Anal. Chem. 1995,67,4487-4490
Direct Count of Bacteria Using Fluorescent Dyes: Application to Assessment of Electrochemical Disinfection Tadashi Matsunaga,* Mina Okochi, and Satoshi Nakasono
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan
A rapid method for direct counting of viable and dead cells using two fluorescent dyes, propidium iodide and 4,6diamidino-2-phenylindole, was developed for estimation of cell viability after electrochemical disinfection. Using this double staining method, dead and viable cells were visualized as red and blue fluorescent cells, respectively, under UV excitation employing epifluorescence microscopy. A good correlation between the red cell ratio and the ratio of dead cells determined by the plate count method was obtained for Vibrio alginolyticus cells exposed to heat (60 "C), 10 ppm of chlorine, 3.7%of formaldehyde, and electrochemical disinfection treatment, Rapid enumeration and estimation of bacterial cell viability is important for assessing the movement of pathogens and for preventing bacterial colonization or biofilm formation on drinking water systems and on cooling towers. The plate count method is the conventional method for assessing the cell viability. However, this method is time consuming (12-24 h for incubation) and may often underestimate bacterial viability. In additip, the plate count method cannot be used to observe bacterial cells directly in situ, especially when the cells are attached on the solid surface. We have investigated the electrochemical disinfection of This method does not generate toxic substances such as trihalomethanes and is useful for disinfection of contaminated bacteria in drinking ~ a t e r . ~This . ~ disinfection method also prevents the formation of biofilm and bi~fouling.~f The efficiency of this disinfection method has been estimated by the plate count method. In the plate count method, the number of disinfected cells and the number of cells that remain adsorbed on the electrode surface were not determined. Therefore, a direct count method that can determine the number of disinfected cells adsorbed on the electrode surface is necessary for accurate estimation of the efficiency of the electrochemical disinfection method. (1) Matsunaga, T.; Namba, Y.; Nakajima, T.Bioelectrochem. Bioenerg. 1984, 13, 393-400. (2) Kitajima, Y.;Shigematsu, A; Nakamura, N.; Matsunaga, T. Denki Kuguku 1988,56,1082-85. (3) Matsunaga, T.; Nakasono, S.; Takamuku, T.; Burgess, J. G.; Nakamura, N.; Sode, IC Appl. Environ. Microbiol. 1992,58,686-89. (4) Matsunaga, T.;Nakasono, S.; Kitajima, Y.; Horiguchi, K Biotechnol. Bioenerg. 1994,43, 429-33. (5) Nakasono, S.; Nakamura, N.; Sode, IC; Matsunaga, T. Bioelectrochem. Bioene?g. 1992,27, 191-98. (6) Nakasono, S.; Burgess, J. G.; Takahashi, K.; Koike, M.; Murayama, C.; Nakamura, S.; Matsunaga, T. Appl. Environ. Microbiol. 1993,59,375762.
0003-2700/95/0367-4487$9.00/0 0 1995 American Chemical Society
Recently, several staining methods have been used for determining the cell viability directly with epifluorescence microscopic observation. Tetrazolium salts7-'0 and fluore~ceinl~-'~ were often used for determining the cell viability on the basis of the cell metabolic activity. However, with tetrazolium salts, it takes more than 1h for the formation of formazan. In the case of fluorescein diacetate, many bacteria could not transport it into the cell, and the esterase activity does not directly provide useful information about the cell viability. Ethidium derivatives such as propidium iodide (PI) are capable of passing through only damaged cell membranes and intercalate with the nucleic acids of injured and dead cells to form a bright red fluorescent ~ o m p l e x . ~ ~ J ~ J ~ In the present study, we have examined PI and 4',&diamidino2-phenylindole (DAPI) for estimation of Vibrio alginolyticus cell viability in various disinfection treatments. V. alginolyticus cell viability was also estimated by the direct count method in the electrochemical disinfection treatment. This method is user friendly and requires only 1min of incubation. With this method, it might be possible to estimate directly the cell viability of biofilm formed on a natural aquatic environment. EXPERIMENTAL SECTION
Materials. Bacto marine broth 2216 was purchased from Difco Laboratories (Detroit, MI). Fluorescent dyes PI and DAPI were obtained from Molecular Probes, Inc. (Pitchford, OR) and Merck (Darmstadt, Germany), respectively. Other reagents were commercially available analytical reagents or laboratory grade materials. Seawater from the Miura Peninsula Uapan) was sterilized by autoclaving for 1min and filtering with a 0.2 pm pore size membrane filter. Preparation of V. alginolyticus. V. alginolyticus ATCC 17749 was cultured aerobically at 25 "C for 10 h in 10 mL of marine broth after preculture for 12 h under the same conditions. The cells were centrifuged at 1600g at room temperature for 10 min, (7)Zimmermann, R.; Ituniaga, R.; Becker-Birck, J. Appl. Environ. Microbiol. 1978,36,926-35. (8) Severin, E.; Stellmach, J.; Nachtigal, H.-M. Anal. Chim. Acta 1985,170, 341-46. (9) Stellmach, J.; Steverin, E. Histochem. j . 1987,19, 21-26. (10) Rodriguez, G. G.; Phipps, D.; Ishiguro, K; Ridgway, H. F. Appl. Environ. Microbiol. 1992,58,1801-08. (11) Lundgren, B. Oikos 1981,36, 17-22. (12)Jones, IC H.; Senft, J. A 1.Histochem. Cytochem. 1985,33,77-79. (13) Schupp, D. G.; Erlandsen. S. L. Appl. Enuiron. Microbiol. 1987,53,70407. (14) Horan, P. K; Kappler, J. W. J Immunol. Methods 1977,18, 309-16. (15) Sauch, J. F.; Flanigan, D.; Galvin, M. L.; Berman, D.; Jakubowski, W. Appl. Environ. Microbiol. 1991,57,3243-47.
Analytical Chemistry, Vol. 67,No. 24,December 15, 1995 4487
washed, and resuspended in sterile seawater (PH 8.0). Cell concentration was determined by a hemacytometer. Disinfection of V. alginolyticus Cells by Several Methods. Electrochemical Disinfection. The electrochemical disinfection system consisted of a potentiostat (Hokuto Denko, Model HA-151,Tokyo, Japan), a basal-plane pyrolytic graphite electrode (surface area, 2.27 cm2) as a working electrode, a saturated calomel electrode (SCE) as a reference electrode, and a carbon fiber as a counter electrode were used. J! alginolyticus cells (5.0 x lo8cells) were immobilized on the membrane filter (pore size, 0.45 pm), and the filter was attached to the electrode. A potential of 1.0 V vs SCE was applied for 30 min in sterile seawater. Exposure to Formaldehyde. J! alginolyticus cells were suspended in sterile seawater at a cell concentration of 105 cells/ mL. One hundred microliters of 37%formaldehyde solution was added to 900 pL of cell suspension, and the mixture was incubated for 5 min at room temperature. Cells were centrifuged at 1600g and resuspended in sterile seawater three times. Exposure to Chlorine. Sodium hypochlorite solution containing 5%available chlorine was added to a V. alginolyticus cell suspension (10: cells/mL), for a final chlorine concentration of 10 ppm. A cell suspension containing sodium hypochlorite solution was incubated for 10 min at room temperature and washed three times by centrifugation in sterile seawater. Heat Exposure. A K alginolyticus cell suspension (lo5 cells/ mL) was incubated at 60 "C for 30 min and then cooled to room temperature. Fluorescent Spectra of V. alginolyticus. The emission spectra of PI and DAPI in viable or dead V. alginolyticus cell suspension were measured with a spectrofluorometer (Shimadzu Co., Ltd., RF-5000,Kyoto, Japan). The fluorescence spectra were obtained at 555 nm excitation for PI and at 355 nm excitation for DAPI. Dead cells were obtained by exposing V. alginolyticus cells to 3.7% formaldehyde as described above. PI and DAPI were dissolved in sterile seawater (pH 8.0) and stored at 4 "C in the dark before use. PI and DAPI concentrations were 100 and 10 pg/mL, respectively. Determination of V. alginolyticus Viability by the Plate Count Method. The numbers of viable cells were determined by plating suitably diluted samples and counting the colonies which appeared on marine broth agar plates after 24 h of incubation at 25 "C. The dead cell ratio was defined as follows: dead cell ratio (%) = [(viable cell number before treatment) (viable cell number after treatment) I / (viable cell number before treatment) x 100
Determination of V. alginolyticus Viability by the Direct Viable Count Method. V. alginolyticus cells were stained first with PI and then with DAPI. The cells stained with PI and DAPI were observed with an epifluorescence microscope (Olympus Optical Co., Ltd., IMT-2, Tokyo, Japan) under W excitation (wavelength, 300-400 nm), and four positions were photographed for each condition. Dead and viable cells were distinct as the red fluoresced cells and bright blue fluoresced cells, respectively. 4488 Analytical Chemistry, Vol. 67,No. 24,December 75, 7995
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Figure 1. Fluorescence emission spectra of V. alginolyficus cell suspension (1.8 x lo9 cells/mL) stained with PI (A) or DAPl (B). Excitation wavelength for the measurement of fluorescence spectra was 555 nm with PI and 355 nm with DAPI. (a) PI (100 pglmL) dead cells. (b) PI (100pglmL) viable cells. (c) PI (100pglmL). (d) DAPl (10 ,uglmL) dead cells. (e) DAPl (10 pg/mL) viable cells. (f) DAPl (10 ,uglmL).
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More than 100 fluorescence cells were then enumerated, and the PI-stained cell ratio was defined as follows: PI-stained cell ratio (%)= (red fluorescence cell number/blue fluorescence cell and red fluorescence cell number) x 100
Direct Determination of V. alginolyticus Viability after Electrochemical Disinfection Using PI and DAPI. The electrochemical disinfection system was constructed as described in the Electrochemical Disinfection section. A basal-plane pyrolytic graphite electrode was immersed in seawater containing lo7 cells/mL of V. alginolyticus stirred at 350 rpm for 2.5 h at room temperature. Cells were adsorbed on the electrode surface. The electrode was then immersed once in sterile seawater to remove unabsorbed cells. The potential was applied for 20 min in sterile seawater. After the potential was applied, cells adsorbed on the electrode were stained with PI (500 pg/mL) and DAPI (10 pg/ mL). Cells adsorbed on the electrode were observed with an epifluorescence microscope under W excitation and photographed. RESULTS AND DISCUSSION Fluorescence Spectra of V. alginolyticus Cell Suspen-
sion Stained with PI or DAPI. The fluorescence spectra of viable and dead V. alginolyticus cells stained with PI are represented in Figure L4. Dead cells were obtained by exposing K alginolyticus cells to 3.7%formaldehyde and washing them with sterile seawater three times. The dead cell ratio was determined to be 100%by the plate count method. The fluorescence intensity of dead cells was very high, while the fluorescence intensity of viable cells was the same as the fluorescence intensity of PI alone. Using PI, selective staining of dead cells was camed out. Figure 2A shows the epifluorescence micrograph of dead cells stained with PI. Dead cells obtained by formaldehyde treatment show red fluorescence under UV excitation. However, viable cells stained with PI were not observed under the same conditions. The concentration of PI necessary for staining V. alginolyticus cells
I Figure 2. Epifluorescence micrographs under UV excitation. (B) DAPI (7 pg/mL)
of V. a/gino/yiicuscells. (A) PI (83@g/mL) + dead cells treated with 3.7% formaldehyde for 5 min
+ viable cells under UV excitation.
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Figure 3. Relationship between dead cell ratio and PI-stained cell ratio of V. alginolyiicus treated with various disinfection methods. COnCentrations of DAPI and PI were 7 and 83 pgmL. respectively. (0)Electrochemica disinfection. (0)Heat (60 "C, 30 min). (0)Chlorine (IO ppm, 5 min). (A)Formaldehyde (3.7%,10 min). (m) Control.
was above 50 Ng/mL. It was confirmed that dead cells were selectively observed when PI was used under UV excitation. Figure 1B shows the fluorescence spectra of viable and dead K alsinolyfinrs cells stained with DAPI. The fluorescence intensities of both viable and dead cells were larger than the fluorescence intensity of DAPI alone. In the epitluorescence micrograph, viable cells stained with DAFl were observed as a bright blue color under UV excitation (Figure 2B). DAPI is an AT-selective DNA stain, and bacteria were identitied from the blue fluorescence under UV excitation?6 Therefore, if the cells were stained with PI and DAPI, the dead cells and viable cells might show red and blue fluorescence. respectively. Estimation of Viibility of Cells Treated With Different Disinfection Methods. Figure 3 shows the viability of cells b a t e d with various disinfection methods, determined by the plate count method and the direct count method with PI and DAFT K alginolytictcs cells were disinfected by exposing cells to heat (60 "C),lO ppm of chlorine, 3.7% of formaldehyde, and a potential of 1.0 V vs SCE. The dead cell ratio and the PI-stained cell ratio were estimated by the plate count method and the direct count method, respectively, using PI and DAPI. The plate count method and the direct count method gave similar results when the cell (16) Kubista
M.:A k e m , B.: Norden, B. Bioehentisto 1987.26.4545-53.
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Potential (V V I . SCE) Figure 4. Effect of applied potential on PI-stained cell ratio of V.
alginolyiicus cells adsorbed on the basal-plane pyrolytic graphite electrode. The potential was applied for 20 min in sterile seawater (PH 8.0). Concentrations of DAPl and PI were 10 and 100 pS/mL, respectively. suspension was exposed to heat, chlorine, formaldehyde, and electrochemicaltreatment. Disinfection using heat, chlorine, and formaldehyde could rapidly change the cell membrane perme ability. As cell viability coordinated with the plate count method, the cell membrane permeability might be also changed by electrochemical treatment. It was shown that rapid determination of cell viability was carried out using the direct count method when cell membrane permeability was changed by disinfection treatment. Estimation ofCell Vnbility affer Electrochemical Disinfection by the Direct Count Method. A constant potential was applied to K alginolyticus cells adsorbed onto the graphite electrode, and cell viability was estimated by the direct count method. Figure 4 shows the effect of applied potential on the PI-stained cell ratio of K alginolyticus. The PI-stained cell ratio was
