Electrical pulse counters – pulse dividers – or shift registers: c – Applications – Counting animate or inanimate entities
Reexamination Certificate
2000-08-28
2002-05-07
Wambach, Margaret R. (Department: 2816)
Electrical pulse counters, pulse dividers, or shift registers: c
Applications
Counting animate or inanimate entities
C377S019000
Reexamination Certificate
active
06385272
ABSTRACT:
TECHNICAL FIELD
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.
BACKGROUND ART
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 (R—R) reaction by using adenosine
5
′-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 R—R 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.
DISCLOSURE OF THE INVENTION
A first mode of the present inv
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Sapporo Breweries Ltd.
Wambach Margaret R.
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