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50 Hz magnetic field effect on the morphology of bacteria
Micron 40 (2009) 918–922
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Short communication
50 Hz magnetic field effect on the morphology of bacteria Luka´sˇ Fojt a,*, Petr Klapetek b, Ludeˇk Strasˇa´k a, Vladimı´r Vetterl a a b
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kra´lovopolska´ 135, Brno 612 65, Czech Republic Czech Metrology Institute, Okruzˇnı´ 31, Brno 638 00, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 April 2009 Received in revised form 19 June 2009 Accepted 20 June 2009
Atomic force microscopy was used to distinguish changes in morphology of bacteria induced by 50 Hz 10 mT magnetic field exposure. It is known that alternating magnetic field exposure causes decrease of viability of different bacterial strains. Previously we found that the viability of rod-like bacteria exposed to magnetic field decreased twice more in comparison with the spherical ones. Motivated by this fact we carried out this study with bacterial cells of both shapes. We used Escherichia coli (rod-like) and Paracoccus denitrificans (spherical) bacteria. As a result we have not observed any change in bacterial morphology neither of rod-like nor of spherical bacteria after 1 h, 50 Hz and 10 mT magnetic field exposure. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: AFM Magnetic field Bacteria
1. Introduction It becomes very important to find out electromagnetic fields environmental and health impact in last years. It is caused by overwhelming spreading of electric and electronic devices and wireless technologies all over the world. It is necessary to distinguish and state safety limits for electromagnetic fields exposure more precisely (we hope that our results might help to it). Lot of studies has been carried out concerning different types of electromagnetic fields from microwaves to static magnetic field effects. Different biological effects were detected such as elevated level of DNA strand brakes in rat brains after low frequency magnetic field exposure (Singh and Lai, 1998; Lai and Singh, 2004) or microwave exposure (Paulraj and Behari, 2006; Lai and Singh, 1996). Increase of DNA strand brakes of fibroblasts and leukaemia cells after 50 Hz magnetic field exposure has been reported in (Wolf et al., 2005). Biological effects on gene transcription, changes in DNA synthesis and transcription have been studied (Repacholi and Greenebaum, 1999; Blank and Goodmann, 2001). Effect of 50 Hz magnetic field on Ca2+ ion channels is discussed in Pessina et al. (2001) and Grassi et al. (2004). Electromagnetic fields affect various enzymatic processes, such as decrease of adenylate kinase activity (Ravera et al., 2004) or decrease of glucose stimulated insulin secretion (Sakurai et al., 2005). In contrary to publications where electromagnetic fields effects are claimed there do exist plenty of studies that do not observe any changes in studied objects. The observation of Lopucki et al. (2005) could be used as an
example where no change in oxidative DNA damage after 50 Hz magnetic field exposure was found. No evidence of DNA strand brakes in rat brain cells after microwave irradiation was reported (Malyapa et al., 1998). Unicellular organisms as experimental subjects are useful tool for electromagnetic fields effects studying (Berg, 1999; Markov et al., 2004). Viability of bacteria (Strasˇa´k et al., 2002, 2005; Babushkina et al., 2005) and yeasts (Ruiz-Go´mez et al., 2004; Nova´k et al., 2007) has been studied. We found that different types of bacteria are affected by the 50 Hz 10 mT magnetic field according to their shape in our previous studies (Fojt et al., 2004, 2007; Strasˇa´k et al., 2005). Gram-negative Escherichia coli and Leclercia adecarboxylata – rod-like bacteria – achieved about 60–70% of CFU number (colony forming units, denotes number of living bacteria able to form a colony) after exposure compared to the control ones. For Gram-positive Paracocuccus denitrificans and Staphylococcus aureus – spherical bacteria – there was about 20% decrease in CFU number. Provoked with this fact we decided to study bacterial morphology after 50 Hz 10 mT magnetic field exposure. Atomic force microscopy (AFM) could be used as a powerful tool for biological objects sensing. Different biological structures were imaged using AFM, like DNA, membranes, photosystems (Fotiadis et al., 2002) and unicellular organisms (Dufrene, 2002; Robichon et al., 1999). AFM was used to study the changes in lymphoblastoid cells under 50 Hz 2 mT magnetic field exposure (Girasole et al., 1998). We used representative of two morphological bacterial groups – E. coli and P. denitrificans for our study. 2. Materials and methods
* Corresponding author. Tel.: +420 541517261; fax: +420 541211293. E-mail addresses: [email protected] (L. Fojt), [email protected] (P. Klapetek), [email protected] (L. Strasˇa´k), [email protected] (V. Vetterl). 0968-4328/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2009.06.009
Magnetic fields were generated in a cylindrical coil (Institute of Scientific Instruments, ASCR, Brno, Czech Republic) powered by an
L. Fojt et al. / Micron 40 (2009) 918–922
919
Santa Barbara, USA) in contact mode and than processed with ˇ MI, Brno, Czech Republic) using subtraction Gwyddion software (C and correlation for to distinguish changes between exposed and control samples. 3. Results
Fig. 1. Dependence of the magnetic field induction Bm in the coil on the distance from the coil axis (which is situated at r = 0 mm) for different current values. Solid line denotes position of Petri dishes, dashed line position of microscopic slides.
autotransformer (50 Hz power net supply, sinusoidal waveform), as described previously (Fojt et al., 2004). The maximum current amplitude at 50 Hz in the coil was Im = 1.9 A, corresponding to a maximum achievable magnetic field amplitude of Bm = 10 mT. The current was checked during the measurements using HC3500-T Multimeter (HC, Seoul, South Korea) and the frequency by oscilloscope PM 3266 (Philips, Eindhoven, The Netherlands). For exposure, the samples were placed in the centre of the coil where uniformity of the magnetic field is optimal (about 5%). For magnetic field distribution inside the coil for different current values see Fig. 1. Magnetic induction was measured using Hall probe, lower magnetic induction levels (under 2 mT) were also checked by magnetic field meter BMM 3000 (EnviroMentor, Go¨teborg, Sweden). Further coil details can be found in Fojt et al. (2004). The temperature inside the coil was measured by thermometer and was maintained by an airflow to laboratory value (both samples, control and exposed ones, were at the same temperature). Control samples were placed away from the coil to avoid magnetic field exposure and one part underwent shamexposure. Both samples (control and exposed ones) originate from the same bacterial culture. Background magnetic field at 50 Hz was determined as 800 nT. Bacteria were exposed either in Petri dishes (diameter of 90 mm) or on microscopic slide on AFM sample holder. The bacteria E. coli (strain K12, Row, genotype 58–161 metB1rpsL 1+ FdefP.Fredericq) were grown in TY broth (8 g tryptone, 5 g yeast extract (Oxoid, UK); 5 g NaCl (Lachema Brno, Czech Republic) diluted in 1 l distilled water). We used basic nutrient agar number 2 (40 g/l, Imuna Sˇarisˇske´ Michal’any, Slovak Republic) as a solid cultivation medium. P. denitrificans CCM 982 (NCIB 8944) were grown in liquid and solid cultivation medium prepared according Kucˇera and Kaplan (1996). Bacteria for AFM measurements were grown for 12 h on agar plates (until the colonies had become visible, bacteria were in log phase of their growth). Two types of 10 mT, 50 Hz and 60 min magnetic field exposures were done: (1) Bacteria were exposed to magnetic field on agar plates and after exposure they were removed to microscopic slide for AFM imaging. (2) Bacteria were exposed directly on microscopic slide (which enables to take AFM picture on the same place before and after exposure). Slide was prepared by covering the glass surface with homogenous layer of bacteria collected from agar plates. Each of the experiment type was at least five times repeated in lower (circa 50 mm 50 mm) and higher (circa 3 mm 3 mm) resolution for both bacterial strains. Scanning region was selected randomly. Data were collected using AFM Explorer head (Veeco,
Both types of used bacterial strains were exposed to 10 mT 50 Hz magnetic field for 60 min. When were bacteria exposed on agar plates (magnetic field exposure 1), only overall information of magnetic field influence on bacterial morphology was acquired. It is not possible to find the same bacteria on images before and after magnetic field treatment. This makes direct comparison between exposed and control samples difficult. We exposed bacteria on microscopic slides (magnetic field exposure 2) to solve this problem. Bacteria grown on agar plates were removed carefully to microscopic slides. In first experimental setup, we made analogous exposure setting as for magnetic field exposure 1 as follows: part of the slides covered with bacteria was exposed to the magnetic field and then imaged with AFM. The other part was imaged with AFM after 60 min of waiting (control samples). We were able to collect AFM images of unexposed and exposed bacteria from these experiments, with no possibility of precise single bacteria (before and after magnetic field treatment) detecting. This data set provides practically the same images as bacteria exposed on agar plates. For making possible the direct comparison of exposed and control samples, we modified slightly the experimental setup. The slide covered with bacteria was put onto the AFM sample holder immediately. In this time, we made AFM image of unexposed glass slice covered with bacteria. The same slice underwent a magnetic field exposure. AFM was employed again after the exposure. We were able to compare images of the same bacteria before and after magnetic field exposure with these experimental setting, because the bacteria did not change their position related to AFM sample holder, thus not in the image itself. For image data acquired by this way, we made mutual assignment of discrete parts of surface – correlation, to determinate lateral changes in exposed sample. Vertical changes were propagated by subtraction of images. These data are shown in Fig. 2 for E. coli in high resolution and in Figs. 3 and 4 for E. coli and P. denitrificans in low resolution AFM images. Each image is set of unexposed sample, exposed sample, subtracted and correlated data. It is not possible to determine any changes in bacterial morphology after magnetic field exposure in all types of our measurements. 4. Discussion Our study was motivated by our previous results (Fojt et al., 2007) where we found differences between exposed Grampositive (about 20% decrease in CFU number for 10 mT, 24 min 50 Hz magnetic field exposure) and Gram-negative (about 30–40% decrease in CFU number, the same exposure conditions, see Fig. 5) bacterial strains. For data acquisition, we choose AFM due to its unchallenged possibility, to image living objects in higher resolution than common light inverse microscopes. Electron microscopy yields high resolution, but requires work in vacuum, staining and other special treatment (Bolshakova et al., 2001). Even environmental electron microscopy is not possible to image living bacteria. We exposed E. coli and P. denitrificans to the magnetic field and focused our attention to the morphological changes of these bacteria. Bacteria were exposed for 60 min, which is the longest possible exposure time which could be from technical and biological conditions achieved (and according to our previous results yields the strongest effects on bacteria). We observed no change in bacterial morphology using AFM. Small changes – strip-
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Fig. 2. AFM contact mode images of E. coli in high resolution. (A) Bacteria before magnetic field exposure, (B) bacteria after magnetic field exposure, (C) correlation, and (D) subtraction.
like and concentric deformation on subtracted and correlated data (vertical and lateral changes, strip like deformation could be observed mostly on lower resolution AFM pictures, concentric on high resolution AFM pictures), are due to the AFM drift during the
time (it was necessary to move and rotate them slightly to get the same position for the same images). Important is that the subtracted and correlated data do not show any changes in lateral and/or vertical dimension in the centre of the sample where the
Fig. 3. AFM contact mode images of E. coli in low resolution. (A) Bacteria before magnetic field exposure, (B) bacteria after magnetic field exposure, (C) correlation, and (D) subtraction.
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Fig. 4. AFM contact mode images of P. denitrificans in low resolution. (A) Bacteria before magnetic field exposure, (B) bacteria after magnetic field exposure, (C) correlation, and (D) subtraction.
Fig. 5. Comparison of magnetic field effect for E. coli (grey column) and P. denitrificans (white column). Data acquired from Fojt et al. (2004). (A) Dependence of the relative number of CFU on the value of the magnetic field induction for t = 12 min. (B) Dependence of the relative number of CFU on the duration of exposure for Bm = 10 mT.
drift is negligible. Figs. 2–4 show an average of five measurements. All images were done on the air, on the microscopic slides. According to our experiments, the bacteria remain alive after our experimental treatment, and this fact is also supported by Bolshakova et al. (2001). We tried to cut down the effect of bacteria drying by cultivating them on agar plates. Beckmann et al. (2006) had reported that bacteria grown on agar plates are more resistant to drying on air than bacteria cultivated in broth. In the experiments where bacteria were treated similarly to the exposed ones we observed very small changes that can be ascribed to sample drift during time. We know that the oscillating magnetic field affects different bacterial strains (in lag-phase of their growth) from our previous results. This fact was found using CFU counting technique (Fojt et al., 2004), electrochemical determination of enzymatic activity (Fojt et al., 2007) and controlling of growth curves using spectrophotometry (Strasˇa´k et al., 2005). Metabolic activity of used bacteria was not affected. We did not find any changes in
bacterial morphology after magnetic field treatment. It seems that bacteria shape does not play any important role in the interaction with used magnetic field. We will focus on possible mechanisms of magnetic field acting in our next experiments. There are a lot of theories that try to explain acting of magnetic fields on living organisms (Panagopoulos et al., 2002; Berg, 1999). But there is still a confusion about which effect or combination of effects is involved in real processes of electromagnetic fields bioeffects. 5. Conclusions We tried to determine 50 Hz 10 mT 60 min magnetic field morphological effect on two different bacterial strains. We were not able to detect any changes in bacteria surface and/or shape according to our different AFM experiments. Further experiments could be done using the scanning near field optical microscopy which could provide other interesting information for our biological objects.
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Acknowledgements This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (Grant KAN 200040651, KAN 311610701), the Ministry of Education, Youth and Sports (project No. 1M0528 and No. LC06035), grant agency of the Czech Republic (Grant 202/08/1688, Grant 310/07/P480), and by the institutional research plans (AVOZ 50040507, AVOZ 50040702). References Babushkina, I.V., Borodin, V.B., Smetkova, N.A., Morrison, V.V., Usanov, A.D., Skripal, A.V., Usanov, D.A., 2005. The influence of alternating magnetic field on Escherichia coli bacterial cells. Pharm. Chem. J. 39, 398–400. Beckmann, M.A., Venkataraman, S., Doktycz, M.J., Nataro, J.P., Sullivan, C.J., MorrellFalvey, J.L., Allison, D.P., 2006. Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM. Ultramicroscopy 106, 695–702. Berg, H., 1999. Problems of weak electromagnetic field effects in cell biology. Bioelectrochem. Bioenerg. 48, 355–360. Blank, M., Goodmann, R., 2001. Electromagnetic initiation of transcription at specific DNA sites. J. Cell. Biochem. 81, 689–692. Bolshakova, A.V., Kiselyova, O.I., Filonov, A.S., Frolova, O.Y., Lyubchenko, Y.L., Yaminsky, I.V., 2001. Comparative studies of bacteria with an atomic force microscopy operating in different modes. Ultramicroscopy 86, 121–128. Dufrene, Y.F., 2002. Atomic force microscopy, a powerful tool in microbiology. J. Bacteriol. 184, 5205–5213. Fojt, L., Strasˇa´k, L., Vetterl, V., Sˇmarda, J., 2004. Comparison of the low-frequency magnetic field effects on bakteria Escherichia coli, Leclercia adecarboxylata and Staphylococcus aureus. Bioelectrochemistry 63, 337–341. Fojt, L., Strasˇa´k, L., Vetterl, V., 2007. Effect of electromagnetic fields on the denitrification activity of Paracoccus denitrificans. Bioelectrochemistry 70, 91–95. Fotiadis, D., Sheuring, S., Muller, S.A., Engel, A., Muller, D.J., 2002. Imaging and manipulation of biological structures with the AFM. Micron 33, 385–397. Girasole, M., Cricenti, A., Generosi, R., Congiu-Castellano, A., Pozzi, D., Pasquali, E., Lisi, A., Santoro, N., Grimaldi, S., 1998. Atomic forcemicroscopy study of lymphoblastoid cells under 50-Hz 2-mT magnetic field irradiation. Appl. Phys. A 67, 219–223. Grassi, C., D’Ascenzo, M., Torsello, A., Martinotti, G., Wolf, F., Cittadini, A., Azzena, G.B., 2004. Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channels and their role in modulation of neuroendocrine cell proliferation and death. Cell Calcium 35, 307–315. Kucˇera, I., Kaplan, P., 1996. A study on the transport and dissimilatory reduction of nitrate in Paracoccus denitrificans using viologen dyes as electron donors. Biochim. Biophys. Acta 1276, 203–209. Lai, H., Singh, N.P., 1996. Single- and double-strand DNA breaks in rat brain cells after acute exposure to radiofrequency electromagnetic radiation. J. Radiat. Biol. 69, 513–521.
Lai, H., Singh, N.P., 2004. Magnetic-field-induced DNA strand breaks in brain cells of the rat. Environ. Health Perspect. 112, 687–694. Lopucki, M., Schmerold, I., Dadak, A., Wiktor, H., Niedermu¨ller, H., Kankofer, M., 2005. Low dose magnetic fields do not cause oxidative DNA damage in human placental cotyledons in vitro. Virchows Arch. 446, 634–639. Malyapa, R.S., Ahern, E.W., Bi, C., Straube, W.L., LaRegina, M., Pickard, W.F., Roti Roti, W.F., 1998. DNA damage in rat brain cells after in vivo exposure to 2450 MHz electromagnetic radiation and various methods of euthanasia. Radiat. Res. 149, 637–645. Markov, M., Hazlewood, C., Ericsson, A., 2004. Systemic effect—a plausible explanation of the benefit of magnetic field therapy: a hypothesis. In: Kostarakis, P. (Ed.), Proceedings of Biological Effects of EMFs, 3rd International Workshop, Kos, Greece, pp. 673–681. Nova´k, J., Strasˇa´k, L., Fojt, L., Slaninova´, I., Vetterl, V., 2007. Effects of low-frequency magnetic fields on the viability of yeast Saccharomyces cerevisia. Bioelectrochemistry 70, 115–121. Panagopoulos, D.J., Karabarbounis, A., Margaritas, L.H., 2002. Mechanism for action of electromagnetic fields on cells. Biochem. Biochem. Biophys. Res. Co. 298, 95–102. Paulraj, R., Behari, J., 2006. Single strand DNA breaks in rat brain cells exposed to microwave radiation. Mutat. Res. 596, 76–80. Pessina, G.P., Aldinucci, C., Palmi, M., Sgaragli, G., Benocci, A., Meini, A., Pessina, F., 2001. Pulsed electromagnetic fields affect the intracellular calcium concentrations in human astrocytoma cells. Bioelectromagnetics 22, 503–510. Ravera, S., Repaci, E., Morelli, A., Pepe, I.M., Botter, R., Beruto, D., 2004. Electromagnetic field of extremely low frequency decreased adenylate kinase activity in retinal rod outer segment membranes. Bioelectrochemistry 63, 317–320. Repacholi, M.H., Greenebaum, B., 1999. Interaction of static and extremely-low frequency electric and magnetic fields with living systems: health effects and research needs. Bioelectromagnetics 20, 133–160. Robichon, D., Girard, J.C., Cenatiempo, Y., Cavallier, J.F., 1999. Atomic force microscopy imaging of dried or living bacteria. C. R. Acad. Sci. Paris/Life Sci. 322, 687–693. Ruiz-Go´mez, M.J., Prieto-Barciab, I., Ristori-Bogajoc, E., Martı´nez-Morillo, M., 2004. Static and 50 Hz magnetic fields of 0.35 and 2.45 mT have no effect on the growth of Saccharomyces cerevisiae. Bioelectrochemistry 64, 151–155. Sakurai, T., Koyama, S., Komatsubara, Y., Jin, W., Miyakoshi, J., 2005. Decrease in glucose-stimulated insulin secretion following exposure to magnetic fields. Biochem. Biophys. Res. Co. 332, 28–32. Singh, N., Lai, H., 1998. 60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells. Mutat. Res. 400, 313–320. Strasˇa´k, L., Vetterl, V., Fojt, L., 2005. Effects of 50 Hz magnetic fields on the viability of different bacterial strains. Electromagn. Biol. Med. 24, 293–300. Strasˇa´k, L., Vetterl, V., Sˇmarda, J., 2002. Effects of low-frequency magnetic fields on the bacteria Escherichia coli. Bioelectrochem. Bioenerg. 55, 161–164. Wolf, F.I., Torsello, A., Tedesco, B., Fasanella, S., Boninsegn, A., D’Ascenzo, M., Grassi, C., Azzena, G.B., Cittadini, A., 2005. 50-Hz extremely low frequency electromagnetic fields enhance cell proliferation and DNA damage: possible involvement of a redox mechanism. BBA-Mol. Cell Res. 1743, 120–129.
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Short communication
50 Hz magnetic field effect on the morphology of bacteria Luka´sˇ Fojt a,*, Petr Klapetek b, Ludeˇk Strasˇa´k a, Vladimı´r Vetterl a a b
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kra´lovopolska´ 135, Brno 612 65, Czech Republic Czech Metrology Institute, Okruzˇnı´ 31, Brno 638 00, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 April 2009 Received in revised form 19 June 2009 Accepted 20 June 2009
Atomic force microscopy was used to distinguish changes in morphology of bacteria induced by 50 Hz 10 mT magnetic field exposure. It is known that alternating magnetic field exposure causes decrease of viability of different bacterial strains. Previously we found that the viability of rod-like bacteria exposed to magnetic field decreased twice more in comparison with the spherical ones. Motivated by this fact we carried out this study with bacterial cells of both shapes. We used Escherichia coli (rod-like) and Paracoccus denitrificans (spherical) bacteria. As a result we have not observed any change in bacterial morphology neither of rod-like nor of spherical bacteria after 1 h, 50 Hz and 10 mT magnetic field exposure. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: AFM Magnetic field Bacteria
1. Introduction It becomes very important to find out electromagnetic fields environmental and health impact in last years. It is caused by overwhelming spreading of electric and electronic devices and wireless technologies all over the world. It is necessary to distinguish and state safety limits for electromagnetic fields exposure more precisely (we hope that our results might help to it). Lot of studies has been carried out concerning different types of electromagnetic fields from microwaves to static magnetic field effects. Different biological effects were detected such as elevated level of DNA strand brakes in rat brains after low frequency magnetic field exposure (Singh and Lai, 1998; Lai and Singh, 2004) or microwave exposure (Paulraj and Behari, 2006; Lai and Singh, 1996). Increase of DNA strand brakes of fibroblasts and leukaemia cells after 50 Hz magnetic field exposure has been reported in (Wolf et al., 2005). Biological effects on gene transcription, changes in DNA synthesis and transcription have been studied (Repacholi and Greenebaum, 1999; Blank and Goodmann, 2001). Effect of 50 Hz magnetic field on Ca2+ ion channels is discussed in Pessina et al. (2001) and Grassi et al. (2004). Electromagnetic fields affect various enzymatic processes, such as decrease of adenylate kinase activity (Ravera et al., 2004) or decrease of glucose stimulated insulin secretion (Sakurai et al., 2005). In contrary to publications where electromagnetic fields effects are claimed there do exist plenty of studies that do not observe any changes in studied objects. The observation of Lopucki et al. (2005) could be used as an
example where no change in oxidative DNA damage after 50 Hz magnetic field exposure was found. No evidence of DNA strand brakes in rat brain cells after microwave irradiation was reported (Malyapa et al., 1998). Unicellular organisms as experimental subjects are useful tool for electromagnetic fields effects studying (Berg, 1999; Markov et al., 2004). Viability of bacteria (Strasˇa´k et al., 2002, 2005; Babushkina et al., 2005) and yeasts (Ruiz-Go´mez et al., 2004; Nova´k et al., 2007) has been studied. We found that different types of bacteria are affected by the 50 Hz 10 mT magnetic field according to their shape in our previous studies (Fojt et al., 2004, 2007; Strasˇa´k et al., 2005). Gram-negative Escherichia coli and Leclercia adecarboxylata – rod-like bacteria – achieved about 60–70% of CFU number (colony forming units, denotes number of living bacteria able to form a colony) after exposure compared to the control ones. For Gram-positive Paracocuccus denitrificans and Staphylococcus aureus – spherical bacteria – there was about 20% decrease in CFU number. Provoked with this fact we decided to study bacterial morphology after 50 Hz 10 mT magnetic field exposure. Atomic force microscopy (AFM) could be used as a powerful tool for biological objects sensing. Different biological structures were imaged using AFM, like DNA, membranes, photosystems (Fotiadis et al., 2002) and unicellular organisms (Dufrene, 2002; Robichon et al., 1999). AFM was used to study the changes in lymphoblastoid cells under 50 Hz 2 mT magnetic field exposure (Girasole et al., 1998). We used representative of two morphological bacterial groups – E. coli and P. denitrificans for our study. 2. Materials and methods
* Corresponding author. Tel.: +420 541517261; fax: +420 541211293. E-mail addresses: [email protected] (L. Fojt), [email protected] (P. Klapetek), [email protected] (L. Strasˇa´k), [email protected] (V. Vetterl). 0968-4328/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2009.06.009
Magnetic fields were generated in a cylindrical coil (Institute of Scientific Instruments, ASCR, Brno, Czech Republic) powered by an
L. Fojt et al. / Micron 40 (2009) 918–922
919
Santa Barbara, USA) in contact mode and than processed with ˇ MI, Brno, Czech Republic) using subtraction Gwyddion software (C and correlation for to distinguish changes between exposed and control samples. 3. Results
Fig. 1. Dependence of the magnetic field induction Bm in the coil on the distance from the coil axis (which is situated at r = 0 mm) for different current values. Solid line denotes position of Petri dishes, dashed line position of microscopic slides.
autotransformer (50 Hz power net supply, sinusoidal waveform), as described previously (Fojt et al., 2004). The maximum current amplitude at 50 Hz in the coil was Im = 1.9 A, corresponding to a maximum achievable magnetic field amplitude of Bm = 10 mT. The current was checked during the measurements using HC3500-T Multimeter (HC, Seoul, South Korea) and the frequency by oscilloscope PM 3266 (Philips, Eindhoven, The Netherlands). For exposure, the samples were placed in the centre of the coil where uniformity of the magnetic field is optimal (about 5%). For magnetic field distribution inside the coil for different current values see Fig. 1. Magnetic induction was measured using Hall probe, lower magnetic induction levels (under 2 mT) were also checked by magnetic field meter BMM 3000 (EnviroMentor, Go¨teborg, Sweden). Further coil details can be found in Fojt et al. (2004). The temperature inside the coil was measured by thermometer and was maintained by an airflow to laboratory value (both samples, control and exposed ones, were at the same temperature). Control samples were placed away from the coil to avoid magnetic field exposure and one part underwent shamexposure. Both samples (control and exposed ones) originate from the same bacterial culture. Background magnetic field at 50 Hz was determined as 800 nT. Bacteria were exposed either in Petri dishes (diameter of 90 mm) or on microscopic slide on AFM sample holder. The bacteria E. coli (strain K12, Row, genotype 58–161 metB1rpsL 1+ FdefP.Fredericq) were grown in TY broth (8 g tryptone, 5 g yeast extract (Oxoid, UK); 5 g NaCl (Lachema Brno, Czech Republic) diluted in 1 l distilled water). We used basic nutrient agar number 2 (40 g/l, Imuna Sˇarisˇske´ Michal’any, Slovak Republic) as a solid cultivation medium. P. denitrificans CCM 982 (NCIB 8944) were grown in liquid and solid cultivation medium prepared according Kucˇera and Kaplan (1996). Bacteria for AFM measurements were grown for 12 h on agar plates (until the colonies had become visible, bacteria were in log phase of their growth). Two types of 10 mT, 50 Hz and 60 min magnetic field exposures were done: (1) Bacteria were exposed to magnetic field on agar plates and after exposure they were removed to microscopic slide for AFM imaging. (2) Bacteria were exposed directly on microscopic slide (which enables to take AFM picture on the same place before and after exposure). Slide was prepared by covering the glass surface with homogenous layer of bacteria collected from agar plates. Each of the experiment type was at least five times repeated in lower (circa 50 mm 50 mm) and higher (circa 3 mm 3 mm) resolution for both bacterial strains. Scanning region was selected randomly. Data were collected using AFM Explorer head (Veeco,
Both types of used bacterial strains were exposed to 10 mT 50 Hz magnetic field for 60 min. When were bacteria exposed on agar plates (magnetic field exposure 1), only overall information of magnetic field influence on bacterial morphology was acquired. It is not possible to find the same bacteria on images before and after magnetic field treatment. This makes direct comparison between exposed and control samples difficult. We exposed bacteria on microscopic slides (magnetic field exposure 2) to solve this problem. Bacteria grown on agar plates were removed carefully to microscopic slides. In first experimental setup, we made analogous exposure setting as for magnetic field exposure 1 as follows: part of the slides covered with bacteria was exposed to the magnetic field and then imaged with AFM. The other part was imaged with AFM after 60 min of waiting (control samples). We were able to collect AFM images of unexposed and exposed bacteria from these experiments, with no possibility of precise single bacteria (before and after magnetic field treatment) detecting. This data set provides practically the same images as bacteria exposed on agar plates. For making possible the direct comparison of exposed and control samples, we modified slightly the experimental setup. The slide covered with bacteria was put onto the AFM sample holder immediately. In this time, we made AFM image of unexposed glass slice covered with bacteria. The same slice underwent a magnetic field exposure. AFM was employed again after the exposure. We were able to compare images of the same bacteria before and after magnetic field exposure with these experimental setting, because the bacteria did not change their position related to AFM sample holder, thus not in the image itself. For image data acquired by this way, we made mutual assignment of discrete parts of surface – correlation, to determinate lateral changes in exposed sample. Vertical changes were propagated by subtraction of images. These data are shown in Fig. 2 for E. coli in high resolution and in Figs. 3 and 4 for E. coli and P. denitrificans in low resolution AFM images. Each image is set of unexposed sample, exposed sample, subtracted and correlated data. It is not possible to determine any changes in bacterial morphology after magnetic field exposure in all types of our measurements. 4. Discussion Our study was motivated by our previous results (Fojt et al., 2007) where we found differences between exposed Grampositive (about 20% decrease in CFU number for 10 mT, 24 min 50 Hz magnetic field exposure) and Gram-negative (about 30–40% decrease in CFU number, the same exposure conditions, see Fig. 5) bacterial strains. For data acquisition, we choose AFM due to its unchallenged possibility, to image living objects in higher resolution than common light inverse microscopes. Electron microscopy yields high resolution, but requires work in vacuum, staining and other special treatment (Bolshakova et al., 2001). Even environmental electron microscopy is not possible to image living bacteria. We exposed E. coli and P. denitrificans to the magnetic field and focused our attention to the morphological changes of these bacteria. Bacteria were exposed for 60 min, which is the longest possible exposure time which could be from technical and biological conditions achieved (and according to our previous results yields the strongest effects on bacteria). We observed no change in bacterial morphology using AFM. Small changes – strip-
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Fig. 2. AFM contact mode images of E. coli in high resolution. (A) Bacteria before magnetic field exposure, (B) bacteria after magnetic field exposure, (C) correlation, and (D) subtraction.
like and concentric deformation on subtracted and correlated data (vertical and lateral changes, strip like deformation could be observed mostly on lower resolution AFM pictures, concentric on high resolution AFM pictures), are due to the AFM drift during the
time (it was necessary to move and rotate them slightly to get the same position for the same images). Important is that the subtracted and correlated data do not show any changes in lateral and/or vertical dimension in the centre of the sample where the
Fig. 3. AFM contact mode images of E. coli in low resolution. (A) Bacteria before magnetic field exposure, (B) bacteria after magnetic field exposure, (C) correlation, and (D) subtraction.
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Fig. 4. AFM contact mode images of P. denitrificans in low resolution. (A) Bacteria before magnetic field exposure, (B) bacteria after magnetic field exposure, (C) correlation, and (D) subtraction.
Fig. 5. Comparison of magnetic field effect for E. coli (grey column) and P. denitrificans (white column). Data acquired from Fojt et al. (2004). (A) Dependence of the relative number of CFU on the value of the magnetic field induction for t = 12 min. (B) Dependence of the relative number of CFU on the duration of exposure for Bm = 10 mT.
drift is negligible. Figs. 2–4 show an average of five measurements. All images were done on the air, on the microscopic slides. According to our experiments, the bacteria remain alive after our experimental treatment, and this fact is also supported by Bolshakova et al. (2001). We tried to cut down the effect of bacteria drying by cultivating them on agar plates. Beckmann et al. (2006) had reported that bacteria grown on agar plates are more resistant to drying on air than bacteria cultivated in broth. In the experiments where bacteria were treated similarly to the exposed ones we observed very small changes that can be ascribed to sample drift during time. We know that the oscillating magnetic field affects different bacterial strains (in lag-phase of their growth) from our previous results. This fact was found using CFU counting technique (Fojt et al., 2004), electrochemical determination of enzymatic activity (Fojt et al., 2007) and controlling of growth curves using spectrophotometry (Strasˇa´k et al., 2005). Metabolic activity of used bacteria was not affected. We did not find any changes in
bacterial morphology after magnetic field treatment. It seems that bacteria shape does not play any important role in the interaction with used magnetic field. We will focus on possible mechanisms of magnetic field acting in our next experiments. There are a lot of theories that try to explain acting of magnetic fields on living organisms (Panagopoulos et al., 2002; Berg, 1999). But there is still a confusion about which effect or combination of effects is involved in real processes of electromagnetic fields bioeffects. 5. Conclusions We tried to determine 50 Hz 10 mT 60 min magnetic field morphological effect on two different bacterial strains. We were not able to detect any changes in bacteria surface and/or shape according to our different AFM experiments. Further experiments could be done using the scanning near field optical microscopy which could provide other interesting information for our biological objects.
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Acknowledgements This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (Grant KAN 200040651, KAN 311610701), the Ministry of Education, Youth and Sports (project No. 1M0528 and No. LC06035), grant agency of the Czech Republic (Grant 202/08/1688, Grant 310/07/P480), and by the institutional research plans (AVOZ 50040507, AVOZ 50040702). References Babushkina, I.V., Borodin, V.B., Smetkova, N.A., Morrison, V.V., Usanov, A.D., Skripal, A.V., Usanov, D.A., 2005. The influence of alternating magnetic field on Escherichia coli bacterial cells. Pharm. Chem. J. 39, 398–400. Beckmann, M.A., Venkataraman, S., Doktycz, M.J., Nataro, J.P., Sullivan, C.J., MorrellFalvey, J.L., Allison, D.P., 2006. Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM. Ultramicroscopy 106, 695–702. Berg, H., 1999. Problems of weak electromagnetic field effects in cell biology. Bioelectrochem. Bioenerg. 48, 355–360. Blank, M., Goodmann, R., 2001. Electromagnetic initiation of transcription at specific DNA sites. J. Cell. Biochem. 81, 689–692. Bolshakova, A.V., Kiselyova, O.I., Filonov, A.S., Frolova, O.Y., Lyubchenko, Y.L., Yaminsky, I.V., 2001. Comparative studies of bacteria with an atomic force microscopy operating in different modes. Ultramicroscopy 86, 121–128. Dufrene, Y.F., 2002. Atomic force microscopy, a powerful tool in microbiology. J. Bacteriol. 184, 5205–5213. Fojt, L., Strasˇa´k, L., Vetterl, V., Sˇmarda, J., 2004. Comparison of the low-frequency magnetic field effects on bakteria Escherichia coli, Leclercia adecarboxylata and Staphylococcus aureus. Bioelectrochemistry 63, 337–341. Fojt, L., Strasˇa´k, L., Vetterl, V., 2007. Effect of electromagnetic fields on the denitrification activity of Paracoccus denitrificans. Bioelectrochemistry 70, 91–95. Fotiadis, D., Sheuring, S., Muller, S.A., Engel, A., Muller, D.J., 2002. Imaging and manipulation of biological structures with the AFM. Micron 33, 385–397. Girasole, M., Cricenti, A., Generosi, R., Congiu-Castellano, A., Pozzi, D., Pasquali, E., Lisi, A., Santoro, N., Grimaldi, S., 1998. Atomic forcemicroscopy study of lymphoblastoid cells under 50-Hz 2-mT magnetic field irradiation. Appl. Phys. A 67, 219–223. Grassi, C., D’Ascenzo, M., Torsello, A., Martinotti, G., Wolf, F., Cittadini, A., Azzena, G.B., 2004. Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channels and their role in modulation of neuroendocrine cell proliferation and death. Cell Calcium 35, 307–315. Kucˇera, I., Kaplan, P., 1996. A study on the transport and dissimilatory reduction of nitrate in Paracoccus denitrificans using viologen dyes as electron donors. Biochim. Biophys. Acta 1276, 203–209. Lai, H., Singh, N.P., 1996. Single- and double-strand DNA breaks in rat brain cells after acute exposure to radiofrequency electromagnetic radiation. J. Radiat. Biol. 69, 513–521.
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