JP2011115148A - Apparatus for bacterial image pickup and apparatus for regulating bacterial liquid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pretreating apparatus for estimating information about bacterial colonies to be collected in the height direction for individual colonies, automatically computing the volume of the colonies, and automatically fishing a required number of the colonies. <P>SOLUTION: An apparatus for bacterial image pickup includes: a camera; a pedestal 113 for carrying the colonies which are the object of the image pickup on a culture medium; a plurality of elevation illuminating units 109 for irradiating the colonies with light from upper different positions; and a transmitting and illuminating unit 111 located on the side opposite to the camera relatively to the pedestal for illuminating the culture medium from the lower side. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a bacterial imaging device and a bacterial solution adjusting device, and more particularly to a bacterial analysis device and a pretreatment device for catching colonies for a method for performing identification / drug sensitivity test.

  In the treatment of infectious diseases, it is important for proper antibacterial treatment to identify pathogenic bacteria, to quickly measure the sensitivity to antibiotics, to determine effective drugs and to establish a treatment strategy. Usually, the submitted specimen is applied to the medium and cultured, and the formed colonies are fished and suspended in physiological saline to prepare a bacterial suspension, which can be used as a measuring device for identification and drug susceptibility testing equipment. Take the flow to inoculate. In the identification / sensitivity test, in order to obtain an accurate result, it is essential that the amount of inoculated bacteria on the device is kept constant at a predetermined concentration with high accuracy and reproducibility. In many cases, in order to obtain a predetermined amount of bacteria, a plurality of colonies of the same species are selected from the colonies grown on the petri dish and fished. Suspend in liquid, measure turbidity or opacity, and adjust to a predetermined concentration (number of bacteria). The types of colonies to be mixed must be the same, but it is normal for multiple types of colonies to grow on the same petri dish, and it is expensive for the laboratory technician to select the same type of colonies from these. Technology is required. In addition, since multiple colonies of various sizes can be selected, it is necessary to add a colony and add physiological saline for dilution after adjustment to obtain a predetermined turbidity. It is not suitable for large-scale processing. As a conventional technique for making the amount of fish per time constant, a simple kit that can obtain a predetermined concentration of fungus by fishing a certain amount and floating in a certain amount of physiological saline has been commercialized. Yes. For example, the method described in Japanese Patent Publication No. 2994675 shows an example in which a certain amount of bacteria can be collected with a simple grooved rod. However, this method has limitations on the concentration and the amount of bacterial solution obtained, and only a small amount of bacterial solution in a relatively low concentration range can be obtained.

  On the other hand, in Patent Document 1, as a colony transfer device, a bacterial colony in a petri dish is imaged with a TV camera, an image displayed on a monitor is visually checked by an inspection engineer, and a colony to be fished is selected and instructed. A method is described in which a fishing fungus jig (fishing fungus tool) is automatically moved to the position of the colony according to the instructions to catch the bacterial colony and transplant it to a test tube or the like. However, in this method, an inspection engineer checks images taken by a television camera on a monitor and selects colonies to be picked one by one, so even if the apparatus is used, the speed-up effect is not so much. In Patent Document 2 and Patent Document 3, similarly as a colony transfer device, an inspection engineer confirms an image captured by a television camera on a monitor, inputs conditions such as the size of a colony to be selected, or instructs a colony to be excluded, and fishing is performed accordingly. A method for automatically transplanting colonies to be fungus into a new medium is shown. In this method, all the bacterial colonies that grow on the petri dish are basically the same species, and all colonies except for exceptions that have grown due to contamination are transplanted. Considerable labor saving. However, the colonies are picked one by one from the petri dish before transplantation and transplanted one by one into a new petri dish medium, so there is no need to mix the colonies and the type of colony is not discriminated. In the preparation of the bacterial solution for the identification test and drug sensitivity test targeted by the present invention, it is necessary to fish many bacteria of the same species from one petri dish, and different types of bacteria are obtained from images obtained with a TV camera. Eliminate the need to select only one type of bacteria. However, with only one image obtained with a television camera, the appearance feature of the colony cannot be extracted well, and there is a concern that an error may occur during selection. On the other hand, not only the surface of a petri dish but a colony counter that measures the number of colonies including colonies in an agar medium is commercially available. In this method, a two-dimensional image captured by a camera is processed, and the number of colonies having a size that meets a condition is measured. Different types of colonies cannot be discriminated. In the above example, the obtained image is based on plane information, and the size of the colony can be measured, but information on the colony shape and the height direction cannot be obtained.

JP 59-11173 A JP 58-201976 A Japanese Patent Laid-Open No. Sho 62-25348 JP-A 62-65700 JP 2000-78999 A JP-A-7-306023

  The problem to be solved by the present invention is that a colony is classified into the same type based on an image of a petri dish obtained by imaging, and after determining and selecting a colony to be fished, a predetermined amount necessary for identification and sensitivity test In order to collect the colonies, information on the height direction of the bacterial colonies to be collected is estimated for each colony, the volume of the colonies is automatically calculated, and the necessary number of colonies are automatically fished.

  Furthermore, by adopting this method, a method and apparatus for automatically preparing a bacterial solution having a predetermined turbidity after automatic fishing according to the obtained information is provided. By providing, it is to reduce the labor cost of the bacteria laboratory.

  The present invention includes a camera, a base on which a colony on a medium to be imaged is placed, a plurality of first illumination units that emit light from different positions above the colony, and a camera with respect to the base. Is located on the opposite side and includes a second illumination unit that illuminates the culture medium from below.

  According to the present invention, the plurality of first illumination units that irradiate light from different positions above the colony, and the second illumination that is positioned on the opposite side of the camera to the base and illuminates the culture medium from below. Since the colony height information is estimated from the part and the colony volume is calculated, it is possible to estimate the quantity necessary to obtain a bacterial solution having a predetermined concentration (turbidity). As a result, the accuracy of the collected amount of colonies to be collected is improved, operations such as addition and dilution of colonies are greatly reduced, and a bacterial solution having a predetermined concentration can be rapidly produced.

  As a result, the time required for the entire bacterial test can be shortened.

The operation principle of the microorganisms automatic analyzer which shows one Example based on this invention. 1 shows an embodiment of an imaging unit based on the present invention. Relationship between colony volume and turbidity. An example of the amount of fishing bacteria necessary to obtain a predetermined turbidity. Monitor display example. Other display examples of the monitor.

  FIG. 1 shows an example of an automatic microorganism analyzer. Prior to use of the apparatus according to the present invention, samples such as sputum, urine, and pus collected from a patient are applied to an agar medium, separated and cultured, and colonies are formed. There is a case where a certain amount of a culture solution in a blood culture container positively determined is collected and inoculated into a new medium to form colonies after culture. In many cases, colonies can be obtained by overnight culture (about 12 to 18 hours). 101 is a petri dish supply stacker, 102 is a colony imaging unit, 103 is an image processing unit, 104 is a colony fishing unit, 105 is a fungus preparation unit, and 106 is a discharge stacker. It is set in the petri dish supply stacker 101 where the colonies have grown, and is conveyed to the imaging unit 102 by the conveying means 107, and is set at a predetermined position suitable for imaging with respect to the illumination units 108, 109, 111, and the camera 110.

  An example of the illumination arrangement of the colony imaging unit 102 is shown in FIG. Reference numeral 108 denotes a low-angle illumination unit, 109 denotes a high-angle illumination unit, 110 denotes a camera, and 111 denotes a transmission illumination unit. 112 is a light shielding plate, 113 is a pedestal, and 114 is a set petri dish. Returning to FIG. 1, bacterial colonies obtained from the specimen are growing on the surface of the agar medium in 114 petri dish.

  The low-angle illumination unit 108, the high-angle illumination unit 109, and the transmission illumination unit 111 are connected to the illumination control unit 115, and can be turned on in any combination. The illumination light is set to illuminate the petri dish 114 when the transmission illumination unit 111 is lit, and the bottom surface of the petri dish 114 is dark when the low-angle illumination unit 108 and the high-angle illumination unit 109 are lit. To be. A colony on the petri dish 114 is picked up by the camera 110, and the obtained image is transferred to the image input means 116. Reference numeral 117 denotes an image processing unit that extracts the bacterial colony region by processing the image data transferred to the image input unit 116, and calculates an image feature amount from each extracted colony using the image processing unit 117. The image feature amount includes, for example, a perimeter of the colony area, an area, color information, brightness information, a difference from the background brightness, and the like. Reference numeral 118 denotes a secondary storage device that can store captured images, processed images, data, colony positions, and areas. Further, the image processing unit 117 analyzes the obtained feature amount, and groups a plurality of types of colonies on the petri dish based on a past database stored in the secondary storage device 118.

  The petri dish after imaging is transported to the colony-fishing part 104 by the transport means 119. 120 is a fishing rod, 121 is a Z stage, and 122 is an XY stage. The captured image information is displayed on the monitor 123, and the inspection engineer selects and determines a plurality of colonies to be fished. The decision information is transmitted to the colony-fishing part 104 together with the colony position. By controlling the Z stage 121 and the XY stage 122, the fungus needle 120 is moved to the coordinates of the colony to be fished in the petri dish 114, and the colony is picked up.

  FIG. 2 shows a detailed configuration example of the imaging unit in FIG. The low-angle illumination unit 108 includes low-angle illumination units 2011, 2012, 2013, and 2014, and lighting control can be individually performed using the illumination control unit 115. The high-angle illumination unit 109 is composed of high-angle illumination units 2021, 2022, 2023, and 2024, which can also perform lighting control individually. Since the surface of the bacterial colony is relatively smooth, a portion that directly reflects is photographed brightly by applying illumination.

  By performing lighting for each azimuth, the brightness in the plane including the illumination, the center of the colony, and the camera optical axis can be obtained. When obtaining the detailed shape of the bacterial colony, four types of images each illuminating each of the low-angle illumination units 2011, 2012, 2013, and 2014 are provided, and each of the high-angle illumination units 2021, 2022, 2023, and 2024 is represented by 1 type. It is desirable to shoot four types of images that are illuminated one by one, images that are illuminated by the transmissive illumination unit 111, and several types of images that are simultaneously illuminated and combined.

  In high-angle illumination, the angle of the bacterial colony at the position of the reflected light can be obtained from the position of the reflected light. The normal direction of the bacterial colony at the position of the bacterial colony from which the direct reflected light is obtained is (Vc + Vi) / 2 using the unit vector Vc from the reflected light position to the camera lens direction and the unit vector Vi from the reflected light position to the illumination. Appears. If the colony is flat, it can be estimated that the position where the reflected light is detected is near the center of the colony, and if the reflected light position is near the colony, the colony has a height from the medium. Further, when the colony has a shape broken from the dome shape, the reflected light is detected at a plurality of positions. After extracting the colonies, the image feature amount is calculated for each colony. High-angle illumination image (illuminated at 109), low-angle illumination image (illuminated at 108), and transmitted illumination image (illuminated at 111) are used to extract features such as size, color, surface unevenness, and shape of the detected colony region. And image processing. For the circularity, the major axis / minor axis when the shape is approximated to an ellipse is used. The brightness, color, transmittance, and three-dimensional shape of the colony are obtained from the transmitted illumination image. Based on the direct reflection of the brightness and the high-angle illumination image, the transmittance and the height from the medium can be obtained.

The transmitted illumination can be used only when the medium transmits light to some extent, but it becomes dark at the position of the colony and becomes dark as the thickness of the colony increases. When a certain lightness inside the colony is IC (x, y) and an average value of the lightness of the medium is IM, the colony thickness D (x, y) at a certain position (x, y) is expressed by the following equation. Desired.
D (x, y) =-G (log IC (x, y) -log IM)

  Here, G is a gain determined for each colony type.

It is assumed that reflected light by high-angle illumination can be detected at a certain position (X, Y). It is assumed that the normal vector at this time is inclined by θ from the vertical direction. At this time, the change in thickness when the position is shifted by Δx is −Δxtanθ. Here, when the difference of D (x, y) is calculated, D (X + Δx, Y) −D (X, Y) = − G (log IC (X + Δx, Y))
−log IC (X, Y)) = − Δxtanθ
Is established.

That is,
G = Δx tan θ / (log IC (X + Δx, Y) −log IC (X, Y))
Thus, the thickness of the colony is obtained at an arbitrary position. This G is constant for the same colony type (or colony). G is obtained at a place where direct reflection of high-angle illumination is obtained. This G can also be used in places where direct reflection is not obtained. The three-dimensional shape is determined by determining the height. The height is determined by the brightness and G in (x, y) obtained from the transmitted illumination image. G is determined by information of direct reflection in a high-angle illumination image. Since G is constant for the same colony type (or colony), one arbitrary point (x, y) reflected may be used.

  Therefore, in the case of a medium having a relatively high permeability, the three-dimensional shape and volume of the colony can be obtained as long as a location where directly reflected light is generated is found. The volume can be obtained by integrating the obtained thickness (height) in the area.

  As described above, logically, for the purpose of obtaining the volume, it can be calculated only by high-angle illumination and transmitted illumination, and low-angle illumination is not essential. However, with high-angle illumination, direct reflection makes it difficult to understand the shape of the colony in some colonies (when the periphery of the colony and the edge are reflected). In such a case, low angle illumination can be used instead of high angle illumination.

FIG. 3 shows the relationship between the colony volume (mm ³ ) of bacteria and turbidity (McF). It can be seen that the turbidity of the suspended bacterial solution varies depending on the bacterial species even in the same volume. This is thought to be due to the difference in the content of polysaccharides such as mucoids that make up the cell wall for each bacterium. According to FIG. 3, when preparing 3 mL of the bacterial solution of McF0.6, it is possible to prepare a higher concentration side than McF0.6 in almost all bacterial species by collecting at least 1 mm ³ or more colonies. By dividing 1 mm ³ by the colony volume calculated by the method of the present invention described above, the minimum number of colonies necessary for the preparation of the bacterial solution can be obtained. On the other hand, as shown in FIG. 3, the turbidity per fixed volume differs depending on the bacterial species, so if the bacterial species of the colonies are known, the number of colonies necessary for the preparation of the bacterial solution can be calculated more accurately. However, the bacterial species has not been identified at this point in normal bacterial testing, and the bacterial species name is not known at the time of fishing. Here, in the present invention, as described above, a colony image with multi-directional illumination is analyzed, feature amounts are extracted, colonies are grouped using a database stored in advance, and the type of colony is estimated. If the grouping is established, the turbidity per fixed volume can be found from the database, and the number of fish required for preparing the bacterial solution can be determined by considering the estimated colony volume obtained from the image and the coefficient for each bacterial species. It can be obtained more accurately. For reference, FIG. 4 shows the number of fishing bacteria necessary to actually obtain the predetermined turbidity.

  If grouping is not possible, a fixed value may be calculated according to the bacterial species having the lowest turbidity. As described above, the individual colony volume is obtained by the image processing means 117, and at the same time, the number of colonies necessary for obtaining a predetermined bacterial solution concentration is calculated. The information is transmitted to the colony fishing fungus unit 104. The colony fishing part 104 catches a plurality of colonies according to the position information of the colonies to be fished and the information on the necessary number of colonies, and suspends them in the physiological saline in the test tube 124. The turbidity of the suspended bacterial solution is measured by a turbidimeter (not shown), the necessary amount of the diluted solution is calculated, the physiological saline as the diluted solution is added, the turbidity is confirmed again, and a predetermined turbidity is obtained. If so, the test tube is discharged.

  On the other hand, if accurate colony thickness information is not obtained, thickness information defined in advance in several stages (high, medium, low, etc.) according to the colony area is stored in the secondary storage device 118. The approximate volume may be estimated by automatically making the thickness information correspond to the colony area obtained by the image processing unit 117 based on the captured image. The thickness information to be stored may be set for each bacterial species according to the characteristics of the colony shape by the bacterial species, or may be a constant value. When the grouping is successful, accurate thickness information unique to the bacterial species is used, and the colony volume can be estimated by multiplying the colony area calculated from the captured image. When the grouping cannot be performed, an approximate volume can be obtained by drawing out from the secondary storage device 118 and using a constant value corresponding to the area, regardless of the bacterial species. Further, instead of automatically calculating the thickness information according to the area, the user may input the information from the image processing unit 103 or select and specify from several stages of candidates.

  FIG. 5 is an example of a screen for input. The monitor 123 has a thickness input area 302 in addition to the colony image area 301. The colonies on the petri dish imaged by the camera 110 are subjected to image processing, and are displayed by being classified into three groups 303 (group α), 304 (group β), and 305 (group γ) on the colony image area. Yes. For example, for the 303 group α colonies, the user inputs a thickness of 0.1 mm for the end thickness A, 0.15 mm for the middle thickness B, and 0.2 mm for the center thickness C. Input is done for each colony group to be fished. Alternatively, the numerical values of A, B, and C may be selected with choices.

  FIG. 6 is an example of another screen. You may input or select the thickness of one place of a colony. The user input is further simplified, and one of low, medium, and high is selected. In this example, Medium is selected. After calculating the volume, the method of calculating the number of colonies to be picked from the relationship between the volume and the turbidity is the same as the process for obtaining the height information accurately. Furthermore, when a specific bacterial species is assumed from the beginning, such as when used in an industrial field, and it is not necessary to perform grouping based on the feature amount of the colony, the low-angle illumination unit 108, the high-angle illumination unit 109, and the transmission A plurality of illumination combinations of the illumination unit 111 are not required, the apparatus configuration is simplified, and the manufacturing cost can be reduced. In this case, the area can be calculated from the captured image by the image processing unit 117, and constant thickness information can be input or selected from a plurality of candidates, and the approximate volume can be obtained by multiplying the area.

101 Petri dish supply stacker 102 Imaging unit (colony imaging unit)
DESCRIPTION OF SYMBOLS 103 Image processing part 104 Colony fishing part 105 Bacteria preparation part 106 Discharge stacker 107,119 Conveying means 108 Low angle illumination unit 109 High angle illumination unit 110 Camera 111 Transmission illumination unit 112 Light shielding plate 113 Base 114 Petri dish 115 Illumination control unit 116 Image Input means 117 Image processing means 118 Secondary storage device 120 Fishing fungus needle 121 Z stage 122 XY stage 123 Monitor 124 Test tube 301 Colony image area 302 Thickness input area 303 Group α
304 Group β
305 group γ
2011, 2012, 2013, 2014 Low angle illumination unit 2021, 2022, 2023, 2024 High angle illumination unit

Claims (16)

  A camera, a base on which a colony on a medium to be imaged is placed, a plurality of first illumination units that emit light from different positions above the colony, and a base opposite to the camera A bacteria imaging apparatus comprising: a second illumination unit that is positioned and illuminates the culture medium from below. In claim 1,
Bacteria characterized in that gain determined for each colony type is obtained from reflected light using the first illumination unit, the brightness of the medium is obtained from the second illumination unit, and the thickness of the colony is obtained from the gain and the brightness of the medium. Imaging device. In claim 2,
A bacterial imaging apparatus characterized in that the thickness of a colony is obtained by the following equation.
D (x, y) =-G (log IC (x, y) -log IM)
(X, y) is position, IC is lightness, IM is lightness average, G is gain In claim 1,
The first imaging unit is provided with a plurality of illuminations having different heights from the base, respectively. In claim 1,
The first imaging unit includes a high-angle illumination that illuminates a colony on a culture medium from an upper high angle, and a low-angle illumination that irradiates from an upper low angle. In claim 1,
A bacterial imaging apparatus, wherein a shape characteristic of a colony is obtained using a first illumination unit and a second illumination unit. A camera,
A base on which a colony on a medium to be imaged is placed;
A plurality of first illumination units that emit light from different positions above the colony;
A second illumination unit that is located on the opposite side of the base from the camera and that illuminates the culture medium from below;
A mechanism for classifying colonies based on information from the camera;
A fishing fungus mechanism that catches colonies,
A bacterial solution adjusting device comprising a bacterial solution adjusting mechanism that prepares a bacterial solution using fish colonies. In claim 7,
A fungus characterized in that a gain determined for each colony type is obtained from reflected light using the first illumination unit, the brightness of the medium is obtained from the second illumination unit, and the thickness of the colony is obtained from the gain and the brightness of the medium. Liquid adjustment device. In claim 8,
The thickness of a colony is calculated | required by following Formula, Bacteria liquid adjustment apparatus characterized by the above-mentioned.
D (x, y) =-G (log IC (x, y) -log IM)
(X, y) is position, IC is lightness, IM is lightness average, G is gain In claim 8,
An apparatus for adjusting a bacterial liquid, wherein the volume of a colony is obtained from the thickness of the colony. In claim 10,
A fungus solution adjusting device, wherein the number of fishing fungi necessary for preparing a fungus solution having a predetermined concentration is calculated from a volume of a colony. In claim 8,
The first illumination unit includes high-angle illumination and low-angle illumination,
Determining which group of the groups prepared in advance belongs to, and based on this determination, calculate the number of fishing bacteria necessary for the preparation of the bacterial solution of a predetermined concentration using the turbidity coefficient per unit volume specific to that group Bacterial fluid adjustment device characterized by A camera,
A base on which a colony on a medium to be imaged is placed;
A mechanism for classifying colonies based on information from the camera;
A fungus adjustment device having a fungus mechanism for catching colonies,
An apparatus for adjusting a bacterial liquid, which calculates the number of fishing bacteria necessary for preparing a bacterial liquid having a predetermined concentration from several stages of colony thickness information prepared in advance according to the size of the colony area.   The colony thickness information can be set by a user.   14. The bacterial solution adjusting device according to claim 13, further comprising a display screen for displaying several stages of colony thickness information.   Furthermore, the thickness information of a colony is prepared for every part of a colony, The fungus liquid adjustment apparatus of Claim 13 characterized by the above-mentioned.

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