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Evaluation of Pseudomonas aeruginosa biofilm formation using Quartz Tuning Forks as impedance sensors
Sensors and Actuators B 189 (2013) 60–65
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Evaluation of Pseudomonas aeruginosa biofilm formation using Quartz Tuning Forks as impedance sensors Tomasz Piasecki a,∗ , Grzegorz Guła b , Karol Nitsch a , Karol Waszczuk a , Zuzanna Drulis-Kawa b , Teodor Gotszalk a a b
Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology, Z. Janiszewskiego 11/17, 50-372 Wroclaw, Poland Institute of Genetics and Microbiology, University of Wroclaw, S. Przybyszewskiego 63/77, 51-148 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Available online 3 January 2013 Keywords: Impedance spectroscopy Bioimpedance Biofilm Equivalent circuit modelling
a b s t r a c t The shape of metallisation on Quartz Tuning Forks (QTFs) allowed to use them as impedance sensors for monitoring biofilm formation of Pseudomonas aeruginosa strains. Electrical equivalent circuits modelling was used. It allowed to detect which electrical properties of the sensor change with biofilm formation. Characteristic frequencies of 20 Hz and 1 MHz were found as suitable for the simple detection of biofilm state. Resistance measured at 1 MHz allowed to detect the changes in media conductivity caused by bacterial culture growth. It was in accordance with the results obtained using another methods. In addition, the conductance at 20 Hz was suitable for detection of biofilm destruction caused by the lack of nutrients. © 2013 Published by Elsevier B.V.
1. Introduction Quartz Tuning Forks (QTFs) apart from typical use as a frequency reference in electronic clocks may be used in many other applications far from their original purpose [1–3]. Previous work pointed that one of them may be to detect the formation of biofilm on its surface by measuring the shift of resonance frequency caused by the mass of adhered biofilm [4,5]. On the other hand the shape of the metallization pattern on the QTF surface (Fig. 1b and c) is similar to the interdigitated electrodes (IDE) on typical impedance sensors used for fast bacteria culture growth monitoring (Fig. 1d) [6,7]. **** The bacterial biofilm represents the most prevalent type of microbial growth in nature and are involved for example in the food contamination during production, preparation or storage, therefore bacterial consortia may be a serious and costly problem in the industry [8]. Biofilms are also crucial for bacterial pathogenicity. They can be a source of persistent infections and are often associated with high-level antimicrobial resistance of the associated organisms [9]. The biofilm forming bacteria produce the extracellular matrix in which cells are embedded causing the resistance to antibiotics or immune defence. The matrix consists of different types of extracellular polymeric substances (EPS) (proteins, lipids, exopolysaccharides, extracellular DNA) and is responsible for adhesion to biotic and abiotic surfaces. Microorganisms that produce
∗ Corresponding author. E-mail address: [email protected] (T. Piasecki). 0925-4005/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.snb.2012.12.087
EPS could colonize indwelling medical devices to form a biofilm. The device-associated infections result in substantial morbidity and mortality, and potentially represent a large increase the treatment cost [10]. The motivation of this work was to determine whether the QTF may be used as an impedance sensor to detect the formation of biofilm. The established methods for biofilm detection such as tube adherence test [11], tissue culture plate [12] or Congo red agar method [13] are very sensitive and reliable however they are are laborious, especially when used to test the dynamics of the biofilm formation. Modern methods based on the the confocal microscopy [14] or PCR [15,16] require the expensive equipment. Proposed method is fast and it allows for in situ detection without interfering with the biofilm growth, similarly to the methods based on the Quartz Crystal Microbalance [17,18]. The positive outcome of such research would also lead to the development of combined mass and impedance biofilm detection as both of these methods use the same sensor.
2. Materials and methods Two strains of Pseusomonas aeruginosa: PAO1 (ATCC 15692) and ATCC 27853 were used as a model of biofilm forming bacteria. The bacteria were stored at −70 ◦ C in Trypticase Soy Broth (Becton Dickinson and Company, Cockeysville, MD, USA) supplemented with 20% glycerol. For the experiments strains were refreshed on Muller Hinton Agar (MHA) (Becton Dickinson and Company, Cockeysville, MD, USA) at 37 ◦ C for 18 h.
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Fig. 1. Quartz Tuning Fork: photograph (a), the 3D model (b) and partial development of the surface (c) and part of IDE sensor with gold electrodes on oxidized silicon (d). Letters A, B, C, D mark sides of the QTF.
For the experiments, the refreshed bacterial culture was diluted in a physiological saline (PS) to an optical density equal to the McFarland No. 0.5. Then the culture was suspended in 2 ml of Muller Hinton Broth (MHB) (Becton Dickinson and Company, Cockeysville, MD, USA) in a 24-well titrate plate to obtain final density of 5 × 105 cells per ml (CFU/ml) in each well. Prior to the experiment the QTFs were soldered to the dedicated holders, sterilized in an autoclave and mounted in an 8-channel switch which allowed for simultaneous monitoring of 8 wells (Fig. 2). The combined results of three experiments are presented in this paper. In first two there were 4 wells with culture of one of the P. aeruginosa strains and 4 wells with pure MHB as the control. In third experiment there were 3 wells with each of the strains and 2 wells with pure MHB as the control. The impedance spectra of QTFs were measured using Solartron 1260 impedance analyser in frequency range from 0.1 Hz to 1 MHz. Amplitude of test signal was 25 mV. During the experiment impedance spectra were measured repeatedly while the cultures were incubated at 37 ◦ C. To minimize the influence of the water
Fig. 2. Photograph of the part of the measurement setup: 8-channel switch and 24-well titrate plate.
evaporation on the results whole experiment was conducted at 100% relative humidity. The circuit board of the switch was covered with a layer of epoxy resin to prevent the condensing water from influencing the measurement results. Impedance spectra obtained during measurements were analysed and parametrised using equivalent circuit modelling with circuit presented in Fig. 3. The equivalent circuit consisted of the resistor Rmed related to the measured resistivity of medium surrounding the sensor and three elements related to the phenomena occurring at the surface of
Fig. 3. The equivalent circuit used for impedance spectra modelling of the QTF in the culture: Rmed – medium conductivity, CPEsurf – polarization at the surface, Rsurf – charge transfer at the surface, CPEd – ion diffusion through the biofilm.
Fig. 4. The changes of the impedance spectra of QTF in P. aeruginosa PAO1 biofilm culture during the incubation.
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Fig. 5. Exemplary results of the equivalent circuit modelling for MHB and P. aeruginosa PAO1 culture: Rmed (a) and capacitance of CPEsurf (b) during first day of incubation and the Q parameter of CPEd for 8 day incubation (c). Arrows mark characteristic differences between culture and control.
the sensor: CPEsurf modelling the polarization of the electric double layer and the aluminium oxide layer at the electrodes, Rsurf was related to the charge transfer resistance through these layers and CPEd modelling the Warburg diffusion impedance in presence of the biofilm [19]. Modelling was performed using Scribner ZView software. To evaluate the adhesion of biofilm to the surface of the QTF during the experiment several QTFs were placed in identical suspension of P. aeruginosa in which impedance measurements were carried out and incubated at 37 ◦ C. After 8, 16, 24, 48, 72 and 120 h of incubation two QTFs were taken out, rinsed using 1 ml and suspended in 2 ml PS (POCh Poland). Thirty minutes later QTFs were rinsed again (using 1 ml PS) and placed into the titration plate wells containing 2 ml of 0.01% CV solution (Aqua-Med ZPAM - KOLASA, Poland). After 20 min the excess CV was removed by washing sensors using 1 ml of PS. Finally, CV stained biofilms on the surface of QTFs were dried in a laminar chamber for 15 min and visualized using Olympus CX31 optical microscope. 3. Results The exemplary impedance spectra obtained during the experiment with P. aeruginosa PAO1 strain are shown in Fig. 4. The significant changes in the electric properties of QTF in the bacterial culture were observed. 3.1. Equivalent circuit analysis After the parametrization of obtained impedance spectra using equivalent circuit modelling it was possible to determine which of the parameters of the equivalent circuit change differently in the culture and the control. Exemplary results of such comparison for PAO1 strain and pure MHB are shown in Fig. 5. The Rmed dropped during first hour of experiment in both culture and control but only in the wells with culture there was another drop of its value after 8–10 h (arrow at Fig. 5a). Lowering the value of the Rmed in the culture pointed that the conductivity of the surrounding of the QTF rose due to the presence of the bacteria. That change was correlated in time with the start of the cells adhesion to the surface of the QTF (Figs. 6 and 7) as well as the lowering of the resonance frequency of QTF caused by biofilm formation presented in the previous work [4]. The changes of the medium conductivity caused by the altering of electric properties of medium also resulted in the increase of capacitance of the CPEsurf (Fig. 5b). On the other hand, after the time in which in the mass measurements of the biofilm showed the gradual lowering of the detected
mass [4] and the thickness of the biofilm decreased (Fig. 6) the significant rise of the Q parameter of CPEd was observed (Fig. 5c). It was concluded that it was caused by the changes of electric properties of the biofilm due its degradation resulting from nutrient deprivation (Fig. 7). 3.2. Simplified measurements Measuring the impedance spectra in wide range of frequency and analysing it using the equivalent circuit modelling requires expensive equipment and sophisticated software. The decomposition of the measured impedance spectra into components according to the structure of the equivalent circuit allows to determine the discrete frequencies at which the value of total impedance was the most influenced by changes of equivalent circuit parameters. These frequencies may be in range suitable for using of simpler equipment such as a RLC meter or dedicated device which are less expensive and may be portable. Such decomposition is presented in Fig. 8. The value of Rmed determines the shape of the impedance spectra above 100 kHz thus the measurement of the real part of impedance at this frequency will be sensitive to changes of that parameter. The frequency that was chosen for analysis was 1 MHz. On the other hand the value of CPEd along with the Rsurf determine the low frequency part of the spectrum, below 1 Hz. However due to the fact that the RLC meters usually do not offer such low frequency measurements the second frequency was set as 20 Hz [20,21]. Such frequency was not optimal but still provided useful informations.
Fig. 6. Photographs of crystal violet stained P. aeruginosa biofilm on the QTF after 8, 16, 24, 48, 72, and 120 h of incubation. (For interpretation of references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. Photographs of the selected places on the the transparent tip of the QTF with crystal violet stained P. aeruginosa biofilm after 8, 16, 24, 48, 72 and 120 h of incubation. At the images for 16 and 24 h upper part is the uniform layer of the EPS covered biofilm while at the lower part there is a scratch made with needle to reveal the bacteria from under the EPS. (For interpretation of references to colour in this figure legend, the reader is referred to the web version of this article.)
Results of such simplified analysis of results obtained during the experiments with P. aeruginosa PAO1 and ATCC 27853 strains and pure MHB as control are shown in Figs. 9 and 10. Both species exhibited similar changes of electric properties of the biofilm but at different times: lowering of the normalized resistance at 1 MHz appears later and the rise of the conductance at 20 Hz is more pronounced for PAO1 strain. Due to the differences in the cell constant of the QTFs acting as the impedance sensors the value of resistance measured at 1 MHz was normalized for more convenient comparison. Plots
were similar to those obtained previously using equivalent circuit modelling showing drop of the resistance after several hour of incubation for both strains. The decomposition of the biofilm was also detected by analysis of the conductance measured at 20 Hz (Fig. 10) but after longer time than by equivalent circuit modelling. In addition the accidental contamination of one of the controls could also be detected. The growth of bacteria caused the drop of the high-frequency resistance however it apparently did not formed biofilm and there was not a rise of the low frequency conductance (dashed line in Figs. 9 and 10).
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4. Conclusions
Fig. 8. The decomposition of the impedance spectra of QTF on separate equivalent circuit elements at 5th day of incubation.
Evaluation of the biofilm formation of two P. aeruginosa strains was presented. The equivalent circuit modelling of impedance spectra obtained using QTFs used as the impedance sensors allowed to determine two parameters which changed in presence of bacteria. Results were in agreement with previously published [4] obtained using classical standard growth curve method and by mass measurements using QTFs and colorimetric assay. Simplified method was also proposed. It relied on the simple measurement of impedance at two characteristic frequencies (20 Hz and 1 MHz) which results were consistent with obtained using equivalent circuit modelling. Electric properties of biofilm formed by both strains changed similarly. Results allowed to conclude that QTFs may be used as impedance sensors of P. aeruginosa biofilm formation thus combined mass and impedance systems may be built. It would detect the formation of the biofilm and would not be sensitive to the planktonic forms of the bacteria. The biofilm mass measurements using QTFs require several steps of preparation [4]. The impedance measurement could be used to determine the moment at which the biofilm formed and at which the mass measurement should be done.
Acknowledgements This work was supported by European Project “Detectors and sensors for measuring factors hazardous to environment – modelling and monitoring of threats”, No. POIG.01.03.0102-002/08-00 and Polish National Science Centre grant, No. 2011/01/N/NZ7/00156.
References
Fig. 9. Changes of the normalized resistance measured at 1 MHz during first day of incubation for two P. aeruginosa strains, control and accidental contamination. Mean value and standard deviation marked by the line and area respectively.
Fig. 10. Changes of the conductance measured at 20 Hz during the incubation for two P. aeruginosa strains, control and accidental contamination. Mean value and standard deviation marked by the line and area respectively.
[1] M. Heyde, M. Kulawik, H.-P. Rust, H.-J. Freund, Double quartz tuning fork sensor for low temperature atomic force and scanning tunneling microscopy, Review of Scientific Instruments 75 (2004) 2446–2450. [2] X. Zhou, T. Jiang, J. Zhang, X. Wang, Z. Zhu, Humidity sensor based on quartz tuning fork coated with sol–gel-derived nanocrystalline zinc oxide thin film, Sensors and Actuators B: Chemical 123 (2007) 299–305. [3] K. Waszczuk, T. Piasecki, K. Nitsch, T. Gotszalk, Application of piezoelectric tuning forks in liquid viscosity and density measurements, Sensors and Actuators B: Chemical 160 (2011) 517–523. [4] K. Waszczuk, G. Gula, M. Swiatkowski, J. Olszewski, W. Herwich, Z. Drulis-Kawa, J. Gutowicz, T. Gotszalk, Evaluation of Pseudomonas aeruginosa biofilm formation using piezoelectric tuning fork mass sensors, Sensors and Actuators B: Chemical 170 (2012) 7–12, Eurosensors XXIV, 2010. [5] G. Gula, K. Waszczuk, T. Olszak, J. Majewska, J. Sarowska, T. Gotszalk, J. Gutowicz, Z. Drulis-Kawa, Piezoelectric tuning fork based mass measurement method as a novel tool for determination of antibiotic activity on bacterial biofilm, Sensors and Actuators B: Chemical 175 (2012) 34–39. [6] J. Atienza, J. Zhu, X. Wang, X. Xu, Y. Abassi, Dynamic monitoring of cell adhesion and spreading on microelectronic sensor arrays, Journal of Biomolecular Screening 10 (2005) 795–805. [7] T. Kim, J. Kang, J.-H. Lee, J. Yoon, Influence of attached bacteria and biofilm on double-layer capacitance during biofilm monitoring by electrochemical impedance spectroscopy, Water Research 45 (2011) 4615–4622. [8] Z. Ali, W. O’Hare, B. Theaker, Detection of bacterial contaminated milk by means of a quartz crystal microbalance based electronic nose, Journal of Thermal Analysis and Calorimetry 71 (2003) 355–362. [9] T. Mah, G.A. O’Toole, Mechanisms of biofilm resistance to antimicrobial agents, Trends in Microbiology 9 (2001) 34–39. [10] H.-C. Flemming, J. Wingender, The biofilm matrix, Nature Reviews Microbiology 8 (2010) 623–633. [11] G.D. Christensen, W.A. Simpson, A.L. Bisno, E.H. Beachey, Adherence of slimeproducing strains of staphylococcus epidermidis to smooth surfaces., Infection and Immunity 37 (1982) 318–326. [12] G.D. Christensen, W.A. Simpson, J.J. Younger, L.M. Baddour, F.F. Barrett, D.M. Melton, E.H. Beachey, Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices, Journal of Clinical Microbiology 22 (1985) 996–1006, PMID: 3905855 PMCID: PMC271866. [13] D.J. Freeman, F.R. Falkiner, C.T. Keane, New method for detecting slime production by coagulase negative staphylococci, Journal of Clinical Pathology 42 (1989) 872–874, PMID: 2475530 PMCID: PMC1142068.
T. Piasecki et al. / Sensors and Actuators B 189 (2013) 60–65 [14] R.J. Palmer Jr., C. Sternberg, Modern microscopy in biofilm research: confocal microscopy and other approaches, Current Opinion in Biotechnology 10 (1999) 263–268. [15] T. Hoshino, N. Noda, S. Tsuneda, A. Hirata, Y. Inamori, Direct detection by in situ PCR of theamoA gene in biofilm resulting from a nitrogen removal process, Applied and Environmental Microbiology 67 (2001) 5261–5266. [16] A.P. Lenz, K.S. Williamson, B. Pitts, P.S. Stewart, M.J. Franklin, Localized gene expression in Pseudomonas aeruginosa biofilms, Applied and Environmental Microbiology 74 (2008) 4463–4471, PMID: 18487401 PMCID: PMC2493172. [17] A.L. Schofield, T.R. Rudd, D. Martin, D.G. Fernig, C. Edwards, Real-time monitoring of the development and stability of biofilms of Streptococcus mutans using the quartz crystal microbalance with dissipation monitoring, Biosensors and Bioelectronics 23 (2007) 407–413. [18] I.M. Marcus, M. Herzberg, S.L. Walker, V. Freger, Pseudomonas aeruginosa attachment on QCM-D sensors: the role of cell and surface hydrophobicities, Langmuir 28 (2012) 6396–6402. [19] E. Barsoukov, J.R. Macdonald (Eds.), Impedance Spectroscopy. Theory, Experiment and Applications, John Wiley & Sons, 2005. [20] Agilent E4980A Precision LCR Meter User’s Guide, Agilent Technologies, fifth edition, 2007. [21] Precision LCR Meter LCR-8101 User Manual, GW Instek, 2011.
Biographies Tomasz Piasecki was born in 1976 in Wroclaw, Poland. He received his PhD degree at the Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology in 2005 where he continues his scientific research. His main area of interest are the measurements of electrical properties of materials, improving the measurement methods, construction of the measuring equipment as well as the novel applications for electric measurements. Grzegorz Guła was born in 1984 in Sroda Slaska, Poland. He graduated from the Faculty of Biology Science at University of Wroclaw in 2008, receiving the MSc degree in biology with a specialization in microbiology and in 2012 the PhD degree
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at the Department of Physicochemistry of Microorganisms at University of Wroclaw, where he continues his research on the application of new methods of observation of bacterial biofilm formation. Karol Nitsch is full professor, PhD, DSc, Professor at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology. His research interests are in area of measurement and analysis of electrical properties of materials, component and sensors. He specializes on the various applications of impedance spectroscopy to microelectronics materials and structures characterization. He has authored over 190 scientific publications. Karol Waszczuk was born in 1984 in Wodzislaw Slaski, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2007, receiving the MSc degree in electronics and telecommunications. Since then, he has been working at Research and Development Department for EBS Inkjet Systems Poland. He is pursuing his PhD degree at Wroclaw University of Technology, working on biochemical and microbiological applications of resonant mass sensors. Zuzanna Drulis-Kawa was born in Wroclaw, Poland. She received the MSc and PhD degrees of biology from University of Wroclaw, Department of Nature Science in 1994 and 2000, respectively. She received first and second clinical microbiology degrees at Medical University of Wroclaw in 1997 and 2000, respectively. She has been honored with bioMerieux-PTM research award (2009) and the prize of Prof. K. Bassalik Contest (2006) for her scientific work. She is the Head of the Department of Pathogen’s Biology and Immunology in Institute of Genetics and Microbiology at University of Wroclaw, Poland. She has authored over 60 scientific publications. Teodor Gotszalk was born in Wroclaw, Poland. He received the MSc degrees from the Faculties of Electronics and of Electrical Engineering of Wroclaw University of Technology in 1989 and 1991, respectively. In 1996, he received the PhD degree from the Institute of Electronic Technology of the Wroclaw University of Technology and the full professor title in 2012. He has been honored with Siemens research award (2000) and the prize of Polish Science Foundation FNP (1997) for his scientific work. He is the head of the Division of Micro- and Nanostructures Metrology at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology. He has authored over 100 scientific publications.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Evaluation of Pseudomonas aeruginosa biofilm formation using Quartz Tuning Forks as impedance sensors Tomasz Piasecki a,∗ , Grzegorz Guła b , Karol Nitsch a , Karol Waszczuk a , Zuzanna Drulis-Kawa b , Teodor Gotszalk a a b
Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology, Z. Janiszewskiego 11/17, 50-372 Wroclaw, Poland Institute of Genetics and Microbiology, University of Wroclaw, S. Przybyszewskiego 63/77, 51-148 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Available online 3 January 2013 Keywords: Impedance spectroscopy Bioimpedance Biofilm Equivalent circuit modelling
a b s t r a c t The shape of metallisation on Quartz Tuning Forks (QTFs) allowed to use them as impedance sensors for monitoring biofilm formation of Pseudomonas aeruginosa strains. Electrical equivalent circuits modelling was used. It allowed to detect which electrical properties of the sensor change with biofilm formation. Characteristic frequencies of 20 Hz and 1 MHz were found as suitable for the simple detection of biofilm state. Resistance measured at 1 MHz allowed to detect the changes in media conductivity caused by bacterial culture growth. It was in accordance with the results obtained using another methods. In addition, the conductance at 20 Hz was suitable for detection of biofilm destruction caused by the lack of nutrients. © 2013 Published by Elsevier B.V.
1. Introduction Quartz Tuning Forks (QTFs) apart from typical use as a frequency reference in electronic clocks may be used in many other applications far from their original purpose [1–3]. Previous work pointed that one of them may be to detect the formation of biofilm on its surface by measuring the shift of resonance frequency caused by the mass of adhered biofilm [4,5]. On the other hand the shape of the metallization pattern on the QTF surface (Fig. 1b and c) is similar to the interdigitated electrodes (IDE) on typical impedance sensors used for fast bacteria culture growth monitoring (Fig. 1d) [6,7]. **** The bacterial biofilm represents the most prevalent type of microbial growth in nature and are involved for example in the food contamination during production, preparation or storage, therefore bacterial consortia may be a serious and costly problem in the industry [8]. Biofilms are also crucial for bacterial pathogenicity. They can be a source of persistent infections and are often associated with high-level antimicrobial resistance of the associated organisms [9]. The biofilm forming bacteria produce the extracellular matrix in which cells are embedded causing the resistance to antibiotics or immune defence. The matrix consists of different types of extracellular polymeric substances (EPS) (proteins, lipids, exopolysaccharides, extracellular DNA) and is responsible for adhesion to biotic and abiotic surfaces. Microorganisms that produce
∗ Corresponding author. E-mail address: [email protected] (T. Piasecki). 0925-4005/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.snb.2012.12.087
EPS could colonize indwelling medical devices to form a biofilm. The device-associated infections result in substantial morbidity and mortality, and potentially represent a large increase the treatment cost [10]. The motivation of this work was to determine whether the QTF may be used as an impedance sensor to detect the formation of biofilm. The established methods for biofilm detection such as tube adherence test [11], tissue culture plate [12] or Congo red agar method [13] are very sensitive and reliable however they are are laborious, especially when used to test the dynamics of the biofilm formation. Modern methods based on the the confocal microscopy [14] or PCR [15,16] require the expensive equipment. Proposed method is fast and it allows for in situ detection without interfering with the biofilm growth, similarly to the methods based on the Quartz Crystal Microbalance [17,18]. The positive outcome of such research would also lead to the development of combined mass and impedance biofilm detection as both of these methods use the same sensor.
2. Materials and methods Two strains of Pseusomonas aeruginosa: PAO1 (ATCC 15692) and ATCC 27853 were used as a model of biofilm forming bacteria. The bacteria were stored at −70 ◦ C in Trypticase Soy Broth (Becton Dickinson and Company, Cockeysville, MD, USA) supplemented with 20% glycerol. For the experiments strains were refreshed on Muller Hinton Agar (MHA) (Becton Dickinson and Company, Cockeysville, MD, USA) at 37 ◦ C for 18 h.
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Fig. 1. Quartz Tuning Fork: photograph (a), the 3D model (b) and partial development of the surface (c) and part of IDE sensor with gold electrodes on oxidized silicon (d). Letters A, B, C, D mark sides of the QTF.
For the experiments, the refreshed bacterial culture was diluted in a physiological saline (PS) to an optical density equal to the McFarland No. 0.5. Then the culture was suspended in 2 ml of Muller Hinton Broth (MHB) (Becton Dickinson and Company, Cockeysville, MD, USA) in a 24-well titrate plate to obtain final density of 5 × 105 cells per ml (CFU/ml) in each well. Prior to the experiment the QTFs were soldered to the dedicated holders, sterilized in an autoclave and mounted in an 8-channel switch which allowed for simultaneous monitoring of 8 wells (Fig. 2). The combined results of three experiments are presented in this paper. In first two there were 4 wells with culture of one of the P. aeruginosa strains and 4 wells with pure MHB as the control. In third experiment there were 3 wells with each of the strains and 2 wells with pure MHB as the control. The impedance spectra of QTFs were measured using Solartron 1260 impedance analyser in frequency range from 0.1 Hz to 1 MHz. Amplitude of test signal was 25 mV. During the experiment impedance spectra were measured repeatedly while the cultures were incubated at 37 ◦ C. To minimize the influence of the water
Fig. 2. Photograph of the part of the measurement setup: 8-channel switch and 24-well titrate plate.
evaporation on the results whole experiment was conducted at 100% relative humidity. The circuit board of the switch was covered with a layer of epoxy resin to prevent the condensing water from influencing the measurement results. Impedance spectra obtained during measurements were analysed and parametrised using equivalent circuit modelling with circuit presented in Fig. 3. The equivalent circuit consisted of the resistor Rmed related to the measured resistivity of medium surrounding the sensor and three elements related to the phenomena occurring at the surface of
Fig. 3. The equivalent circuit used for impedance spectra modelling of the QTF in the culture: Rmed – medium conductivity, CPEsurf – polarization at the surface, Rsurf – charge transfer at the surface, CPEd – ion diffusion through the biofilm.
Fig. 4. The changes of the impedance spectra of QTF in P. aeruginosa PAO1 biofilm culture during the incubation.
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Fig. 5. Exemplary results of the equivalent circuit modelling for MHB and P. aeruginosa PAO1 culture: Rmed (a) and capacitance of CPEsurf (b) during first day of incubation and the Q parameter of CPEd for 8 day incubation (c). Arrows mark characteristic differences between culture and control.
the sensor: CPEsurf modelling the polarization of the electric double layer and the aluminium oxide layer at the electrodes, Rsurf was related to the charge transfer resistance through these layers and CPEd modelling the Warburg diffusion impedance in presence of the biofilm [19]. Modelling was performed using Scribner ZView software. To evaluate the adhesion of biofilm to the surface of the QTF during the experiment several QTFs were placed in identical suspension of P. aeruginosa in which impedance measurements were carried out and incubated at 37 ◦ C. After 8, 16, 24, 48, 72 and 120 h of incubation two QTFs were taken out, rinsed using 1 ml and suspended in 2 ml PS (POCh Poland). Thirty minutes later QTFs were rinsed again (using 1 ml PS) and placed into the titration plate wells containing 2 ml of 0.01% CV solution (Aqua-Med ZPAM - KOLASA, Poland). After 20 min the excess CV was removed by washing sensors using 1 ml of PS. Finally, CV stained biofilms on the surface of QTFs were dried in a laminar chamber for 15 min and visualized using Olympus CX31 optical microscope. 3. Results The exemplary impedance spectra obtained during the experiment with P. aeruginosa PAO1 strain are shown in Fig. 4. The significant changes in the electric properties of QTF in the bacterial culture were observed. 3.1. Equivalent circuit analysis After the parametrization of obtained impedance spectra using equivalent circuit modelling it was possible to determine which of the parameters of the equivalent circuit change differently in the culture and the control. Exemplary results of such comparison for PAO1 strain and pure MHB are shown in Fig. 5. The Rmed dropped during first hour of experiment in both culture and control but only in the wells with culture there was another drop of its value after 8–10 h (arrow at Fig. 5a). Lowering the value of the Rmed in the culture pointed that the conductivity of the surrounding of the QTF rose due to the presence of the bacteria. That change was correlated in time with the start of the cells adhesion to the surface of the QTF (Figs. 6 and 7) as well as the lowering of the resonance frequency of QTF caused by biofilm formation presented in the previous work [4]. The changes of the medium conductivity caused by the altering of electric properties of medium also resulted in the increase of capacitance of the CPEsurf (Fig. 5b). On the other hand, after the time in which in the mass measurements of the biofilm showed the gradual lowering of the detected
mass [4] and the thickness of the biofilm decreased (Fig. 6) the significant rise of the Q parameter of CPEd was observed (Fig. 5c). It was concluded that it was caused by the changes of electric properties of the biofilm due its degradation resulting from nutrient deprivation (Fig. 7). 3.2. Simplified measurements Measuring the impedance spectra in wide range of frequency and analysing it using the equivalent circuit modelling requires expensive equipment and sophisticated software. The decomposition of the measured impedance spectra into components according to the structure of the equivalent circuit allows to determine the discrete frequencies at which the value of total impedance was the most influenced by changes of equivalent circuit parameters. These frequencies may be in range suitable for using of simpler equipment such as a RLC meter or dedicated device which are less expensive and may be portable. Such decomposition is presented in Fig. 8. The value of Rmed determines the shape of the impedance spectra above 100 kHz thus the measurement of the real part of impedance at this frequency will be sensitive to changes of that parameter. The frequency that was chosen for analysis was 1 MHz. On the other hand the value of CPEd along with the Rsurf determine the low frequency part of the spectrum, below 1 Hz. However due to the fact that the RLC meters usually do not offer such low frequency measurements the second frequency was set as 20 Hz [20,21]. Such frequency was not optimal but still provided useful informations.
Fig. 6. Photographs of crystal violet stained P. aeruginosa biofilm on the QTF after 8, 16, 24, 48, 72, and 120 h of incubation. (For interpretation of references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 7. Photographs of the selected places on the the transparent tip of the QTF with crystal violet stained P. aeruginosa biofilm after 8, 16, 24, 48, 72 and 120 h of incubation. At the images for 16 and 24 h upper part is the uniform layer of the EPS covered biofilm while at the lower part there is a scratch made with needle to reveal the bacteria from under the EPS. (For interpretation of references to colour in this figure legend, the reader is referred to the web version of this article.)
Results of such simplified analysis of results obtained during the experiments with P. aeruginosa PAO1 and ATCC 27853 strains and pure MHB as control are shown in Figs. 9 and 10. Both species exhibited similar changes of electric properties of the biofilm but at different times: lowering of the normalized resistance at 1 MHz appears later and the rise of the conductance at 20 Hz is more pronounced for PAO1 strain. Due to the differences in the cell constant of the QTFs acting as the impedance sensors the value of resistance measured at 1 MHz was normalized for more convenient comparison. Plots
were similar to those obtained previously using equivalent circuit modelling showing drop of the resistance after several hour of incubation for both strains. The decomposition of the biofilm was also detected by analysis of the conductance measured at 20 Hz (Fig. 10) but after longer time than by equivalent circuit modelling. In addition the accidental contamination of one of the controls could also be detected. The growth of bacteria caused the drop of the high-frequency resistance however it apparently did not formed biofilm and there was not a rise of the low frequency conductance (dashed line in Figs. 9 and 10).
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4. Conclusions
Fig. 8. The decomposition of the impedance spectra of QTF on separate equivalent circuit elements at 5th day of incubation.
Evaluation of the biofilm formation of two P. aeruginosa strains was presented. The equivalent circuit modelling of impedance spectra obtained using QTFs used as the impedance sensors allowed to determine two parameters which changed in presence of bacteria. Results were in agreement with previously published [4] obtained using classical standard growth curve method and by mass measurements using QTFs and colorimetric assay. Simplified method was also proposed. It relied on the simple measurement of impedance at two characteristic frequencies (20 Hz and 1 MHz) which results were consistent with obtained using equivalent circuit modelling. Electric properties of biofilm formed by both strains changed similarly. Results allowed to conclude that QTFs may be used as impedance sensors of P. aeruginosa biofilm formation thus combined mass and impedance systems may be built. It would detect the formation of the biofilm and would not be sensitive to the planktonic forms of the bacteria. The biofilm mass measurements using QTFs require several steps of preparation [4]. The impedance measurement could be used to determine the moment at which the biofilm formed and at which the mass measurement should be done.
Acknowledgements This work was supported by European Project “Detectors and sensors for measuring factors hazardous to environment – modelling and monitoring of threats”, No. POIG.01.03.0102-002/08-00 and Polish National Science Centre grant, No. 2011/01/N/NZ7/00156.
References
Fig. 9. Changes of the normalized resistance measured at 1 MHz during first day of incubation for two P. aeruginosa strains, control and accidental contamination. Mean value and standard deviation marked by the line and area respectively.
Fig. 10. Changes of the conductance measured at 20 Hz during the incubation for two P. aeruginosa strains, control and accidental contamination. Mean value and standard deviation marked by the line and area respectively.
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Biographies Tomasz Piasecki was born in 1976 in Wroclaw, Poland. He received his PhD degree at the Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology in 2005 where he continues his scientific research. His main area of interest are the measurements of electrical properties of materials, improving the measurement methods, construction of the measuring equipment as well as the novel applications for electric measurements. Grzegorz Guła was born in 1984 in Sroda Slaska, Poland. He graduated from the Faculty of Biology Science at University of Wroclaw in 2008, receiving the MSc degree in biology with a specialization in microbiology and in 2012 the PhD degree
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at the Department of Physicochemistry of Microorganisms at University of Wroclaw, where he continues his research on the application of new methods of observation of bacterial biofilm formation. Karol Nitsch is full professor, PhD, DSc, Professor at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology. His research interests are in area of measurement and analysis of electrical properties of materials, component and sensors. He specializes on the various applications of impedance spectroscopy to microelectronics materials and structures characterization. He has authored over 190 scientific publications. Karol Waszczuk was born in 1984 in Wodzislaw Slaski, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2007, receiving the MSc degree in electronics and telecommunications. Since then, he has been working at Research and Development Department for EBS Inkjet Systems Poland. He is pursuing his PhD degree at Wroclaw University of Technology, working on biochemical and microbiological applications of resonant mass sensors. Zuzanna Drulis-Kawa was born in Wroclaw, Poland. She received the MSc and PhD degrees of biology from University of Wroclaw, Department of Nature Science in 1994 and 2000, respectively. She received first and second clinical microbiology degrees at Medical University of Wroclaw in 1997 and 2000, respectively. She has been honored with bioMerieux-PTM research award (2009) and the prize of Prof. K. Bassalik Contest (2006) for her scientific work. She is the Head of the Department of Pathogen’s Biology and Immunology in Institute of Genetics and Microbiology at University of Wroclaw, Poland. She has authored over 60 scientific publications. Teodor Gotszalk was born in Wroclaw, Poland. He received the MSc degrees from the Faculties of Electronics and of Electrical Engineering of Wroclaw University of Technology in 1989 and 1991, respectively. In 1996, he received the PhD degree from the Institute of Electronic Technology of the Wroclaw University of Technology and the full professor title in 2012. He has been honored with Siemens research award (2000) and the prize of Polish Science Foundation FNP (1997) for his scientific work. He is the head of the Division of Micro- and Nanostructures Metrology at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology. He has authored over 100 scientific publications.