for 18 h. The cells were then harvested by centrifugation

for 10 min at 5,000g. [3H]-thymidine uptake was stopped

by precipitation of each portion with ice-cold 10% tri-

chloroacetic acid (TCA), washed 2 times and then again

harvested by centrifugation. Then, 3 ml of Bray’s scintil-

lation cocktail counting solution was added to each sample

and the radioactivity was measured by using a Wallac

1,450 liquid scintillation counter.

2.7 Statistical analysis

Statistical analyses were conducted by SPSS 17 software.

The differences among the means of treatment were tested

by using the paired sample t test and expressed in the

figures with the help of asterisks (*). All experiments were

repeated at least three times. Values shown below are the

means of these experiments ± SE.

3 Results

To investigate the effect of 30-min exposure of MV and

ELF-EMF (2, 4, 6, 8, 10 Hz frequency, 30 dB intensity and

B = 0.4 mT intensity correspondingly) on the bacterial

growth, the bacterial cell proliferation was studied by using

radioactive [3H]-thymidine assay and by calculating a CPM

(count per minute) of each sample by liquid scintillation

counter (Fig. 3).

As the frequency ‘‘windows’’ are the 4 and 8 or 10 Hz,

the next series of experiments have been done at these

frequencies.

The classical microbiological study of the bacterial

growth was shown that 0.4 mT EMF at 4 and 10 Hz has

inhibition effect of cell growth (Fig. 4). Similar effects

were observed when EMF-treated nutrient medium was

inoculated with untreated cells. At 4 Hz EMF espoused

nutrient medium 20% of cells survived, while in the case of

10 Hz the survival increased up to 30%.

At these frequencies of EMF treatment, the cells lost

their capacity to divide and to grow as filaments, i.e., they

were unable to form colonies on the solid media. The

microscopic study of microbe cultures showed that

Fig. 3 Frequency-dependent effect of EMF and MV treatment of the

bacterial growth. In the graph, the bacteria culture treated with culture

media marked as EMF, MV and beforehand treated culture media

marked as EMF-M and MV-M. The incubation time after treat-

ments was 18 h. Values in the graph are the means of these

experiments ± SE

Environmentalist

123

Author's personal copy

significant number of cells treated by EMF lost their ability

to divide and formed long filaments (Fig. 5). These fila-

ments were much longer than the ones in control.

The classical microbiological study of the bacterial

growth has shown that 30 dB MV at 4 Hz has stimulation

effect of cell growth (Fig. 6). The exposure time of the

treatment was 30 min. Namely, 4 Hz vibrations induced a

threefold increase in the number of cells as compared to the

control (p \ 0.01); 10 Hz vibrations did not induce any

significant change in the number of cells (Fig. 6a). After

3 h of incubation under stationary conditions (Fig. 6b),

4 Hz exposed culture showed better growth than the 10 Hz

exposed culture.

Bearing in mind that MV can affect cells not only

directly but also indirectly (through changes in the med-

ium), we attempted to evaluate the medium-mediated

effect of MV on the cells. For this, the NEB pretreated with

MV was inoculated with intact cells and incubated at 37�C

(Fig. 7). After 30 min of incubation, the concentration of

cells did not significantly change (Fig. 7a). However, after

3 h of incubation (Fig. 7b), the NEB pretreated with 4 and

10 Hz vibrations provided for, respectively, 1.75- and

*1.5-fold better growth than the control medium

(p \ 0.05 for the experiments with both frequencies).

The microscopic observation of the cells exposed to MV

either directly or indirectly (through the treatment of the

culture medium) showed a drastic increase in their motility.

The treatment of cultures with 4 and 10 Hz vibrations

stimulated the fragmentation of the cell filaments sponta-

neously. It should be noted that filament cells, which are

50–100 times as long as normal cells and comprise 1–2%

of the population of the culture, can also contribute to the

increase in the culture density in response to exposure to

MV. The division of filament cells exposed to 4 Hz

vibrations was found to increase the culture density by

1.5–2.0 times.

Changes in the motility of cells exposed to MV were

also studied in the device described above by measuring

the optical densities of cell suspensions in the cuvettes

(Fig. 8). The measurements showed that the MV-treated

cells more rapidly migrated from cuvette 1 to cuvette 2

than did the control cells. The migration rate of cells’ was

particularly higher 60 min after their exposure to 4 Hz

vibrations (p \ 0.01).

As follows from the experimental data presented, MV

substantially influenced the growth rate and the division of

bacterial cells through both the direct and indirect (med-

ium-mediated) action on the cells. In both cases, MV

stimulate (4 and 10 Hz vibrations) the growth of E. coli

cells. When the cells were exposed to 4 Hz vibrations for

30 min, the culture grew three times faster than the control

culture. Hence, the culture treated with 4 Hz vibrations for

30 min at 37�C produced 2–3 new cell generations, while

the untreated culture produced no more than one new cell

generation over the same time period.

It should be noted also that the main reason of the above

mentioned changes of behaviors of microbial cells are

conditioned by physical properties of water. Thus, as has

been mentioned above, MV and ELF-EMF could change

the structure and ionization of water molecule (Klassen

1982; Stepanyan et al. 1999; Gudkova et al. 2005; Ayra-

petyan and Markov 2006; Voikov 2006). Such treatments

change the solubility of CO2 in water and promote the

formation of reactive oxygen species and H2O2, through

which the biological effects of this treatment are realized

Fig. 4 Quantity of E. coli K-12 able to form macro colonies under

the influence of ELF-EMF

Fig. 5 The morphological changes of E. coli K-12 cells after ELF-EMF treatment

Environmentalist

123

Author's personal copy

(Chaplin 2011). So, it is interesting to find the effect of

H2O2 and CO2 on the cell morphological changes at the

same time to find the indirect effect of these treatments on

the cells.

The electronmicroscopic study of the action of CO2 and

H2O2 on the E. coli K-12 there was used the methods of

negative contrasting and ultrathin section.

Electronmicroscopic study of the culture of the E. coli

K-12 has shown that cells have an ultrastructure, typical for

gram-negative bacteria (Fig. 9a, b).

Ultrastructural analysis of E. coli K-12 has shown that

effect of different biocides: such as CO2 and H2O2 lead to

the ultrastructural changes: bacterial cells membranes,

cytoplasm and nucleotide. Electronmicroscopic study

action of CO2 on the E. coli K-12 leads to the destruction of

cellular wall and the removal of external membrane of wall

of bacteria (Figs. 10a, b, 11a–c).

It was discovered that the bactericide high activity of

H2O2 and its derivatives leads to the ultra structural

changes: removal of external membrane of wall and

Fig. 6 The effect of the 30-min exposure of cells to different

vibrations on their reproduction: (a) the cell concentration was

estimated immediately after the ending of the MV treatment and

(b) 3 h afterward. The number of cells in the control culture (C) was

arbitrarily taken to be unity

Fig. 7 The effect of the MV-pretreated nutrient medium on the

reproduction of bacterial cells: the concentration of cells was

estimated (a) 30 min and (b) 3 h after they were placed in the

MV-treated medium. The number of cells in the control culture

(C) was taken to be unity

Fig. 8 Changes in the optical density of medium in cuvette 2 caused

by the passage of motile cells from cuvette 1 with the MV-treated or

control (C) culture

Fig. 9 Transmission of electronmicroscopy (TEM). Intact cell of

E. coli K-12: (a) negative staining, (b) ultrathin section. Scale bar(a, b) = 1 lm

Environmentalist

123

Author's personal copy

increase of the transparency of cytoplasmatic membrane

(Fig. 12a, b).

4 Discussion

It has been shown that MV and ELF-EMF (2, 4, 6, 8, 10 Hz

frequencies, 30 dB intensity and B = 0.4 mT intensity

correspondingly, for 30 min) on the bacterial cell prolif-

eration has stimulation or inhibition effect depending on

the frequency of these treatments (Fig. 3).

It has been shown that 2 Hz ELF-EMF and MV stim-

ulate bacterial growth (Fig. 3). But in the case of MV the

treatment of the culture media has stimulation effect

(7.0%), while in case of EMF exposure the preliminary

treatment of the culture media shows the inhibition effect

on the cell proliferation (25.8%). It meant that the nature of

the stimulation at 2 Hz is different: probably the stimula-

tion by MV leads to the direct and non-direct effect by

changes the physicochemical properties of culture media,

and stimulation by EMF leads to the direct effect on bac-

teria themselves (Fig. 3).

At 4 Hz MV treatment has observed an inhibition effect

and EMF has shown stimulation effect on the bacterial

growth (Fig. 3). The preliminary treatment of the bacterial

media by MV (12.6%) and EMF (77.2%) has inhibition

effect on the cell proliferation (Fig. 3), but in both cases

probably the effect of 4 Hz is directly on bacterial culture.

At 6 Hz MV and EMF treatments stimulation effects

had been observed (Fig. 3). The preliminary treatment of

the bacterial media in MV (10.6%) and EMF (8.6%) has

inhibition effect on the cell proliferation (Fig. 3), and in

both cases, probably the effect of 6 Hz is directly on bac-

terial culture.

At 8 Hz MV and EMF treatments have shown inhibition

effect (Fig. 3). The preliminary treatment of the bacterial

media in MV (34.3%) and EMF (13.1%) has stimulation

effect on the cell proliferation (Fig. 3). It meant that the

nature of the inhibition at 8 Hz is the same in both cases:

probably the inhibition by MV and EMF leads to the direct

and non-direct effect by changes the physicochemical

properties of culture media (Fig. 7).

At 10 Hz MV and EMF treatments have shown inhibi-

tion effect (Fig. 6). The preliminary treatment of the bac-

terial media in MV (3.4%) and EMF (18.6%) has

stimulation effect on the cell proliferation (Fig. 7). And the

data are similar to the 8 Hz, it means that MV and EMF at

10 Hz have direct and non-direct effect to the bacterial

culture.

So, it was found that, depending on their frequency, MV

and ELF-EMF can either stimulate or inhibit the growth of

E. coli K-12.

The nature of MV- and ELF-EMF-treated nutrient

medium was not always similar to the one observed in the

case of the exposure of microbes themselves (Figs. 4, 6, 7,

8, 9).

The performed investigation demonstrated that EMF

leads to frequency-dependent inhibition of the growth

(CFUs) and division of E. coli K-12 cells. The effect of

EMF-treated nutrient medium was similar to the one

observed in the case of the exposure of microbes them-

selves (Fig. 4). The obtained data are interpreted from the

point of view of the factor-induced water structure changes

of cell bathing aqua solutions.

It is well documented that ELF-EMF and MV have a

specific frequency and modulation ‘‘windows,’’ which in

turn depend on the chemical and physical composition of

environmental medium and initial states of water and

organisms (Adey 1981; Belyaev 2005).

In the present work, 4 and 8 or 10 Hz are the main

‘‘windows’’ that have shown biological effects (Figs. 3, 4,

6, 7, 8, 9).

It is concluded that the ultrastructural visualizations

could provide valuable data for correction of cytopatho-

logic characteristics of mechanism of the biocides: CO2,

H2O2 actions on E. coli K-12, which is provided the indi-

rect effect of these treatments (EMF and MV) (Figs. 9, 10,

11, 12).

So, in this study, as the main windows are 4 (direct

effect) and 8 or 10 Hz (non-direct and direct effects) and

Fig. 10 TEM. The action of

CO2 on the E. coli K-12.

Negative staining (a). Ultrathin

section (b): the external

membrane of the cellular wall

with expansion of the

periplasmatic dimension has

been taken. Scale bar(a, b) = 1 lm

Environmentalist

123

Author's personal copy

they have different nature of biological effects on bacterial

culture, it is suggested that the frequency-depending

stimulation or inhibition effects of MV and ELF-EMF on

the growth of E. coli K-12 cannot be explained by the

changes in the aqua media and it is not only the mechanism

which is in response for this kind of treatments and the

treatments have also direct effect to microbes.

Acknowledgments We thank to Ms. Anna Abrahamyan for her

technical assistance.

References

Alvarez DC et al. (2006) Process Biochemistry. 41:1967–1973

Adey W (1981) Tissue interactions with non-ionizing electromagnetic

field. Physiol Rev 61:435–514

Ayrapetyan S, Markov M (2006) Bioelectromagnetics current con-

cepts: the mechanisms of the biological effect of extremely high

power pulses. Springer, Berlin

Baureus K, Sommarin M, Persson B, Salford L, Eberhardt J (2003)

Interaction between weak low frequency magnetic fields and cell

membranes. Bioelectromagnetics 24:395–402

Belyaev I (2005) Non-thermal biological effects of microwaves.

Microw Rev 11:13–29

Blackman C, Benane S, Elliott D, Pollock M (1988) Influence of

electromagnetic fields on the efflux of calcium ions from brain

tissue in vitro: a three-model analysis consistent with the frequency

response up to 510 Hz. Bioelectromagnetics 9:215–227

Ceon R, Martin J, Powell M (1987) Low-level, magnetic-field-

induced growth modification of Bacillus subtilis. Bioelectro-

magnetics 8:275–282

Chacon D, Haber V, Fong A, Mas S, Serguera M, Rodriguez O (1996)

Influence of the electromagnetic field in the growth of Candidautilis Y- 660 yeast. Tecnol Quım 16:52–60

Chaplin M (2011) Water structure and science. http://www.lsbu.ac.

uk/water/

Dang W, Wang S, Tian S, Chen B, Sun F, Li W, Jiao Y, He L (2007)

Effects of infrasound on activities of 3beta hydroxysteroid

dehydrogenase and acid phosphatase of polygonal cells in

adrenal cortex zona fasciculate in mice. Zhonghua Lao Dong

Wei Sheng Zhi Ye Bing Za Zhi Feb 25(2):91–95

Fojt L, Strasak L, Vetterl V, Smarda J (2004) Comparison of the low-

frequency magnetic field effects on bacteria Escherichia coli,Leclercia adecarboxylata and Staphylococcus aureus. Bioelec-

trochemistry 63:337–341

Gudkova O, Gudkov S, Gapeyev A, Bruskov V, Rubanik A, Chemeris

N (2005) The study of the mechanisms of formation of reactive

oxygen species in aqueous solutions an exposure to high peak-

power pulsed electromagnetic radiation of extremely high

frequencies. Biofizica 50:773–779

Ivanova V, Hristov J, Dobreva E (1996) Performance of a magnet-

ically stabilized bed reactor with immobilized yeast cells. Appl

Biochem Biotechnol 59:187–198

Klassen V (1982) Magnetized water systems. ‘‘Chemistry’’ Press,

296 pp (in Russian), English translation: European Biology and

Bioelectromagnetics (2006): 201–220

Kohane M, Tiller W (2000) Energy, fitness, and information-

augmented electromagnetic fields in Drosophila melanogaster.

J Sci Explor 14(2):217–231

Lei C, Berg H (1998) Electromagnetic window effects on prolifer-

ation rate of Corynebacterium glutamicum. Biolectroch Bioener

45:261–265

Moore R (1979) Biological effects of magnetic fields: studies with

microorganisms. Can J Microbiol 25:1145–1151

Nakasono S, Saiki H (2000) Effect of ELF magnetic fields on protein

synthesis in Escherichia coli K12. Radiat Res 154:208–216

Fig. 11 TEM. Action of CO2 on E. coli K-12: (a) negative contrasting, (b) the inner cellular contact with the external membrane of cellular wall

of E. coli, (c) ultrathin section. Scale bar 1 lm (a, b, c)

Fig. 12 TEM. The effects of

H2O2 on E. coli K-12. Negative

contrasting (a) and ultrathin

section (b): the external

membrane cellular wall with

expansion of periplasmatic

space has been taken. Scale bar(a, b) = 1 lm

Environmentalist

123

Author's personal copy

Naruse Y (2002) Mechanical vibration model for chromosomes in

metaphase of mitosis and possible application to the interruption

of cell division. Biosystems 66:55–63

Pei Zh, Sang H, Li R, Xiao P, He J, Zhuang Zh, Zhu M, Chen J, Ma H

(2006) Infrasound-induced hemodynamics, ultrastructure, and

molecular changes in the rat myocardium. Environ Toxicol

22(2):169–175

Polk C, Postow E (1996) Handbook of biological effects of

electromagnetic field, 2nd edn. CRS Press, New York

Presser K, Ratkowsky D, Ross T (1997) Modeling the growth rate of

Escherichia coli as a function of pH and lactic acid concentra-

tion. Appl Environ Microbiol 63(6):2355–2360

Rao T, Sonolikar R, Saheb S (1997) Influence of magnetic field on the

performance of bubble columns and airlift bioreactor with

submerged microorganisms. Chem Eng Sci 52:4155–4160

Stepanyan R, Ayrapetyan G, Arakelyan A, Ayrapetyan S (1999)

Effect of mechanical oscillations on electrical conductivity of

water. Biophys J 44:199–204

Steplewski Z, Stoklosa E, Brazegowy A (1964) On the effect of

horizontal mechanical vibrations on the enzyme activity of the

reticuloendothelial system (RES) in the liver of white rats.

PubMed 5(20):580–586

Voikov V (2006) Biological significance of active oxygen-dependent

processes in aqueous systems. In: Pollack G, Cameron I,

Wheatley D (eds) Water and the cell. Springer, Dordrecht,

pp 285–298

Wang B, Long X, Liu Y, Duan C, Sakanishi A (2002) The effects of

mechanical vibration on the microstructure of Gerbera jamesonii

acrocarpous callus. Colloids Surf B Biointerfaces 23(1):1–5

Wei Y, Liu J, Shu Q, Huang X, Chen J (2007) Effects of infrasound

on ultrastructure of testis cell in mice. Environ Toxicol

Pharmacol 23(1):111–114

Zhu W, Zhou N, Qu J, Xu W, Kong L (2006) Effects of mechanical

vibration on cell density and cell morphology in the dynamic

microcellular foaming process. J Cell Plast 13:42–49

Environmentalist

123

Author's personal copy