The present invention relates to methods of rapidly examining microbes employing ATP-luciferase method, and more particularly to a method of rapidly examining microbes allowing the exact number of microbes or colonies thereof to be electrically counted from the image of a luminescence phenomenon representing the presence of the microbes and an apparatus for achieving such a method.
ATP-luciferase method is known for a method of determining the presence of microbes. This ATP-luciferase method attracts attention as a method of rapidly examining microbes, which determines the presence of microbes by causing luciferin-luciferase (Rxe2x80x94R) reaction by using adenosine 5xe2x80x2-triphosphate (ATP) existing peculiarly in a mass in a living cell so as to detect a faint luminescence generated in proportion to the content of ATP by using a high-sensitivity detector.
Japanese Laid-Open Patent Application No. 6-237793, for example, discloses a method of and an apparatus for performing the method of examining microbes. According to the method, a specimen liquid is first filtered so as to capture living microbes on a filter, and the filter is detected by using a system for analyzing image of microbe luminescence. According to the system, the filter, on which the living microbes are captured, is processed with an extraction reagent and a luminescence reagent, and is set on a specimen holder. Then, a television camera including an optical system and an image acquisition means such as a charge coupled device is set as closely to the filter as possible in order to photograph the state of luminescence of the filter. Data for the photographed image are shown on a display through an image processing device and a data-analyzing device for observation, and the result of analysis is printed out.
FIG. 1 is a schematic diagram of the system having a high-sensitivity television camera 1 including a tapered fiber, an optical amplifier portion and a camera tube, a camera controller 2, an image processor 3, a data-analyzing device 4 and a television monitor 5. The measurement is made as follows: A filter 6 having living bacteria thereon, on which luminescence treatment is performed, is set closely to the high-sensitivity television camera 1. The image of luminescence from the bacteria is acquired by accumulating two-dimensional photons for a predetermined period of time, for example, 30 to 180 seconds by employing the camera controller 2 and the image processor 3. Luminescence noises are erased by the data-analyzing device 4, so that only intense luminescence from the bacteria remains to be displayed on the television monitor 5. This process erases other luminescence than that from the bacteria as noise, and the number of the measured luminous points becomes the number of the living bacteria or colonies thereof. The luminous points are the image representing the state of luminescence of the microbes. Bright lights are emitted around from positions in which the microbes exist by causing the Rxe2x80x94R reaction on a medium. The number of these luminous points corresponds to that of the microbes.
As described above, there already exists the apparatus for automatically detecting the number and presence of microbes by means of image analysis. However, there is a disadvantage in a conventional detecting apparatus. FIG. 2 shows a photographed image of the state of luminescence of ATP on a filter. When a detecting apparatus recognizes an intense light, the intense light takes the form of a high peak and is indicated on a monitor as points of pseudo-colors corresponding to the height of the peak.
The luminous points are indicated as white luminous points as luminous points in an upper window (a white square) of FIG. 2. The lower part of FIG. 2 shows one of the luminous points in a three-dimensional way. In the three-dimensional image, a waving sea surface-like portion indicates a group of blue points serving as a background (BKG) for the data for the image, and a bundle of high peaks in the center indicates the spreading of the luminous point. All of these peaks are converted into numerical values, and the peak levels of necessary coordinates are stored as data. A peak is counted as one luminous point when it is recognized that the peak has a height and an area equal to or more than a certain value (a threshold) on the basis of the average value of peak levels waving at the lower levels of the data for the image.
In order to judge whether a luminous point shown in the image is a luminous point originating in ATP or the BKG, a threshold to distinguish a luminous point from the BKG is defined depending on the kind of bacterium to be detected and the height of the BKG.
As previously described, the conventional detecting apparatus defining a threshold to make a count is effective in distinguishing luminous points. However, in some cases, there is a difference between the number of luminous points counted by the conventional detecting apparatus and that visually counted. This is because the conventional counting method is performed only on the basis of the height and area of a peak, which prevents the number of luminous points in a variety of shapes and sizes from being exactly counted. The followings are two possible causes thereof.
(1) In some cases, one luminous point has peaks and valleys, which causes the counted number to differ from the real number.
One luminous point does not always include only one peak. A luminous point, in some cases, emits light in a distorted way depending on the extracted state of ATP or the applied state of a luminescence reagent to a single microbe or the colonies of microbes. Therefore, when a three-dimensional analysis is performed, it is discovered ,in some cases, that a peak has shoulders or a number of peaks overlap. In such cases, the number of the peaks, which are substantially luminous points, is recognized as the number of luminous points when there are valleys among the peaks. Therefore, the number of the luminous points differs from that visually counted. FIGS. 3(A) and 3(B) show an original image and a count result according to a counting method before improvement displayed on a television monitor, respectively. The topmost luminous point in the original image is visually counted as one, while the luminous point is counted as four according to the counting method before improvement. Further, the second topmost luminous point is counted as two and the total of ten luminous points are counted according to the conventional counting method. This is because the topmost luminous point has a shape as typically shown in an enlarged fragmentary view of FIG. 3(B), so that each of protruding portions a, b, c, and d is electrically counted as one individual luminous point. (2) When generated is a luminous point of such intense luminance that a light diffused therefrom causes the great fluctuation of the peak of a background around the luminous point, a peak recognized as a luminous point, in some cases, is generated in a part where ATP of microbes does not exist.
FIG. 4 shows data for the image of a large luminous point. The luminance thereof is so intense that a light emitted therefrom is so shown as to be diffused therearound. There is a part where the diffused light is intense (a cross-like luminous point situated in the lower left from the luminous point), and the part is recognized as a luminous point. Further, because of the presence of the intense diffused light, the number of the luminous points is counted as three in the example of FIG. 4, which should correctly be counted as one.
The present invention is made in the light of the above disadvantage, and the object thereof is to provide a method of counting the number of bacteria, in which errors in the above count resulting from the shape of a luminous point originating in ATP and from intense luminance are eliminated when the number of the luminous points is electrically counted on the basis of an image signal.
A first mode of the present invention includes a method of counting the number of microbes by counting the number of the luminous points of the image, acquired through an image acquisition device, of fluorescence from the microbes on which luminescence treatment is performed by using a reagent, including the steps of reading data for the luminance of the image acquired through the image acquisition device into memories corresponding to pixels in a two-dimensional matrix-like form; correcting obtained data for the luminance on the basis of a background value; binarizing corrected data for the luminance stored in the memories on the basis of a defined threshold so that a judgment is made, with respect to each of the corrected data for the luminance, as to whether the corrected data for the luminance has a luminance value higher than a predetermined level; counting the number of luminous points of luminance equal to or greater than the threshold; and judging, with respect to each of the luminous points of the luminance equal to or greater than the threshold, whether there exists a luminous point of luminance equal to or greater than a predetermined value within a predetermined range adjacent to the luminous point of the luminance equal to or greater than the threshold so as to group and count adjacent luminous points as one luminous point when there exists the luminous point of the luminance equal to or greater than the predetermined value.
A second mode of the present invention includes an apparatus for counting the number of microbes by counting the number of the luminous points of the image, acquired through an image acquisition device, of fluorescence from the microbes on which luminescence treatment is performed by using a reagent, including a means for reading data for the luminance of the image acquired through the image acquisition device into memories corresponding to coordinates in a two-dimensional matrix-like form; a means for correcting obtained data for the luminance on the basis of a background value; a means for binarizing corrected data for the luminance stored in the memories on the basis of a defined threshold so that a judgment is made, with respect to each of the corrected data for the luminance, as to whether the corrected data for the luminance has a luminance value higher than a predetermined level; a means for counting the number of luminous points of luminance equal to or greater than the threshold; and a means for judging, with respect to each of the luminous points of the luminance equal to or greater than the threshold, whether there exists a luminous point of luminance equal to or greater than a predetermined value within a predetermined range adjacent to the luminous point of the luminance equal to or greater than the threshold so as to group and count adjacent luminous points as one luminous point when there exists the luminous point of the luminance equal to or greater than the predetermined value.
According to the above described modes of the present invention, in electrically counting the number of luminous points of fluorescence generated from microbes by image analysis, an error in the count caused by misjudging one luminous point of fluorescence generated from one microbe to be fluorescence generated from a plurality of microbes due to an irregular shape of the microbe can be eliminated. Thereby, the number thereof can exactly be counted.
Further, when one luminous point has a plurality of peaks and valleys, in order to avoid counting the plurality of peaks as so many luminous points, a first luminous point and a second luminous point existing adjacently to the first luminous point within a given range around the first luminous point are grouped and counted as one luminous point instead of being separately counted as independent luminous points.
Moreover, when there exists a luminous point of great luminance, in order to avoid recognizing a light diffused therefrom as a luminous point, the luminous point of great luminance and a luminous point generated within a given range around the luminous point of great luminance are grouped and counted as one luminous point.
A third mode of the present invention includes a method of counting the number of microbes by counting the number of luminous points of the image, acquired through an image acquisition device, of fluorescence from the microbes on which luminescence treatment is performed by using a reagent, including the steps of reading data for the luminance of the image acquired through the image acquisition device into memories corresponding to coordinates in a two-dimensional matrix-like form; correcting obtained data for the luminance on the basis of a background value; binarizing corrected data for the luminance stored in the memories on the basis of a defined first threshold and a defined second threshold, which is greater than the first threshold, so that a judgment is made, with respect to each of the corrected data for the luminance, as to whether the corrected data for the luminance has a luminance value higher than a predetermined level; and counting the number of luminous points of luminance equal to or greater than the predetermined level, characterized in judging, with respect to each of the luminous points of the luminance equal to or greater than the first threshold, whether there exists a luminous point of luminance equal to or greater than the first threshold within a first predetermined range of pixels adjacent to the luminous point of the luminance equal to or greater than the first threshold, and adjacent luminous points are grouped and counted as one luminous point when there exists the luminous point of the luminance equal to or greater than the first threshold; and judging, with respect to each of the luminous points of the luminance equal to or greater than the second threshold, whether there exists a luminous point of luminance equal to or greater than the first threshold and smaller than the second threshold within a second predetermined range of pixels adjacent to the luminous point of the luminance equal to or greater than the second threshold, and adjacent luminous points are grouped and counted as one luminous point when there exists the luminous point of the luminance equal to or greater than the first threshold and smaller than the second threshold.
A fourth mode of the present invention includes an apparatus for counting the number of microbes by counting the number of luminous points of the image, acquired through an image acquisition device, of fluorescence from the microbes on which luminescence treatment is performed by using a reagent, including a means for reading data for the luminance of the image acquired through the image acquisition device into memories corresponding to coordinates in a two-dimensional matrix-like form; a means for correcting obtained data for the luminance on the basis of a background value; a means for binarizing corrected data for the luminance stored in the memories on the basis of a defined first threshold and a defined second threshold, which is greater than the first threshold, so that a judgment is made, with respect to each of the corrected luminance data, as to whether the corrected data for the luminance has a luminance value higher than a predetermined level; and a means for counting the number of luminous points of luminance equal to or greater than the predetermined level, characterized in judging, with respect to each of the luminous points of the luminance equal to or greater than the first threshold, whether there exists a luminous point of luminance equal to or greater than the first threshold within a first predetermined range of pixels adjacent to the luminous point of the luminance equal to or greater than the first threshold, and adjacent luminous points are grouped and counted as one luminous point when there exists the luminous point of the luminance equal to or greater than the first threshold; and judging, with respect to each of the luminous points of the luminance equal to or greater than the second threshold, whether there exists a luminous point of luminance equal to or greater than the first threshold and smaller than the second threshold within a second predetermined range of pixels adjacent to the luminous point of the luminance equal to or greater than the second threshold, and adjacent luminous points are grouped and counted as one luminous point when there exists the luminous point of the luminance equal to or greater than the first threshold and smaller than the second threshold.
According to the above described modes of the present invention, in electrically counting the number of luminous points of fluorescence generated from microbes by image analysis, an error in the count caused by misjudging one luminous point of fluorescence..generated from one microbe to be fluorescence generated from a plurality of microbes due to an irregular shape of the microbe can be eliminated. Further, when there is a luminous point of great luminance, it can be avoided to count a light diffused therefrom as one independent luminous point.