A new rapid technique for detection of microorganisms using bioluminescence and fluorescence microscope method

JOURNALOF BIOSCIENCE AND BIOENGINEERING Vol. 89, No. 5, 509-513. 2000

A New Rapid Technique for Detection of M icroorganisms Using Biolum inescence and FluorescenceM icroscope Method TOSHIHIRO TAKAHASHI,* YASUKAZU NAKAKITA, JUNJI WATARI,

AND

KEN SHINOTSUKA

Brewing Research Laboratories, Sapporo Breweries Ltd., 10 Okatome, Yaizu, Shizuoka 425-0013, Japan Received 1 September 1999/Accepted 3 February 2000

The MicroStar-RMDS-SPS (RMDS; Rapid Microbe Detection System) enables detection and determination of the number of microorganisms trapped on a membrane filter based on the ATP-bioluminescence method. However, this system could not provide information about the identity of contaminating speciesbased on the measurement results. Therefore, we developed a new technique for observing microcolonies of contaminants using a fluorescence microscope (Micro Scanner). The coordinates in the image data of the RMDS were converted to the coordinates of the sample stage of the microscope, and a microcolony could be easily observed. A single yeast cell and a microcolony consisting of 20-30 lactic acid bacterial cells could be observed on a membrane filter after measurement using the RMDS. [Key words:

MicroStar-RMDS-SPS,

bioluminescence, Micro Scanner, rapid detection]

The M icroStar-RMDS-SPS (RMDS, developed by Sapporo Breweries Ltd., Tokyo, in cooperation with Nihon M illipore Ltd., Tokyo), including the membrane filter technique, ATP-bioluminescencemethod and photon counting technique,was developedfor rapid quantification of m icrobial contaminants in food products (1, 2). The RMDS was designed for industrial use, i.e., it can analyze many samples in a short period with high operation stability (2). Moreover, the RMDS is a very highly sensitive detection system that can, within 24 h, detect lactic acid bacteria, which form colonies between 2 and 4 d (1). The effectiveness of the system was confirmed by a test for its practicality and it is already being used in our brewing plant (1, 2). The RMDS detects ATP regardlessof its origin, so it cannot provide information about the identity of m icroorganisms detected. The system guaranteesthe detection and determination of the number or population of m icrobial contaminants in food products. However, it is more desirable to be able to identify the contaminants with respect to the process control of the product line. The RMDS should be used in combination with other methods to discriminate the speciesof contaminants. Some methods that can fulfill the above requirement have been reported; the use of selective medium for specific bacteria (Application sheet, Nihon M illipore Ltd.), the PCR technique (3), and the enzyme-linkedimmunoassay method (4, 5). Although these methods can be applied to the identification of certain species of m icroorganisms, it was difficult to use them in an industrial scale because of the complexity of operation and the insufficient accuracy, e.g., the false positive results and the low detection lim it. On the other hand, the m icrocolony fluorescencestaining method (5) is simpler than other rapid detection methods and the morphology of m icrobial cells can be observed for a short time. However, the system has an incomplete image processing to discriminate the m icrocolonies of m icroorganisms * Corresponding author. Abbreviations: RMDS, MicroStar-RMDS-SPS; RLU, Relative Light Unit; SBC, Sapporo Breweries Culture Collection, Yaizu, Japan.

509

from other fine particles, thus the m isinterpretation occasionally occurred (5). If a person observesthe samples instead of an automated inspector, fine distinction between colors and of forms is possible. But, it is physically impossible to observe many samples on an ordinary 47 mm-diameter membranefilter in a short time. We consider that this method is effective for observation of the morphology of m icroorganisms in a lim ited field of view. Therefore, we combined two simple methods (RMDS and fluorescencem icroscope method) and developed a new and simple technique that can distinguish the morphology of unknown contaminants, which are detected by RMDS, in products, using a fluorescencem icroscope with an automatic sample stage (Micro Scanner, Daiichi Seimitsu Ltd., Yaizu, Shizuoka) (5). Our results indicate that this combined system can compensatefor deficiencies of each system and will be a more practical system for a brewing plant. Principle of analysis of luminous spot in image data of RMDS The cells were trapped on a polycarbonate membrane filter (Isoporefilter with ring, Nihon M illipore), and ATP was extracted using an extracting reagent (RMD reagent kit, Nihon M illipore) on the filter. The filter was put on the sample stage of the luminous detector after spraying it with a luminous reagent (RMD reagent kit, Nihon M illipore). ATP bioluminescence spots were obtained by a 90-s photon counting. These spots indicated the number of living cells (1, 2). The position of the spot on the filter could be shown as (x, y) coordinates on the image display. The method of changing the coordinates of the RMDS to those of the sample stage of the M icro Scanneris as follows. To prepare the “standard filter”, spot of a O.Zmicroliters ATP solution (1 X lo-l4 mol) containing 0.00005% methylene blue (Wako Pure Chem. Ind. Ltd., Osaka) was applied at any three points on a membrane filter. Points were marked on the edge of the filter and along the sample holders of the luminous detector of the RMDS and of the M icro Scanner. These points were the origins of the filter and both the devices. The origin of the filter was then set to a constant position on the sample stage of each device. The filter was applied to RMDS and the

510

TAKAHASHI

J. BIOSCI.BIOENG.,

ET AL.

coordinates of the bioluminescent spots were measured by the RMDS software. The coordinates of these three points were defined as (xi, yi), (x2, ~2) and (x3, ~3). The filter was then set on the sample stageof the M icro Scanner with the (X, Y) coordinate. A spot containing methylene blue was moved under the view of the m icroscope and the (X, Y) coordinate of this position was recorded. The coordinates of the stage of the M icro Scanner, which correspondedto (xi, yr), (x2, ye) and (x3, y3 of the RMDS were defined as (X1, Yr), (X2, Y,) and (X3, Y3). The coordinates, (x3, yj) and (X3, Y,), were set as the temporary origin of each device and the relative distances of (xi, yr) and (x2, yz) from (x3, y3), and of (X1, Y,) and (X2, Y,) from (X3, Y,) were used. These coordinates, (XI, ~1) and (Xl, YI), (x2, ~2) and (X2, Yz), were

substituted into Expression 1, and the coefficients a, b, c, and d were determined. ($) = (z $cIG:)

+ (2)

Expression 1

Expression 1 is used for conversion of the surveyed coordinates of RMDS, (xi, yi) to calculated coordinates of M icro Scanner, (Xi’, xc). Next, the method of sample measurementis as follows. After measurementusing the RMDS, the filter was stained with 0.2% acridine orange (Wako Pure Chem.) for observation with the fluorescencem icroscope and set on the sample stage of the M icro Scanner. The surveyed coordinate (Xi, yi) of the luminous spot in the image data was then substituted into Expression 1 and converted to the calculated coordinate (Xi, YJ of the M icro Scanner. The area around the coordinate (Xi, YJ in the filter was usually observed under the fluorescencem icroscope at 100x magnification. In cooperation with Daiichi Seim itsu Ltd., we developed the calculation software that converted the coordinates of the RMDS to those of the M icro Scanner based on Expression 1. The coordinate (Xi, yi) of the RMDS was input to the M icro Scannerand the sample stage of the M icro Scanner automatically moved the position of the calculated coordinate (Xi, YJ under the view of the fluorescencem icroscope. Next, the sample stage of the M icro Scanner then automatically started wheel moving (wheel scanning) around (Xi, Y). Operators stopped wheel scanning when the stained m icrocolony was under the view and observed the mor-

FIG. 1. Image data of brewing yeast using RMDS. Thirteen luminous spots are single brewing yeast cells.

phology of cells using a higher magnification lens (400x or 600 x magnification). Observation of brewing yeast and lactic acid bacteria

The yeast cells were suspendedin sterilized water, and a certain volume of suspensioncontaining about 20 cells was filtered using the membrane filter. The number of cells on the filter was measuredwith the RMDS without cultivation and the image data is shown in Fig. 1 in which 13 yeast cells were detected. On the other hand, the coefficients a, b, c and d of Expression 1 was determ ined using the following coordinates which were measured using the standard filter; (386, 134), (235, 255) and (226, 94) of the RMDS, (50.81, 16.34), (35.88, 28.88) and (34.93, 12.63)of the M icro Scanner, and were calculated to be 0.099, 0, -0.002 and 0.101, respectively.The coordinates of the luminous spot in the image data of the RMDS, (xi, yi), are presentedin Table 1. The calculated coordinates of the M icro Scanner, (X,“, Yi’), were obtained using Expression 1. The views around (X,“, Y:) were observedwith the M icro Scanner and all of the 13 single yeast cells could be easily seen (Fig. 2). The cell emitted an orange fluorescencein the green background. The cells maintained their oval shape and were not deformed by treatment with the ATP extracting reagent, which consisted of alcohol and ammonia. The coordinates of the M icro Scanner, (Xi, YJ, on which the single yeast cells could be actually observed are shown on the next row of (Xi”, Y:). Differencesbetween the calculated coordinates and actual ones were calculated as (XiXi”, yi- yic). The maximum differencesin (Xi-X:) and (YiYic)were 1.43m m and 0.53 m m , respectively.Fundamentally, the cells or the m icrocolonies were searched for under 100x magnification; one view of this magnification was within 0.76mm radius. Based on the above maxim u m difference, i.e., (Xi-Xf, Yi-Yi’)=(l?r 1.43m m , to.53 mm), the yeast cell could be searchedinside the area of 2.86 x 1.06m m , which corresponds to about 8 fields of view (0.76 x 0.76 mm) under 100x magnification. In order to search for a single yeast cell on a 47-mm-diameter filter under 100x magnification, it is necessaryto observe 3000 fields of view, i.e., 1734mm2.As long as the observation views are considerably restricted, it is extremely difficult to find a single yeast cell within a short time with any current techniques. Such high sensitivity and accuracy would not be possible by fluorescence m icroscopy alone. Lactobacillus brevis SBC 8008 suspendedin beer (350 m l) was filtered with the membrane filter. The bacteria

FIG. 2. A single yeast cell observed under a fluorescence microscope ( x 600).

VOL.

89, 2000

TECHNICAL TABLE

Spot no.

1.

Coordinates of spot in image data xi

1 2 3 4 5 6 7 8 9 10 11 12 13

25.57 24.69 28.72 27.37 32.79 23.86 27.28 26.82 36.71 30.39 20.87 16.87 16.83

25.05 28.61 28.82 35.35 37.54 39.31 40.11 42.48 43.09 45.85 46.63 44.54 46.62

on the filter were anaerobically cultivated on M-NBB m e d ium (modified Nachweismedium fur biershadlich Bakterien), containing 0.5% caseinpeptone, 0.5% yeast extract, 0.2% meat extract, 0.05% Tween 80, 0.6% CHrCOOK, 0.05% L-malic acid, 0.2% K2HPOI, 0.02% L-cysteine-HCl,1.5% glucoseand 1.5% m a ltose, (pH 5.2) (6), at 30°C for 24 h, and their number was determined using the RMDS. Forty-four bioluminescencespots were detected (image data not shown) and 34 coordinates, which were randomly selected,out of 44 spots, were analyzed (Table 2). Although fine particles in the beer existTABLE spot no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Coordinates of spot in image data

2.

Practical coordinates of Micro Scanner

Differences (mm)

xi

yi

xj-Xi’

yi-yic

26.11 28.96 29.76 35.07 37.17 39.24 40.33 43.75 44.52 45.91 46.78 44.71 46.66

25.31 25.22 28.73 26.87 32.50 23.81 26.91 27.21 37.11 30.38 21.04 16.97 16.88

1.06 0.35 0.94 -0.28 -0.37 -0.07 0.22 1.27 1.43 0.06 0.15 0.17 0.04

-0.26 0.53 0.01 -0.50 -0.29 -0.05 -0.37 0.39 0.40 -0.01 0.17 0.10 0.05

ed on the filter surface, the m icrocolonies could be observedunder a fluorescencem icroscopearound the calculated coordinate of the M icro Scanner, except for spot no. 28. The reasonwhy this spot was not identified is discussedin the next paragraph. The maximum differences in (Xi-Xi’) and (Yi-Yi’) were 1.55m m and 1.59m m , respectively, i.e., these m icrocolonies could be found within an area of 3.2 x 3.2 m m , in which the calculated coordinates of the M icro Scanner were located as the centerpoint. The area correspondsto 16 fields of view at 100x m a g n ification. The apparentnumber of cells form-

Image data analysis of luminous spots of L. brevis SBC 8008

Calculated coordinates of Micro Scanner

Xi

Yi

.

XiC

y;c

186 208 262 300 320 358 312 324 298 228 160 148 58 156 192 204 236 280 314 328 338 408 342 284 252 202 220 124 142 242 254 298 314 200

54 56 66 56 68 96 114 130 144 167 156 184 260 244 248 212 236 236 200 190 178 232 270 266 258 306 338 360 378 428 358 334 312 282

30.96

8.67 8.83 9.73 8.64 9.81 12.56 14.47 16.06 17.53 20.00 19.03 21.89 29.75 27.93 28.26 24.60 26.96 26.87 23.16 22.12 20.89 26.20 30.17 29.89 29.15 34.10 37.30

33.14 38.49 42.25 44.23 48.00 43.45 44.64 42.07 35.14 28.41 27.22 18.32 28.03 31.59 32.77 35.95 40.30 43.66 45.05 46.04 52.98 46.45 40.70 37.53 32.59 34.38 24.88 26.66 36.57 37.75 42.10 43.68 32.39

511

Image data analysis of luminous spots of brewing yeast

Calculated coordinates of Micro Scanner Xi’ Yf

Yi 220 212 252 240 294 206 240 236 334 272 178 138 138

126 162 164 230 252 270 278 302 308 336 344 323 344

NOTES

39.72 41.50 46.35 39.25 36.73 34.48 31.68

Practical coordinates of Micro Scanner xi

Y;

32.14 9.20 34.21 9.23 39.26 9.78 42.99 9.20 44.88 9.79 48.96 12.48 44.52 14.38 45.57 15.82 43.11 17.08 36.29 19.39 29.08 18.85 28.16 21.65 19.70 29.88 28.65 27.59 32.64 28.24 33.61 24.24 36.74 26.77 41.43 26.56 44.64 22.87 46.06 21.57 47.32 20.78 54.33 25.78 47.85 29.84 41.85 29.78 38.35 28.94 34.07 34.28 35.67 37.03 Unidentified 27.95 41.31 38.05 46.11 38.91 39.05 43.48 36.45 42.43 33.27 33.94 33.27

Differences (mm) yi- yic xi-xc 1.18 1.07 0.77 0.74 0.65 0.96 1.07 0.93 1.04 1.15 0.67 0.94 1.38 0.62 1.05 0.84 0.79 1.13 0.98 1.01 1.28 1.35 1.40 1.15 0.82 1.48 1.29 1.29 1.48 1.16 1.38 -1.25 1.55

0.53 0.40 0.05 0.56 -0.02 -0.08 -0.09 -0.24 -0.45 -0.61 -0.18 -0.24 0.13 -0.34 -0.02 -0.36 -0.19 -0.31 -0.29 -0.55 -0.11 -0.42 -0.33 -0.11 -0.21 0.18 -0.27 -0.19 -0.24 -0.20 -0.28 -1.21 1.59

Number of cells

Luminescence intensity WU)

220 80 100 80 30 180 200 290 350 300 350 40 200 500 90 90 80 130 20 35 100 40 400 90 260 370 380 400 190 250 170 220 30

8479 1593 3957 3641 1821 11198 4896 8830 10021 9714 13120 1771 10252 10597 6374 6852 7560 6305 1088 4784 3699 1101 13085 3159 6682 11660 5692 1106 13926 4425 5048 7522 3244 4120

512

TAKAHASHI

J. BIOSCI. BIOENG.,

ET AL.

y=21.692x +2433.8

2 14000 [z

I

R* = 0.6045 a 0

‘; 12000 .* : 10000 s .’ 8000

0” 5 6000 : .-2 4000 3 2000 0 0

100

200

300

400

500

600

Number of cells

FIG. 3. Relationship between the number of cells in a microcolony and luminescence intensity of a spot in image data of RMDS. Symbols: 0, spot identified by Micro Scanner; 0, unidentified spot.

ing each m icrocolony as counted at 600 x fluorescence m icroscope magnification (Table 2). Their numbers were between20 and 400 cells. Up to now, a m icrocolony consisting of less than 20 bacterial cells could not be identified with this measurementsystem, suggestingthat the detection lim it of the RMDS was 20-30 L. brevis cells. In a previous report (2), the detection lim it of the RMDS under our measurementconditions was 6.3 x lo-l6 mol of ATP and this value corresponded to one-half or quarter yeast cell or 50 cells of lactic acid bacteria according to the data of ATP contents per one cell of these m icroorganisms (7). The theoretical detection lim it roughly agreed with the practical one using the direct observation method. However, these m icrocolonies may consist of more cells than those counted through the M icro Scanner, becauseonly the cells on the surface of m icrocolonies can be observed. The luminescence intensities of 34 spots were measured as follows. Set the window of the 30 diameter pixel circle contoured for a luminous spot and then count the total pulses (Relative Light Unit; RLU) accumulated in the window, using the RMDS software (Table 2) (8). The relationship between the luminous intensity and the “apparent” number of cells in the m icrocolonies are shown in Fig. 3. An approximate cell number was pre-

FIG. 4. Image data of mixed culture with RMDS.

sumed from the luminous intensity of the spot (R’== 0.61); the luminous intensities of 50, 100, 200 and 400 cells were 1000-4000RLU, 4000-7000RLU, 5000-10,000 RLU, and about 14,00ORLU, respectively. The value of less than 1000RLU was the detection lim it and it correspondedto 20-30 L. brevis cells. Faint luminous spots like spot no. 28 in Table 2 would consist of 20-30 cells and this m icrocolony would be so small that it was overlooked. In order to improve accuracy, other fluorescence reagents should be substituted for acridine orange and the fluorescenceof m icrocolonies should be intensified. Measurementof m ixed culture sample Sewagesludge was added to bottled beer and the beer was filtered with the membrane filter. The sample on the filter was incubated on the medium for 20 h at 30°C and applied to RMDS. The image data (Fig. 4) were analyzed and results are presentedin Table 3. The m icroorganisms, including the mould (Fig. 5a), the wild yeast (Fig. 5b), and the rod-shaped bacteria (Fig. 5c), were observed around the calculated coordinates for all the 16 luminous spots. By using this system, we could visualize the cell morphology not only of cells in pure culture but also in m ixed culture. For detailed identification, the addition of the PCR

TABLE 3. Image data analysis of luminous spots of unknown sample spot no.

Coordinates of spot in image data xi

Yi

16

1

150

2 3 4 5 6 7 8 9

328 82 104 188 278 404 98 170

10 11 12 13 14 15 16

158 170 182 126 218 236 284

82 178 156 160 194 194 288 252 214 300 336 382 322 364 278

Calculated coordinates of Micro Scanner Yi’ Xf 27.40 45.03 20.69 22.86 31.18 40.10 52.57 22.29 29.41 28.23 29.42 30.62 25.08 34.18 35.97 40.71

10.97

11.21 21.42 19.15 19.38 22.63 22.37 32.50 28.71 30.96 33.56 37.17 41.94 35.69 39.89 31.10

Practical coordinates of Micro Scanner X K 26.19 10.80 44.76 19.31 22.28 30.49 40.22 52.44 22.10 29.00 27.65 29.09 29.93 24.29 33.76 35.54 40.40

10.59 21.88 18.28 18.64 21.80 22.02 32.12 28.57 30.62 33.10 36.39 41.50 35.12 40.70 30.70

Differences (mm) Xi-Xi’ Y,- Yf ~0.61 -0.17 ~0.27 -1.38 -0.58 ~0.69 0.12 PO.13 -0.19 -0.41 -0.58 PO.33 -0.69 --0.79 -0.42 PO.43 -0.31

-0.62 0.46 -0.87 -0.74 ~0.83 -0.35 -0.38 PO.14 --0.34 -0.46 -0.78

-0.44 PO.57 0.81 -0.40

The analysis of spots no. 17-21 were omitted, because these spots would be surely recognized based on their size.

Morphology Wild yeast Mould Mould Wild yeast Rod-shaped bacteria Wild yeast Wild yeast Rod-shaped bacteria Wild yeast Wild yeast Rod-shaped bacteria Rod-shaped bacteria Rod-shaped bacteria Wild yeast Wild yeast Rod-shaped bacteria

TECHNICAL

VOL. 89. 2000

NOTES

513

FIG. 5. Microorganisms observed under a fluorescence microscope ( x 600) in mixed culture. (a) Mould (spot no. 2); (b) wild yeast (spot no. 4); (c) rod-shaped bacterium (spot no. 5).

method would be effective (3,9,10), except for the problems of the high running cost and complicated operation for daily tests in a plant. We believe that a system of the ATP-bioluminescencemethod (MicroStar-RMDSSPS) in combination with fluorescence m icroscopy (Micro Scanner) can be developed for practical application. This system will improve the reliability of the ATP-bioluminescence method and will have widespread application because it affords direct observation of a m icrocolony.

4.

5.

6. REFERENCES 1. Takahashi, T., Nakakita, Y., Monji, Y., Watari, J., and Shinotsuka, K.: Application of new automatic MicroStarRMDS-SPS (ATP-bioluminescence system) for rapid quantitative detection of brewery contaminants. EBC Congress, 259266 (1999). 2. Takahashi, T., Nakakita, Y., Nara, Y., Uehara, A., Monji, Y., Watari, J., and Shinotsuka, K.: Application of automatic MicroStar-RMDS-SPS (ATP-bioluminescence method) for product testing in brewery. Antibac. Antifug. Agents, 27, 759-764 (1999). (in Japanese) 3. Yamauchi, I-I., Yamamoto, H., Shibano, Y., Amaya, N., and Saeki, T.: Rapid methods for detecting Saccharomyces diastati-

7. 8. 9. 10.

cus, a beer spoilage yeast, using the polymerase chain reaction. J. Am. Sot. Brew. Chem., 56, 58-63 (1998). Masuko, M., Kataoka, T., Sugiyama, N., Uchiyama, S., Sugiyama, H., Tan& K., Kamiya, K., and Kawai, S.: A photon counting TV camera equipped with an image guide for rapid detection and counting of single bacteria in a wide field. Photochem. Photobiol., 56, 107-111 (1992). Yasui, T.: Recent advance in rapid detection and identification methods for beer spoilage lactic acid bacteria. 24th Convention of the Institute of Brewing (March/AP section), Singapore, 24, 228-231 (1996). Nishikawa, N. and Kohgo, M.: Microbial control in the brewery. MBAA Tech. Quart., 22, 61-66 (1985). Kodaka, H., Fukuda, K., Mizuochi, S., and Horigoe, K.: Adenosine triphosphate content of microorganisms related with food spoilage. Jpn. J. Food Microbial., 13, 29-34 (1996). Masuko, K., Hosoi, S., and Hayakawa, T.: Rapid detection and counting of single bacteria in a wide field using a photon-counting TV camera. FEMS Microbial. Lett., 83, 231-238 (1991). Tsuchira. Y.. Kaneda. H.. Kano. Y.. and Koshino. S.: Detection of beer ‘spoilage ‘organisms by polymerase chain reaction technology. J. Am. Sot. Brew. Chem., 50, 64-67 (1992). Tsuchiya, Y., Kano, Y., and Koshino, S.: Detection of Lactobacillus brevis in beer using polymerase chain reaction technology. J. Am. Sot. Brew. Chem., 51, 40-41 (1993).