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Intracellular analysis of Saccharomyces cerevisiae using CLSM after ultrasonic treatments
Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 – 163 www.nanomedjournal.com
Diagnostics
Intracellular analysis of Saccharomyces cerevisiae using CLSM after ultrasonic treatments Takuo Kon,a Shinya Nakakura,a Kohji Mitsubayashi, PhDb,T a Tokai University, Kanagawa, Japan Tokyo Medical and Dental University, Tokyo, Japan Received 25 February 2005; accepted 30 March 2005 b
Abstract
The effect of ultrasonic treatments (28, 45, and 100 kHz) as sterilization on Saccharomyces cerevisiae was investigated both by colony-forming ability and by a confocal laser-scanning microscope (CLSM), using a fluorescence staining approach with MDY-64 for endoplasmic reticula and Rhodamine B for mitochondria. The ultrasonic treatments, especially at the lower frequency of 28 kHz, were effective for sterilizing S cerevisiae, thus inducing the remarkable decrease in colony counts of S cerevisiae on YPD plate medium, but with some inactive (dead) cells. The CLSM images of fluorescence-stained organelles in the cell showed the intracellular fracture and the increase in fluorescent intensities of MDY-64 for endoplasmic reticula and Rhodamine B for mitochondria without cell membrane collapse by the ultrasonic treatments, especially at 28 kHz. The effect of the conditions (frequency, power, medium, and so on) of the ultrasonic treatments on cell components such as biological membranes would be different, thus inducing the effective and selective sterilization of some types of microorganisms. D 2005 Elsevier Inc. All rights reserved.
Key words:
CLSM (confocal laser scanning microscope); Ultrasonic treatment; Saccharomyces cerevisiae; Cell collapse; Organelle fracture; Selective sterilization
Introduction Microorganisms (such as Saccharomyces cerevisiae and Bacillus subtilis) are commonly used in the food industry and purifying facilities [1- 4]. The selective sterilization of microorganisms by external stimuli is extremely useful in meeting sanitation requirements and quality control measures [5,6]. Ultrasonic treatments are among mechanically convenient approaches for sterilization, as they do not involve detailed disinfect mechanisms [7,8]. We have investigated a biosensing system that observed the effects of ultrasonic treatments on cells by means of both intracellular and extracellular approaches [9,10]. Confocal laser scanning
No financial conflict of interest was reported by the authors of this paper. T Corresponding author. Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail address: [email protected] (K. Mitsubayashi). 1549-9634/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2005.03.006
microscopy (CLSM) is used to record individual images of fluorescence-stained organelles in cells. We used CLSM analysis (intracellular) for the evaluation of ultrasonic treatment on S cerevisiae as a model analyte of eukaryotic cells, thus taking the CLSM imaging of simultaneous stained organelles with fluorescence reagent. Then the internal damage at various ultrasonic frequencies was assessed for effectiveness as a disinfectant and as selective sterilization.
Methods S cerevisiae (YSC-2 yeast, Sigma, St. Louis, Mo), the model analyte of eukaryotic cells, was cultured by standard procedures in YPD medium (yeast extract, 10 g, polypeptone, 20 g, and glucose, 20 g in 1 L of distilled water). Cultures were derived from isolated colonies picked from culture YPD plates (with 20 g of agar) grown for 48 hours at 308C. The experimental step is shown in Figure 1. S cerevisiae was cultured in a vibrating incubator at 308C for 24 hours,
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T. Kon et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 –163
Fig 1. Procedure of microorganism analysis with (A) colony counting and (B) CLSM imaging after the ultrasonic treatments (28, 45, 100 kHz).
and then subjected to the ultrasonic treatment (VS-100III, 100 W, As-One Ltd, Japan) at frequencies of 28, 45, and 100 kHz. Effectiveness of the ultrasonic treatment was evaluated by colony counting on the plate culture after 24 hours and CLSM imaging analysis [11]. Cell deposition after centrifugation at 6400 rpm for 3 minutes (GMC-060, 100w, LMS Ltd, Japan) was mixed with buffer solution (HEPES, pH 7.4, 5% glucose). The S cerevisiae suspensions were labeled with fluorescence reagent MDY-64 (excitation wavelength 456 nm, fluorescent wavelength 505 nm) as a vacuole membrane marker for
Fig 2. Photographs of S cerevisiae colonies on YPD plate medium (A) before, (B) 10, (C) 20, and (D) 30 minutes after ultrasonic treatment (45 kHz).
endoplasmic reticula (ER), and with Rhodamine B (excitation wavelength 556 nm, fluorescent wavelength 578 nm) for mitochondria. The endoplasmic reticulum serves many general functions, including the facilitation of protein folding and transport of proteins. The primary function of mitochondria is to manufacture adenosine triphosphate, which is used as a source of energy [12]. The effect of the ultrasonic treatment at various frequencies and times on the sterilization of S cerevisiae was evaluated by changes in colony number on the plate medium. A differential interference contrast (DIC) and fluorescent (505 nm, 578 nm, and superimposed) images
Fig 3. Effect of the ultrasonic treatments (28, 45, 100 kHz) on colony counts of S cerevisiae on YPD plate medium.
T. Kon et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 –163
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Fig 4. Differential interference contrast images in S cerevisiae (A) before, (B) 30, (C) 40, (D) 60, and (E) 90 minutes after ultrasonic treatment (45 kHz).
Fig 5. Typical images of DIC, endoplasmic reticula (505 nm), mitochondria (578 nm), and superimposed in S cerevisiae (A) before, (B) 10, (C) 20, and (D) 30 minutes after ultrasonic treatment (100 kHz).
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T. Kon et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 –163
Fig 6. Fluorescence changes in MDY-64 (505 nm, ER) and Rhodamine B (578 nm, mitochondria) in S cerevisiae by the ultrasonic treatments (filled, 28 kHz; hatched, 45 kHz; open, 100 kHz).
were used for the intracellular assessment after the ultrasonic treatment. Results and discussion Figure 2 shows photographs of S cerevisiae colonies on YPD plate medium (A) before, (B) 10, (C) 20, and (D) 30 minutes after ultrasonic treatment (45 kHz). As seen in Figure 2, the number of colonies on the plate medium decreased with increasing time for the ultrasonic treatment at 45 kHz. Figure 3 illustrates the effect of ultrasonic treatments (square: 28, circle: 45, triangle: 100 kHz, n = 10) on colony counts of S cerevisiae. The colony number declined rapidly within 2 minutes, and decreased gradually with time thereafter, especially at the lower frequency, 28 kHz. Shortexposure ultrasonic treatments showed inhibitory effects on colony-forming activity, and thus sterilization of S cerevisiae.
Many nondestructive cells were, however, observed in the suspensions of S cerevisiae even after ultrasonic treatments. Figure 4 indicates the DIC images in S cerevisiae (A) before, (B) 30, (C) 40, (D) 60, and (E) 90 minutes after ultrasonic treatment (45 kHz). The intracellular organelle was destroyed with the collapse of the cell membrane in Figure 4, E, for 90 minutes, but organelle damage without cell collapse was also observed at 30, 40, and 60 minutes (Figure 4, B, C, and D, respectively). Figure 5 shows typical DIC images of ER (505 nm), mitochondria (578 nm), and superimposed in S cerevisiae (A) before, (B) 10, (C) 20, and (D) 30 minutes after ultrasonic treatment (100 kHz). Organelle damage with the scattered and brightly fluorescent light (ER and mitochondria) without cell collapse was observed. Fluorescent intensity in S cerevisiae was calculated by the operating software in CLSM (LSM5 PASCAL, Carl Zeiss Ltd) as the sum of 10 cells. Results of various fluorescence changes in MDY-64 (ER) and Rhodamine B (mitochondria) in S cerevisiae after ultrasonic treatments are shown in Figure 6. In contrast to colony count decline in Figure 3, fluorescent intensity in ER and mitochondria increase with treatment time. The increase in fluorescent intensity was correlated with dye scattering caused by the segmentation of the stained organelle membrane, especially at the lower frequency of 28 kHz (filled bar). Figure 7 shows the change in the rate of fluorescence in MDY-64 (ER) and Rhodamine B (mitochondria) in S cerevisiae at 45 kHz. Although the fluorescent rate change increased until 30 minutes for both, intensity in the ER jumped after 40 minutes because of additional dissolution of the ER. Conclusions
Fig 7. Fluorescence rate changes in MDY-64 (505 nm, ER) and Rhodamine B (578 nm, mitochondria) in S cerevisiae after ultrasonic treatment (45 kHz).
The effectiveness of ultrasonic treatment in sterilizing S cerevisiae was observed both by reduced colony-forming ability and via the CLSM using a fluorescence staining
T. Kon et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 –163
approach with MDY-64 for ER and Rhodamine B for mitochondria. In particular, ultrasonic treatments at 28 kHz were effective in sterilizing S cerevisiae, thus inducing a remarkable decrease in colony counts on YPD plate medium. The CLSM images of fluorescence-stained organelles showed intracellular breakdown and an increase in fluorescence intensities of MDY-64 for ER and Rhodamine B for mitochondria without cell membrane collapse. References [1] Senda M, Takeda J, Abe S, Nakamura T. Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol 1979; 20:1441-3. [2] Scheurich P, Zimmermann U, Schnabl H. Electrically stimulated fusion of different plant cell protoplasts. Plant Physiol 1981;67:849-53. [3] Pareilleux A, Sicard N. Lethal effects of electric current on Escherichia coli. Appl Microbiol 1970;19:421-4.
163
[4] Matsunaga T, Namba Y. Detection of microbial cells by cyclic voltammetry. Anal Chem 1984;56:798-801. [5] Matsunaga T, Namba Y, Nakajima T. Electrochemical sterilization of microbial cells. Bioelectrochem Bioenerg 1984;13:393-400. [6] Kitajima Y, Shigematsu A, Nakamura N, Matsunaga T. Electrochemical sterilization using carbon fiber. Electrochemistry 1988;56: 1082-5. [7] Fujishima A, Honda K. Electrochemical photolysis of water at a semiconductor electrode. Nature 1972;238:37-8. [8] Kawai T, Sakata T. Hydrogen evolution from water using solid carbon and light energy. Nature 1979;282:283. [9] Mitsubayashi K, Yokoyama K, Takeuchi T, Karube I. A flexible biosensor for glucose. Electroanalysis 1995;(7/1):83 - 7. [10] Mitsubayashi K, Wakabayashi Y, Ohya Y, Endo T. Optical-transparent and flexible glucose sensor with ITO electrode. Biosens Bioelectron 2003;19:67 - 71. [11] Yamamoto M, Ohya Y. Manual book for yeast experiment [Japanese]. Tokyo7 Springer-Verlag; 1998. [12] Maeno M, Isokawa K. Biochemistry and molecular biology [Japanese]. Tokyo7 Yodosya Ltd; 1999.
Diagnostics
Intracellular analysis of Saccharomyces cerevisiae using CLSM after ultrasonic treatments Takuo Kon,a Shinya Nakakura,a Kohji Mitsubayashi, PhDb,T a Tokai University, Kanagawa, Japan Tokyo Medical and Dental University, Tokyo, Japan Received 25 February 2005; accepted 30 March 2005 b
Abstract
The effect of ultrasonic treatments (28, 45, and 100 kHz) as sterilization on Saccharomyces cerevisiae was investigated both by colony-forming ability and by a confocal laser-scanning microscope (CLSM), using a fluorescence staining approach with MDY-64 for endoplasmic reticula and Rhodamine B for mitochondria. The ultrasonic treatments, especially at the lower frequency of 28 kHz, were effective for sterilizing S cerevisiae, thus inducing the remarkable decrease in colony counts of S cerevisiae on YPD plate medium, but with some inactive (dead) cells. The CLSM images of fluorescence-stained organelles in the cell showed the intracellular fracture and the increase in fluorescent intensities of MDY-64 for endoplasmic reticula and Rhodamine B for mitochondria without cell membrane collapse by the ultrasonic treatments, especially at 28 kHz. The effect of the conditions (frequency, power, medium, and so on) of the ultrasonic treatments on cell components such as biological membranes would be different, thus inducing the effective and selective sterilization of some types of microorganisms. D 2005 Elsevier Inc. All rights reserved.
Key words:
CLSM (confocal laser scanning microscope); Ultrasonic treatment; Saccharomyces cerevisiae; Cell collapse; Organelle fracture; Selective sterilization
Introduction Microorganisms (such as Saccharomyces cerevisiae and Bacillus subtilis) are commonly used in the food industry and purifying facilities [1- 4]. The selective sterilization of microorganisms by external stimuli is extremely useful in meeting sanitation requirements and quality control measures [5,6]. Ultrasonic treatments are among mechanically convenient approaches for sterilization, as they do not involve detailed disinfect mechanisms [7,8]. We have investigated a biosensing system that observed the effects of ultrasonic treatments on cells by means of both intracellular and extracellular approaches [9,10]. Confocal laser scanning
No financial conflict of interest was reported by the authors of this paper. T Corresponding author. Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. E-mail address: [email protected] (K. Mitsubayashi). 1549-9634/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2005.03.006
microscopy (CLSM) is used to record individual images of fluorescence-stained organelles in cells. We used CLSM analysis (intracellular) for the evaluation of ultrasonic treatment on S cerevisiae as a model analyte of eukaryotic cells, thus taking the CLSM imaging of simultaneous stained organelles with fluorescence reagent. Then the internal damage at various ultrasonic frequencies was assessed for effectiveness as a disinfectant and as selective sterilization.
Methods S cerevisiae (YSC-2 yeast, Sigma, St. Louis, Mo), the model analyte of eukaryotic cells, was cultured by standard procedures in YPD medium (yeast extract, 10 g, polypeptone, 20 g, and glucose, 20 g in 1 L of distilled water). Cultures were derived from isolated colonies picked from culture YPD plates (with 20 g of agar) grown for 48 hours at 308C. The experimental step is shown in Figure 1. S cerevisiae was cultured in a vibrating incubator at 308C for 24 hours,
160
T. Kon et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 –163
Fig 1. Procedure of microorganism analysis with (A) colony counting and (B) CLSM imaging after the ultrasonic treatments (28, 45, 100 kHz).
and then subjected to the ultrasonic treatment (VS-100III, 100 W, As-One Ltd, Japan) at frequencies of 28, 45, and 100 kHz. Effectiveness of the ultrasonic treatment was evaluated by colony counting on the plate culture after 24 hours and CLSM imaging analysis [11]. Cell deposition after centrifugation at 6400 rpm for 3 minutes (GMC-060, 100w, LMS Ltd, Japan) was mixed with buffer solution (HEPES, pH 7.4, 5% glucose). The S cerevisiae suspensions were labeled with fluorescence reagent MDY-64 (excitation wavelength 456 nm, fluorescent wavelength 505 nm) as a vacuole membrane marker for
Fig 2. Photographs of S cerevisiae colonies on YPD plate medium (A) before, (B) 10, (C) 20, and (D) 30 minutes after ultrasonic treatment (45 kHz).
endoplasmic reticula (ER), and with Rhodamine B (excitation wavelength 556 nm, fluorescent wavelength 578 nm) for mitochondria. The endoplasmic reticulum serves many general functions, including the facilitation of protein folding and transport of proteins. The primary function of mitochondria is to manufacture adenosine triphosphate, which is used as a source of energy [12]. The effect of the ultrasonic treatment at various frequencies and times on the sterilization of S cerevisiae was evaluated by changes in colony number on the plate medium. A differential interference contrast (DIC) and fluorescent (505 nm, 578 nm, and superimposed) images
Fig 3. Effect of the ultrasonic treatments (28, 45, 100 kHz) on colony counts of S cerevisiae on YPD plate medium.
T. Kon et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 –163
161
Fig 4. Differential interference contrast images in S cerevisiae (A) before, (B) 30, (C) 40, (D) 60, and (E) 90 minutes after ultrasonic treatment (45 kHz).
Fig 5. Typical images of DIC, endoplasmic reticula (505 nm), mitochondria (578 nm), and superimposed in S cerevisiae (A) before, (B) 10, (C) 20, and (D) 30 minutes after ultrasonic treatment (100 kHz).
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T. Kon et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 –163
Fig 6. Fluorescence changes in MDY-64 (505 nm, ER) and Rhodamine B (578 nm, mitochondria) in S cerevisiae by the ultrasonic treatments (filled, 28 kHz; hatched, 45 kHz; open, 100 kHz).
were used for the intracellular assessment after the ultrasonic treatment. Results and discussion Figure 2 shows photographs of S cerevisiae colonies on YPD plate medium (A) before, (B) 10, (C) 20, and (D) 30 minutes after ultrasonic treatment (45 kHz). As seen in Figure 2, the number of colonies on the plate medium decreased with increasing time for the ultrasonic treatment at 45 kHz. Figure 3 illustrates the effect of ultrasonic treatments (square: 28, circle: 45, triangle: 100 kHz, n = 10) on colony counts of S cerevisiae. The colony number declined rapidly within 2 minutes, and decreased gradually with time thereafter, especially at the lower frequency, 28 kHz. Shortexposure ultrasonic treatments showed inhibitory effects on colony-forming activity, and thus sterilization of S cerevisiae.
Many nondestructive cells were, however, observed in the suspensions of S cerevisiae even after ultrasonic treatments. Figure 4 indicates the DIC images in S cerevisiae (A) before, (B) 30, (C) 40, (D) 60, and (E) 90 minutes after ultrasonic treatment (45 kHz). The intracellular organelle was destroyed with the collapse of the cell membrane in Figure 4, E, for 90 minutes, but organelle damage without cell collapse was also observed at 30, 40, and 60 minutes (Figure 4, B, C, and D, respectively). Figure 5 shows typical DIC images of ER (505 nm), mitochondria (578 nm), and superimposed in S cerevisiae (A) before, (B) 10, (C) 20, and (D) 30 minutes after ultrasonic treatment (100 kHz). Organelle damage with the scattered and brightly fluorescent light (ER and mitochondria) without cell collapse was observed. Fluorescent intensity in S cerevisiae was calculated by the operating software in CLSM (LSM5 PASCAL, Carl Zeiss Ltd) as the sum of 10 cells. Results of various fluorescence changes in MDY-64 (ER) and Rhodamine B (mitochondria) in S cerevisiae after ultrasonic treatments are shown in Figure 6. In contrast to colony count decline in Figure 3, fluorescent intensity in ER and mitochondria increase with treatment time. The increase in fluorescent intensity was correlated with dye scattering caused by the segmentation of the stained organelle membrane, especially at the lower frequency of 28 kHz (filled bar). Figure 7 shows the change in the rate of fluorescence in MDY-64 (ER) and Rhodamine B (mitochondria) in S cerevisiae at 45 kHz. Although the fluorescent rate change increased until 30 minutes for both, intensity in the ER jumped after 40 minutes because of additional dissolution of the ER. Conclusions
Fig 7. Fluorescence rate changes in MDY-64 (505 nm, ER) and Rhodamine B (578 nm, mitochondria) in S cerevisiae after ultrasonic treatment (45 kHz).
The effectiveness of ultrasonic treatment in sterilizing S cerevisiae was observed both by reduced colony-forming ability and via the CLSM using a fluorescence staining
T. Kon et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 159 –163
approach with MDY-64 for ER and Rhodamine B for mitochondria. In particular, ultrasonic treatments at 28 kHz were effective in sterilizing S cerevisiae, thus inducing a remarkable decrease in colony counts on YPD plate medium. The CLSM images of fluorescence-stained organelles showed intracellular breakdown and an increase in fluorescence intensities of MDY-64 for ER and Rhodamine B for mitochondria without cell membrane collapse. References [1] Senda M, Takeda J, Abe S, Nakamura T. Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol 1979; 20:1441-3. [2] Scheurich P, Zimmermann U, Schnabl H. Electrically stimulated fusion of different plant cell protoplasts. Plant Physiol 1981;67:849-53. [3] Pareilleux A, Sicard N. Lethal effects of electric current on Escherichia coli. Appl Microbiol 1970;19:421-4.
163
[4] Matsunaga T, Namba Y. Detection of microbial cells by cyclic voltammetry. Anal Chem 1984;56:798-801. [5] Matsunaga T, Namba Y, Nakajima T. Electrochemical sterilization of microbial cells. Bioelectrochem Bioenerg 1984;13:393-400. [6] Kitajima Y, Shigematsu A, Nakamura N, Matsunaga T. Electrochemical sterilization using carbon fiber. Electrochemistry 1988;56: 1082-5. [7] Fujishima A, Honda K. Electrochemical photolysis of water at a semiconductor electrode. Nature 1972;238:37-8. [8] Kawai T, Sakata T. Hydrogen evolution from water using solid carbon and light energy. Nature 1979;282:283. [9] Mitsubayashi K, Yokoyama K, Takeuchi T, Karube I. A flexible biosensor for glucose. Electroanalysis 1995;(7/1):83 - 7. [10] Mitsubayashi K, Wakabayashi Y, Ohya Y, Endo T. Optical-transparent and flexible glucose sensor with ITO electrode. Biosens Bioelectron 2003;19:67 - 71. [11] Yamamoto M, Ohya Y. Manual book for yeast experiment [Japanese]. Tokyo7 Springer-Verlag; 1998. [12] Maeno M, Isokawa K. Biochemistry and molecular biology [Japanese]. Tokyo7 Yodosya Ltd; 1999.