JP5713826B2 - Culture observation equipment - Google Patents

Description

  The present invention relates to an apparatus for culturing microorganisms, and more particularly to an apparatus for accurately observing microorganisms when culturing microorganisms in a medium.

  Traditionally, microorganisms are cultured on an agar medium, and the microbial contamination of food is examined by detecting the number of viable bacteria after a certain period of time (hereinafter sometimes referred to as the number of colonies). Yes. In this inspection method, the number of viable bacteria living in 1 g or 1 ml of a food inspection sample is used as an indicator of microbial contamination of the food. Counting the number of viable bacteria in such an inspection method is performed, for example, by the pour method. In the pour method, a sample of viable bacteria and a standard agar medium are mixed well in a petri dish, cultured under conditions suitable for culture (at 35 ° C. for 48 hours), and then the number of colonies formed by proliferation is counted. Such a counting method has a problem that colonies overlap as a result of long-term culture, and accurate counting is difficult.

  Japanese Patent Laying-Open No. 2003-85533 (Patent Document 1) discloses a number counting method for solving such a problem. This number counting method is a reference with a binarization step that receives light that has passed through a measurement target by an area sensor, binarizes an image acquired by the area sensor every predetermined time, and sequentially obtains a binarized image. An image storage step for storing the binarized image in the image storage means, and a determination area that includes a predetermined pixel connection area in the binarized image obtained after obtaining the reference binarized image An area setting step, an intra-area counting step for counting the number of pixel connection areas existing in a comparison area corresponding to the determination area in the reference binarized image, and the number counted in the intra-area counting step. When 0 or 1, the comparison area is replaced with the determination area, and when the number counted in the in-area counting step is 2 or more, the comparison area is maintained and the binarized image stored in the image storage means is updated. Update step , And a number counting step of counting the number of pixels connected area in the stored binarized image in the image storage means.

JP 2003-85533 A

  In the number counting method disclosed in Patent Document 1 described above, the pour method is employed as a method for counting the number of viable bacteria, the colony growth process is sequentially measured with a microscope, and the number of colonies is obtained by image processing with a computer. Count. This counting method employs a method of irradiating colonies with light such as a laser and magnifying and observing the shadow image of the colonies with a CCD area sensor, and a structure that prevents dew condensation in the container in which the medium is placed. In this counting method, a sample is magnified and observed with a microscope from the start of culture, and image information is sequentially taken into a computer and image processing is performed at regular time intervals. In this case, the number of viable bacteria is determined when the colonies are sequentially detected from the grown and enlarged colonies and there are no newly detected colonies. Therefore, it is possible to determine the viable cell count in a short time from the culture start time. Furthermore, since counting is performed from the stage where colonies do not overlap, accurate counting is possible.

  In this number counting method, a device for growing a colony in an incubator is devised. In the case of an ordinary petri dish used in the pour method, accurate measurement becomes difficult due to the formation of condensation inside the upper lid of the petri dish. In order to solve this problem, in this number counting method, the medium surface of the area to be measured is sealed so as not to contact the air layer, and a medium area that is in contact with the air layer is formed without sealing all the medium surfaces. In addition, a medium container is adopted. According to such a structure of the medium container, the medium area in contact with the air layer is condensed, but the closed medium area can be continuously and continuously measured without dew condensation. In this way, both drying of the medium and condensation on the petri dish are prevented, and continuous automatic measurement for a long time is realized.

However, in this medium container, since the surface of the medium in the measurement area does not contact the air layer, even if it is possible to prevent the inside of the petri dish lid from condensing, if air is required for growth of viable bacteria Viable bacteria will not grow and the number of colonies cannot be measured accurately.
On the other hand, when the number of colonies is measured by imaging the state of viable bacteria in the petri dish with a two-dimensional image sensor without using such a specially shaped medium container, The effect of condensation cannot be excluded. In order to eliminate this effect with an ordinary petri dish, the medium is imaged after opening the upper lid of the petri dish and wiping off the condensation, or the medium is imaged by opening the upper lid of the petri dish. When taking an image in this way, the top lid of the petri dish must be opened each time the image is taken. The culture conditions change, which may interfere with culture, making automatic measurement difficult, or the petri dish position changes when the top lid is opened. Therefore, accurate counting becomes difficult. Further, if illumination is performed from above the petri dish with strong illuminance in order to eliminate the influence of dew condensation, halation occurs at the center of the petri dish, making accurate counting difficult. As a result, neither the number counting method disclosed in Patent Document 1 nor a known technique can achieve both the growth of viable bacteria and the accurate counting of the number of viable bacteria.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a culture observation apparatus that can achieve both proliferation of viable bacteria and accurate counting of the number of viable bacteria.

In order to achieve the above-mentioned object, the present invention takes the following technical means.
The culture observation apparatus according to the present invention cultures microorganisms in a flat medium in a petri dish. The culture observation apparatus includes a mounting table on which the petri dish is placed, an illumination unit that is provided on the side of the petri dish and emits light from the side of the medium toward the medium, and the bottom plate side of the petri dish through the placing table. An imaging means for imaging the culture medium, a mounting table, a lighting unit, and an imaging means, and a housing capable of forming an environment suitable for culturing microorganisms.

  Preferably, the illumination unit can be configured to be illumination provided discretely around the petri dish. More preferably, the illumination unit can be configured to be a cylindrical illumination having an inner diameter larger than the outer diameter of the petri dish. More preferably, the light source of the illumination unit can be configured to be an LED that emits blue-white light toward the culture medium.

More preferably, the imaging means can be configured to be a two-dimensional area sensor made of CMOS.
More preferably, it can be configured to have a temperature adjustment unit that adjusts the temperature of the space in which the petri dish inside the casing is placed.
More preferably, the housing can be configured to include a main body including an opening having an upper opening and a lid that closes the opening. In this case, the mounting table is provided in the opening, the imaging unit is arranged on the inner bottom of the main body so as to image the upper side, and the illumination unit is located on the side of the petri dish when the lid is closed. It is provided inside the lid, and can be configured to be able to irradiate light from the side of the medium toward the medium when the lid is closed.

  According to the culture observation apparatus according to the present invention, it is possible to achieve both proliferation of viable bacteria and accurate counting of the number of viable bacteria.

1 is a perspective view of a culture observation apparatus according to a first embodiment of the present invention. It is a permeation | transmission perspective view of the culture observation apparatus of FIG. It is sectional drawing of the arrow C direction shown to FIG. 1 (A). It is sectional drawing of the arrow A direction shown to FIG. 1 (A). It is sectional drawing of the arrow B direction shown to FIG. 1 (A). It is a side view of the detailed positional relationship between the petri dish and the illumination device in the culture observation apparatus according to the first embodiment of the present invention. It is a perspective view of the culture observation apparatus which concerns on the 2nd Embodiment of this invention. FIG. 5 is a cross-sectional view corresponding to FIG. 4. FIG. 7 is a side view corresponding to FIG. 6.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
In the following description, the same parts are denoted by the same reference numerals. Their names and functions are also the same. Therefore, detailed description thereof will not be repeated.
<First Embodiment>
[Device configuration]
FIG. 1 shows an overall perspective view of the culture observation apparatus 100. This culture observation apparatus 100 has a three-dimensional housing in which a semi-cylinder and a rectangular parallelepiped are combined, and is mainly composed of an upper case 102 and a lower case 104. FIG. 1A shows a state where the upper case 102 is closed, and FIG. 1B shows a state where the upper case 102 is opened. As shown in FIG. 1B, the lighting unit 120 is attached to the inner side of the upper case 102. Although the detailed structure is mentioned later, the illumination unit 120 irradiates light to the agar medium in the petri dish 200 mounted on the transparent glass plate 110. The lower case 104 is provided with a first plate 106 that also serves as a positioning guide for the petri dish 200 placed on the glass plate 110. The first plate 106 includes a through portion 108. The petri dish 200 is placed on air heated by an electric heater (for example, a film heater attached to the side surface of the lower case 104) provided in the lower case 104 (air having a temperature suitable for culture). In addition, it flows through the through-hole 108 into the space in the upper case 102.

  In FIG. 2, the permeation | transmission perspective view of the culture observation apparatus 100 is shown. This transparent perspective view corresponds to FIG. In FIG. 2, a partial external view is shown superimposed on a transparent perspective view. As shown in FIG. 2, the upper case 102 and the lower case 104 of the culture observation apparatus 100 are joined to each other by two flat torque hinges 132 so as to be opened and closed, and the upper case 102 is held in a closed state by a cuff latch fastener 130. Can do. The upper case 102 is kept closed, and the microorganisms are cultured.

3 is a cross-sectional view in the direction of arrow C shown in FIG. 1 (A), FIG. 4 is a cross-sectional view in the direction of arrow A shown in FIG. 1 (A), and FIG. Cross-sectional views in the direction of arrow B are shown respectively. The internal structure of the culture observation apparatus 100 will be described with reference to FIGS.
Inside the culture observation apparatus 100, a transparent glass plate 110 on which the petri dish 200 is placed, an illumination unit 120 that illuminates the medium in the petri dish 200, and a medium provided on the bottom dish of the petri dish 200 are imaged from below. And a camera unit 150.

  The glass plate 110 has a circular shape with a diameter D (2) larger than the diameter D (1) of the bottom dish of the petri dish 200. The glass plate 110 is placed on a second plate 112 having a circular hole having a diameter D (3) smaller than the diameter D (2). Above the glass plate 110, a first plate 106 having a circular hole portion having a diameter D (4) larger than the diameter D (1) and smaller than the diameter D (2) is placed. By placing the petri dish 200 in the circular hole of the first plate 106, the position of the petri dish 200 on the glass plate 110 can be regulated. Since it is configured in this manner, the petri dish 200 on the glass plate 110 can be easily positioned, and the entire culture medium spread on the bottom dish of the petri dish 200 can be imaged by the camera unit 150.

As shown in FIG. 1, the first plate 106 includes a temperature adjusting through-hole 108. The second plate 112 also includes a penetrating portion having the same shape at the same position as the first plate 106.
The lighting unit 120 is provided in four directions so as to surround the petri dish 200. Each lighting unit 120 includes an LED unit 122 and a holding unit 124 that holds the LED unit 122. The LED unit 122 uses LEDs that generate white light to blue light. The illumination unit 120 can irradiate white light to blue light (hereinafter, collectively referred to as blue white light) from the side toward the medium of the petri dish 200 to illuminate the medium substantially uniformly. What is necessary is not limited to four places.

The illuminated culture medium is imaged through the glass plate 110 by the camera unit 150 provided in the lower case 104. At this time, colonies of viable bacteria cultured on the medium are shaded and imaged by the camera unit 150.
The camera unit 150 includes a camera sensor 152 composed of a CMOS image sensor and a camera plate 154 that holds the camera sensor 152. A CMOS image sensor is an example of a two-dimensional area sensor.

  The size of viable bacteria to be counted is 0.1 mm. Here, the number of pixels of the camera sensor 152 is 1.3 million pixels. Since the size of the petri dish 200 is 90 mm, it becomes 0.0872 mm per pixel. If the resolution is higher than this, the surface irregularities of the medium are imaged, and the accurate count is obstructed. An imaging signal from the camera sensor 152 is taken into, for example, a video capture board via the USB harness 140. At this time, the number of pixels of the acquired image is, for example, 640 × 480, and each pixel has 8-bit color gradation. Such specifications are not limited as long as viable bacteria or colonies cultured in the medium of the petri dish 200 can be observed. It should be noted that the use of CMOS for the camera sensor 152 is advantageous in that it is less expensive than a CCD, has a small element, consumes less power, and in principle does not generate smear or blooming and can be read at high speed. .

  Furthermore, the lower case 104 is provided with an electric heater 160 to warm the air in the lower case 104. The warm air rises and passes through the through portion 108 and flows into the space in the upper case 102 on which the petri dish 200 is placed. A temperature sensor (for example, a thermocouple) (not shown) is provided in the space in the upper case 102 on which the petri dish 200 is placed, and the temperature sensor is connected to the temperature adjustment unit. The temperature adjustment unit performs on / off control of the electric heater so as to be a set temperature (for example, 35 ° C.) ± 2.5 ° C., or performs PID control of the electric heater with the set temperature as a target value.

  Furthermore, a detailed positional relationship between the illumination unit 120 and the petri dish 200, which is a characteristic configuration of the culture observation apparatus 100 according to the present embodiment, will be described with reference to FIG. FIG. 6 is an enlarged view of the illumination unit 120 and the petri dish 200. As shown in FIG. 6, the LED unit 122 of the lighting unit 120 is configured such that the medium 300 on the flat plate spread on the bottom plate 220 of the petri dish 200 is placed on the side slightly above or on the side (collectively these To blue-white light. The medium 300 is an agar medium, for example, and contains nutrients for growing live bacteria. For this reason, the kind of culture medium changes with the kind of living microbe to culture | cultivate. There are various types of the culture medium 300, but there are many culture media 300 that express the function of the light guide plate by irradiating the white light from the side of the culture medium 300 from the LED unit 122. By irradiating the blue-white light from the four LED units 122 and the medium 300 exhibiting the function of the light guide plate, the medium 300 can be illuminated uniformly. As shown in FIG. 3, the diameter D (1) of the bottom plate 220 of the petri dish 200 is smaller than the diameter D (4) of the circular hole portion of the first plate 106, and the circular hole portion of the first plate 106 Since there is a space between the end and the bottom end of the bottom plate 220, the light irradiated from the LED unit 122 reaches the culture medium 300 through this space.

  The position (relative position with respect to the culture medium), the irradiation angle, the wavelength, and the illuminance of the LED unit 122 may be such that the culture medium 300 functions as a light guide plate and the culture medium 300 can be illuminated uniformly. In this way, the LED unit 122 is provided around the petri dish 200 so that the culture medium 300 functions as a light guide plate, and emits light from the side of the culture medium 300 toward the culture medium 300. That is, the side includes all directions in which the culture medium 300 functions as a light guide plate. In addition, you may make it change at least one of the wavelength and the illumination intensity of the light irradiated from the LED unit 122 with the kind of culture medium.

  In this way, by imaging the culture medium 300 irradiated with blue-white light from the LED unit 122 with the camera unit 150 through the glass plate 110, it is possible to completely eliminate the influence of dew condensation that occurs inside the upper lid 210 of the petri dish 200. . For this reason, it is possible to accurately count the number of colonies grown on the medium. In particular, since it is not necessary to open the top cover of the petri dish 200 and wipe off the dew condensation, it is not necessary to move the position of the petri dish 200, and accurate and continuous automatic counting can be performed. Specifically, since it is not necessary to move the position of the petri dish 200, it is possible to accurately compare the state of the colony before a certain time and the state of the current colony when observing (counting) at a certain time interval. .

The position of the LED unit 122 illustrated in FIG. 6 is an example, and the position of the LED unit 122 may be lower than the position illustrated in FIG. 6 as long as the position does not interfere with the first plate 106. If it exists below, the LED unit 122 can irradiate the culture medium 300 with blue-white light from the side.
Further, the LED unit 122 may be energized continuously except when there is an influence of light on the culture of microorganisms or the like. By doing in this way, it becomes possible to reduce the power consumption of an electric heater with the heat which generate | occur | produces from the LED unit 122. FIG.

[Mode of operation]
A mode of observing an increase state of microorganisms (counting the number of colonies) while culturing microorganisms using the culture observation apparatus 100 according to the present embodiment described above will be described.
Microorganisms are applied to the bottom dish 220 of the petri dish 200 on which the medium 300 is spread. At this time, the medium 300 suitable for the bacteria cultured in the petri dish 200 is selected.

The petri dish 200 coated with the bacteria is placed on the glass plate 110 of the culture observation apparatus 100. At this time, the hole of the first plate 106 serves as a guide for the bottom plate 220 of the petri dish 200. Thereafter, the upper case 102 is closed, and the upper case 102 is held in a closed state by the cuff latch fastener 130.
The lighting unit 120 is turned on and an initial image is captured by the camera unit 150. When the electric heater 160 is energized, the air in the lower case 104 is warmed, and the warmed air rises and flows through the through portion 108 to the space in the upper case 102 on which the petri dish 200 is placed. Is maintained at a temperature suitable for culture. At this time, the lighting unit 120 may be turned on as long as the culture is not inhibited.

The camera unit 150 images the state of the bacteria (colony state) grown on the medium 300 at a preset time interval (for example, every 3 hours). Image processing is performed on the captured image, and the number of colonies is counted.
At this time, as a process for counting the number of colonies from an image captured by the camera unit 150 (image processing), those skilled in the art can adopt a standard method. For example, the initial image is subtracted from the images taken at regular time intervals to eliminate the influence of dust or the like attached to the petri dish 200 from the beginning. The subtracted image is binarized, consecutive pixel regions in the binarized image are continuously processed, the pixel regions are labeled, and the connected pixel regions are counted as one colony.

  By the above processing, the number of colonies can be counted. This counting process may be executed by a personal computer outside the culture observation apparatus 100, or an image processing apparatus is provided in the culture observation apparatus 100 and processed, and the result of the image processing is transferred to the external computer. It doesn't matter. As described above, the camera unit 150 and the external computer or the like are connected via, for example, the USB harness 140 or the LAN communication unit.

[effect]
As described above, according to the culture observation apparatus according to the present embodiment, desired viable bacteria can be cultured in the petri dish, and the number of colonies cultured in the petri dish can be accurately determined without being affected by dew condensation inside the upper lid. In addition, automatic counting can be continuously performed. In this way, online continuous observation is possible.

<Second Embodiment>
The culture observation apparatus 400 according to the second embodiment of the present invention will be described below with reference to FIGS. 7 is a perspective view of the culture observation apparatus 400 corresponding to FIG. 1B, FIG. 8 is a sectional view corresponding to FIG. 4, and FIG. 9 is a side view corresponding to FIG.
As shown in these drawings, the culture observation apparatus 400 according to the present embodiment includes an illumination unit 420 different from the culture observation apparatus 100 according to the first embodiment. Since other configurations are the same as those of the first embodiment, detailed description thereof will not be repeated.

  The illumination unit 420 is provided in the upper case 402 of the culture observation apparatus 400, and includes a hollow cylindrical portion 426 that surrounds the petri dish 200 and a holding portion 424 that holds the hollow cylindrical portion 426 in the upper case 402. An LED unit 422 is arranged inside the hollow cylindrical portion 426 so as to uniformly illuminate the culture medium 300 in the petri dish 200. The hollow cylindrical portion 426 and the holding portion 424 are made of, for example, an integrally molded resin.

The hollow cylindrical portion 426 is formed of polypropylene or the like and serves as a diffusion plate that diffuses light from the LED unit 422.
The LED unit 422 uses the same LED as the LED unit 122 of the first embodiment. Here, the illumination unit 420 only needs to be able to irradiate the culture medium of the petri dish 200 with blue-white light from the side to illuminate the culture medium substantially uniformly. It is not limited.

  Furthermore, a detailed positional relationship between the illumination unit 420 and the petri dish 200, which is a characteristic configuration of the culture observation apparatus 400 according to the present embodiment, will be described with reference to FIG. FIG. 9 is an enlarged view of the lighting unit 420 and the petri dish 200. As shown in FIG. 9, the LED unit 422 of the illumination unit 420 irradiates the culture medium 300 spread on the bottom plate 220 of the petri dish 200 with blue-white light from above or from the side. By irradiating blue-white light from the LED unit 422, many culture media 300 express the function of a light guide plate, and by irradiating the blue-white light from the LED unit 422, the culture media 300 can be illuminated uniformly.

In this way, by imaging the culture medium 300 irradiated with blue-white light from the LED unit 422 with the camera unit 150 through the glass plate 110, it is possible to completely eliminate the influence of dew condensation occurring inside the upper lid 210 of the petri dish 200. .
The position of the LED unit 422 shown in FIG. 9 is an example, and the position of the LED unit 422 is more than the position shown in FIG. 9 as long as it does not interfere with the first plate 106 as in the first embodiment. It may be down. If it exists below, the LED unit 422 will irradiate the culture medium 300 with blue-white light from the side. Here, the lateral meaning is the same as in the first embodiment.

The operation mode of the culture observation apparatus 400 according to the present embodiment is the same as that of the culture observation apparatus 100 according to the first embodiment.
As described above, according to the culture observation apparatus according to the present embodiment, the desired viable bacteria can be cultured in the petri dish as in the first embodiment, and the petri dish is not affected by the dew condensation inside the upper lid. It is possible to accurately and continuously automatically count the number of viable bacteria or colonies cultured therein.

  As mentioned above, it should be thought that embodiment disclosed this time is an illustration and restrictive at no points. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

100, 400 Culture observation apparatus 102 Upper case 104 Lower case 106 First plate 108 Through part 110 Glass plate 112 Second plate 120, 420 Illumination unit 122, 422 LED unit 124 Holding unit 130 Cuff latch fastener 132 Flat torque hinge 140 USB harness 150 Camera unit 152 Camera sensor 154 Camera plate 200 Petri dish 300 Medium

Claims (6)

A culture observation apparatus for culturing microorganisms in a flat medium in a petri dish,
A mounting table for mounting the petri dish;
An illumination unit that is provided on the side of the petri dish and irradiates light from the side of the medium toward the medium; and
Imaging means for imaging the medium from the bottom dish side of the petri dish through the mounting table,
A housing containing the mounting table, the lighting unit, and the imaging means, and capable of forming an environment suitable for culturing microorganisms;
I have a,
The housing is
A main body provided with an opening having an upper opening; and a lid for closing the opening;
The mounting table is provided in the opening,
The imaging means is arranged on the inner bottom of the main body so as to image the upper side,
The illumination unit is provided inside the lid that is located on the side of the petri dish when the lid is closed, and emits light from the side of the medium toward the medium when the lid is closed. A culture observation apparatus characterized by being arranged in a possible manner .   The culture observation apparatus according to claim 1, wherein the illumination unit is illumination provided discretely around the petri dish.   The culture observation apparatus according to claim 1, wherein the illumination unit is a cylindrical illumination having an inner diameter larger than an outer diameter of the petri dish.   The culture observation apparatus according to any one of claims 1 to 3, wherein a light source of the illumination unit is an LED that emits blue-white light toward the culture medium.   The culture observation apparatus according to claim 1, wherein the imaging unit is a two-dimensional area sensor made of CMOS.   The culture observation apparatus according to any one of claims 1 to 5, further comprising a temperature adjustment unit that adjusts a temperature of a space in which the petri dish inside the housing is placed.

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