JP5860254B2 - Mount for fixing microorganism culture sheet and microorganism inspection method - Google Patents

Description

The present invention is a microbial culture sheet fixed microbial cultures seat locking mount for securing a plurality of microbial culture sheets, and the mount to form the image data of a plurality of microbial cultures sheet by the imaging, based on the image data The present invention relates to a microorganism testing method for counting the number of colonies.

  In order to confirm the presence of microorganisms or measure the number of microorganisms, various microorganism culture sheets have been developed in place of conventional agar media. For example, a sheet medium having a structure in which a medium laminate composed of a porous matrix layer and a water-soluble polymer compound layer is bonded to an adhesive sheet and a transparent film is covered thereon is disclosed (Patent Document 1). The culture medium laminate is adhered to the center of the pressure-sensitive adhesive sheet that is larger than the culture medium laminate, and a transparent film is placed on the culture medium laminate, and the portion of the transparent film that protrudes from the culture medium laminate is the culture medium laminate of the adhesive sheet. Is bonded to a portion that is not bonded. In order to inoculate the bacterial solution, the transparent film is peeled off from the pressure-sensitive adhesive sheet, the bacterial solution is added to the medium laminate, and the transparent film is applied again and cultured.

  A base material having an upper surface; a pedestal mounted on the upper surface of the base material and including a non-absorbable culture surface; a flexible cover sheet attached to the base material and having a bottom surface; There is also a microorganism sheet including a culture medium deposited on one or a plurality of surfaces selected from the culture surface and the bottom surface of the cover sheet (Patent Document 2). The diffusion of the liquid sample can be limited so that the sample does not spill, resulting in a diffusion sample of uniform shape and size.

  The culture medium layer used for the microorganism culture sheet is a dry culture layer, and inoculates the bacterial solution to add water to the dry culture layer so that the microorganism can be grown while forming a culture environment of the microorganism. Next, after the microorganism sheet inoculated with the bacterial solution is cultured at a predetermined temperature for a predetermined time, the presence of bacteria can be measured or identified. By changing the composition of the dry culture layer, microbial culture sheets for different applications such as aerobic bacteria, E. coli, coliforms, enteric bacteria, yeast, Staphylococcus aureus, and general bacteria are prepared. be able to.

  The Food Sanitation Law sets bacteriological component standards for specific foods such as meat products and fish paste products. For example, for meat products, the number of general viable bacteria, Escherichia coli, Staphylococcus aureus, etc. meet the prescribed standards. Demands that. In any case, it is preferable to use two culture media for the sake of accuracy. In order to measure the number of general viable bacteria, two plates are evaluated at different dilution ratios. For example, using four microbial culture sheets, inoculate two of the 100-fold diluted solution, inoculate two of the 1,000-fold diluted solution, and calculate the average number of colonies in consideration of the dilution rate. Therefore, when evaluating the number of general viable bacteria and Escherichia coli and Staphylococcus aureus for a certain sample, four microorganism culture sheets for the general viable bacteria count, two microorganism culture sheets for Escherichia coli, and for Staphylococcus aureus Two microbial culture sheets were prepared, spread on a desk, inoculated with a specimen bacterial solution, and cultured according to each standard, and then colonies were observed.

  On the other hand, there is also a technique for automating the measurement of microbial colonies and thereby reducing human error (Patent Document 3). Receiving one or more images of a biological growth medium, identifying a first number of colonies associated with the interior of the biological growth medium, and a periphery of the biological growth medium Identifying a second number of colonies associated with said one or more colonies within a defined distance from a growth region edge of said growth medium if said first number is less than a threshold value And a step of excluding it from the second number. The biological growth medium is put into the biological scanning device one by one, an image is formed, and the obtained image is communicated to an external computer including a processor for performing image analysis. Is to measure. The biological growth medium is labeled with a bar code or other identification marking used to identify the growth plate.

JP 2001-245893 A JP-T-2004-515236 Special table 2007-533296

The microorganism culture sheet described in Patent Document 1 or Patent Document 2 is a sheet-like material, and is thin and compact, unlike the agar medium, and has an advantage that the storage volume and culture volume can be made compact. However, since the microorganism culture sheet uses a dry culture layer, the bacterial solution gradually spreads on the medium and soaks into the medium. The cover sheet is pressed for about 1 to 3 minutes until the bacterial solution after inoculation is pressed with a spreader having a circular pressing portion so that the bacterial solution spreads in a predetermined range, or until the bacterial solution after inoculation is soaked in the medium. After coating, it is necessary to leave it horizontally . The number of specimens of whether to meet fine bacteriological element standard evaluation is required very often, the development of microbial cultures sheet capable more efficient operation is desired.

  Moreover, when the colony of a culture medium is evaluated, as disclosed in Patent Document 3, if a colony can be automatically measured, a manual measurement process can be avoided. When a microorganism culture sheet is used as the biological growth medium, it is easy to form image data (image file such as jpg.jpeg.bmp.tiff) by the image forming apparatus. However, the method described in Patent Document 3 is to measure the number of colonies by forming images one by one from a microorganism culture sheet. Each sheet must be inserted into the scanning unit, and a predetermined time is required for image formation. Therefore, development of a microorganism culture sheet capable of forming image data more efficiently is desired.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a mounting board for fixing a microorganism culture sheet capable of easily fixing a microorganism culture sheet.

In addition, an object of the present invention is to provide a microorganism testing method using the microorganism culture sheet with a mount.

The present inventors have made study in detail the microorganism culture sheet, to secure the plurality of microbial cultures sheet base paper or to have it automatically count the number of colonies by images processed once for a plurality of microbial cultures sheet by forming an image on a short time we found that you can form an image different from the case of the image forming microbial culture sheet one by one, thereby completing the present invention.

That is, the present invention is a microbial culture sheet fixing mount made of a synthetic resin for fixing a plurality of microbial culture sheets ,
A mounting board for fixing a microorganism culture sheet, characterized in that the microorganism culture sheet can be detachably fixed, a partition indicating a fixing position of each microorganism culture sheet is formed, and a frame is formed with a low-lightness color. To do.

In addition, the present invention is inoculated with Kin'eki the microorganism culture sheet, and cultured to express colony, to secure the plurality of microbial cultures sheet was expressed the colonies to the microbial cultures sheet fixing mount,
Forming image data of a mount on which the microorganism culture sheet is fixed ;
The present invention provides a microorganism testing method , wherein the number of colonies for each microorganism culture sheet is counted using the image data of the microorganism culture sheet included in the image data.

  According to the mounting board for fixing a microorganism culture sheet of the present invention, since a plurality of microorganism culture sheets can be fixed, the operability is excellent.

According to mount with microbial cultures sheet of the present invention, since a plurality of microbial cultures sheet is fixed, it is possible to form an image data at a time for a plurality of microbial culture sheet, the image data forming an easily Yes, it is possible to automatically count the number of colonies efficiently.

It is a figure explaining an example of the mounting board for microorganisms culture sheets concerning the present invention. It is a figure explaining an example of the color sample arrangement | sequence table which can be displayed on the microbe culture sheet fixing mount which concerns on this invention, and is a figure which shows the aspect containing a color chart, a gray scale chart, and a position recognition marker as a color sample arrangement | sequence table. is there. It is a figure explaining the aspect which fixed the microorganisms culture sheet fixation board | substrate which concerns on this invention to the two steps | paragraphs of 8 microorganism culture sheets at a time. It is a figure explaining the microorganisms culture sheet with a mount concerning the present invention, Drawing 4 (a) is a top view and Drawing 4 (b) is a XX line sectional view of Drawing 4 (a). The microorganism culture sheet shows an embodiment in which a circular dry culture layer is formed on a base sheet, and is detachably fixed by two strips of adhesive layers formed on the mount. Fig.5 (a) is a top view of the microorganism culture sheet with a mount based on this invention, FIG.5 (b) is XX sectional drawing of Fig.5 (a). The microorganism culture sheet shows an embodiment in which a recess is formed in a part of the base sheet, and a dry culture layer is formed in the recess. It is a top view of the microorganism culture sheet with a mount concerning the present invention, and is a figure explaining the mode by which two microorganism culture sheets are fixed to the mount with a serial number indication display. Fig.7 (a) is a top view of the microbe culture sheet | seat with a mount based on this invention, FIG.7 (b) is XX sectional drawing of Fig.7 (a). It is a figure explaining the aspect in which eight microbe culture sheets were formed in 2 steps | paragraphs at a time on the A4 size mount. FIG. 8 (a) is a plan view of the continuous microorganism culture sheet according to the present invention, and FIG. 8 (b) is a cross-sectional view taken along the line XX of FIG. 8 (a). It is a figure explaining the aspect by which the eight microorganism culture sheets were formed in two steps | paragraphs at a time on the A4 size base material sheet. It is a perspective view of the storage tray which concerns on this invention. It is a perspective view of the storage tray which concerns on this invention, and is a figure explaining the aspect comprised from the shelf step part which laminates | stacks a some shelf, and the cover part which coat | covers the upper part. It is a figure explaining the shelf which comprises the storage tray which concerns on this invention, FIG.11 (a) is a perspective view, FIG.11 (b) is a top view, FIG.11 (c) is a bottom view, FIG.11 (d) is FIG. A side view is shown. It is a figure explaining the method of laminating | stacking a some shelf by fitting and coat | covering a cover part on the upper part, and assembling the storage tray which concerns on this invention. It is a figure explaining the other aspect of the shelf which comprises the storage tray which concerns on this invention, Fig.13 (a) is a perspective view, FIG.13 (b) is a top view, FIG.13 (c) is a bottom view, FIG. (D) shows a side view. It is a figure explaining inclination correction processing of original layer data. 6 is a diagram showing original layer data obtained in Example 2. FIG.

First aspect of the present invention, microbial cultures sheet fixing mount der made of a synthetic resin for fixing the plurality of microbial culture sheet,
A mounting board for fixing a microorganism culture sheet, wherein the microorganism culture sheet can be detachably fixed, a partition indicating a fixing position of each microorganism culture sheet is formed, and a frame is formed with a low-lightness color. The Wherein the base sheet, may have notches for inserting and fixing the corner portions of the microbial culture sheet, or a pressure-sensitive adhesive layer for fixing the microorganism culture sheet is formed, a color chart for color correction Alternatively, gray scale and / or position recognition markers may be displayed.

The second of the present invention were inoculated with Kin'eki the microorganism culture sheet, cultured to express colonies, fixed a plurality of microbial cultures sheet was expressed the colonies to the microbial cultures sheet fixing mount And
Forming image data of a mount on which the microorganism culture sheet is fixed ;
It is a microorganism testing method , wherein the number of colonies for each microorganism culture sheet is counted using image data of the microorganism culture sheet included in the image data. The position of the image data may be corrected using a position recognition marker included in the image data, and then color correction may be performed using a color chart and a gray scale to count pixels corresponding to colonies included in the culture medium. .
Hereinafter, the present invention will be described in detail.

(1) Mount for fixing microorganism culture sheet The mount for fixing microorganism culture sheet of the present invention is a flat plate for fixing a microorganism culture sheet. FIG. 1 is a plan view of the microorganism culture sheet fixing mount (A) of the present invention. It is preferable that the microbial culture sheet fixing mount (A) has a frame (F) with a low-lightness color such as straw, dark brown, or black at least on the outer periphery. This is because it is easy to recognize the outer frame of the mount (A) in the image analysis after taking the image of the mount (A) with a flatbed scanner or the like. The mount (A) used in the present invention may further be formed with a partition or the like so that each fixing position for fixing a plurality of microorganism culture sheets can be understood. The microorganism culture sheet fixing mount shown in FIG. 1 shows a mode in which sections (a) divided by eight dividing lines (L) are formed in two stages of four inside each black border. A microorganism culture sheet can be fixed to each of the compartments (a). The mounting plate (A) for fixing the microorganism culture sheet only needs to know the position to fix the microorganism culture sheet, and the partition line (L) may not be provided. For example, when a notch (b) or the like for inserting a corner of the microorganism culture sheet is formed corresponding to the position to be fixed, the fixing position can be easily understood without the partition line (L). be able to. In addition, in FIG. 1, the aspect which has both a partition line (L) and a notch part (b) is shown.

  The material for the microorganism culture sheet fixing mount (A) may be paper, synthetic resin, metal, or a composite material thereof. The method for fixing the microorganism culture sheet is not particularly limited, but it can be used multiple times as long as it can be fixed detachably, and can be appropriately selected according to the material of the mount (A). In FIG. 1, an embodiment is shown in which a notch (b) into which a corner of the microorganism culture sheet can be inserted is formed on the mount (A), but instead of such a notch (b), For example, a photo corner for fixing a corner may be attached. Further, an adhesive layer may be formed on the mount (A), and the microorganism culture sheet may be fixed through the adhesive layer. The notched portion (b) and the partition line (L) can be appropriately selected according to the material of the mount (A). For example, the partition line (L) is formed by embossed unevenness in addition to printing. May be.

  Further, the mount (A) may display a color chart for color correction, a color sample array table (70) such as a gray scale, a position recognition marker, a serial number (80), and the like according to the application. When the microorganism culture sheet (B) is fixed on the mount, inoculated with the bacterial solution, and cultured to form colonies, colony-containing image data can be formed to automatically count the number of colonies. it can. At this time, if the color sample arrangement table (70) is displayed on the mount (A), the color correction of the image data can be performed by the color sample arrangement table (70) included in the image data, and the measurement accuracy can be improved. .

  In the present invention, examples of the color sample array table (70) include a color chart (71) and a gray scale chart (73). FIG. 2 shows an example of such a color sample arrangement table (70). The color chart (71) is a color array including 12 basic colors excluding gray scale, and in FIG. 2, a color chart of 18 colors is displayed. The gray scale chart (73) is a gray scale display of achromatic colors from white to black. As such a color sample arrangement table (70), as shown in FIG. 2, position recognition markers (75a, 75b, 75c, 75d) may be displayed at the four corners of the color sample arrangement table. At the time of image analysis, the position of the mount, and thus the position of each microorganism culture sheet (B) can be aligned using the position recognition markers displayed in the color sample arrangement table (70) included in the image data. The colonies of each microorganism culture sheet (B) can be observed accurately and the number of colonies can be counted.

  The mount (A) of the present invention may be one in which other characters or patterns are further displayed at the fixed position of each microorganism culture sheet (B). For example, if characters such as “missing” are displayed, missing characters will be displayed at the location where the microorganism culture sheet has been removed, so if there are “missing” characters when automatically counting the number of colonies Can be counted in correspondence with programs that do not measure.

  There are no particular limitations on the number of microorganism culture sheets to be fixed to the microorganism culture sheet fixing mount (A) as long as it is plural. Examples include one in which the microorganism culture sheets are fixed in two stages in the longitudinal direction of the mount, in addition to one in which three sheets are fixed in two stages. When using a large microbial culture sheet, one sheet may be fixed to this mount. When the mount (A) is larger than the microorganism culture sheet (B), it is easy to ensure the horizontal level after inoculating the bacterial solution, and the culture is maintained while maintaining the horizontal level until the bacterial contact is soaked into the dry culture layer. It can be conveyed to the apparatus, and the time from the inoculation of the bacterial solution to the culture can be shortened.

  In the present invention, the size of the mount (A) can be appropriately selected in consideration of the microorganism culture sheet (B) to be fixed. When fixing a plurality of microorganism culture sheets (B) to the mount (A), it is possible to use A series size A4 of Japanese Industrial Standards (JIS), or mount series (A) of B series B5 or B4 size. preferable. This is because it is easy to obtain the material of the mount (A), and even when colonies are automatically counted after image processing, image formation can be easily performed using a flatbed scanner. Further, since the number of dots corresponding to A size paper and B size paper is generally defined as the resolution, the resolution can be easily selected.

(2) Microorganism culture sheet with mount
The base paper with a microorganism culture sheet (100), a plurality of microbial cultures sheet (B) is fixed to the microorganism culture sheet fixing mount (A). FIG. 3 shows an embodiment in which eight microorganism culture sheets (B) are fixed to the microorganism culture sheet fixing mount (A) shown in FIG.

  In the present invention, it is sufficient that a plurality of microorganism culture sheets (B) can be fixed to the mount (A). Referring to FIG. 4, the mount (A) and the microorganism culture sheet (B) are bonded to the adhesive layer (C). A mode of fixing via the above will be described. The plan view of FIG. 4A is a plan view in which the microorganism culture sheet (B) is fixed to the mount (A). The microorganism culture sheet (B) may be a commercially available product, and is generally formed on at least one surface of the substrate sheet (10) and the substrate sheet, as shown in the cross-sectional view along the line XX in FIG. A dry culture layer (20) and a cover sheet (30) covering the dry culture layer. The pressure-sensitive adhesive layer (C) formed on the mount (A) may be formed in one line so that the upper part and the central part of the back surface of the microorganism culture sheet (B) are fixed, and the upper part and the lower part. A linear pressure-sensitive adhesive layer (C) may be formed at two locations. Furthermore, you may form the adhesive layer (C) of the same area as a back surface so that the whole back surface of a microorganisms culture sheet (B) may be fixed. FIG.4 (b) is a side view of the microorganism culture sheet (100) with the mount of this invention which fixed the microorganism culture sheet (B) with the two adhesive layers (C). When two linear pressure-sensitive adhesive layers (C) are formed on the mount (A), the microorganism culture sheet (B) can be stably fixed, and curling of the microorganism culture sheet (B) can be avoided.

  The microorganism culture sheet (B) used in the present invention generally covers the base sheet (10), the dry culture layer (20) formed on one surface of the base sheet, and the dry culture layer. A cover sheet (30). Furthermore, the microorganism culture sheet containing another structure may be sufficient. As shown in FIG. 4 (a), a dilution factor entry field (40) may be displayed on the base sheet (10), the cover sheet (30), etc., and a medium identification color bar (50) and the like may be displayed. It may be displayed. The dilution factor entry column (40) is a column describing the dilution factor of the bacterial solution. In the case of a 10-fold diluted solution, check the column below -1, and in the case of a 100-fold diluted solution, -2. A check can be put in the lower column, and the dilution factor can be easily entered. The medium identification color bar (50) can be displayed in red for general viable count, green for Escherichia coli, yellow for Staphylococcus aureus, etc., and the type of medium can be visually determined from the appearance.

  Further, an identifier (60) such as a barcode may be displayed. FIG. 4A shows a two-dimensional barcode, but a one-dimensional barcode may be used. If the individual information of the microorganism culture sheet (B), for example, the medium type, serial number, etc. is input to such an identifier, it can be read by a barcode reader to obtain the individual information of the microorganism culture sheet (B). It can be made to correspond to the specimen number described in the microorganism culture sheet (B).

  The microorganism culture sheet (B) may further display a date entry column, a sample number entry column, and the like.

  In addition, although the microorganism culture sheet | seat (B) of FIG. 4 shows the aspect by which a circular dry culture layer (20) is formed on a base material sheet (10), dry culture is carried out on the whole surface of a base material sheet (10). A layer (20) may be formed. Moreover, a culture layer may be formed also on the surface where the cover sheet (30) contacts the dry culture layer (20). Furthermore, you may form a recessed part in a part of base material sheet (10), and form a dry culture layer (20) in this recessed part. Such an embodiment is shown in FIG. FIG. 5 (a) is a plan view of a microorganism culture sheet (100) with a mount of the present invention in which such a microorganism culture sheet (B) is fixed to the mount (A), and FIG. 5 (b) is a plan view of FIG. It is XX sectional drawing of a).

The microorganism culture sheet (100) with a mount of the present invention has a plurality of microorganism culture sheets (B) fixed thereon, preferably 2 to 10 sheets, more preferably 4 to 10 sheets, particularly preferably 4 to 8 canes. is there. FIG. 6 shows an aspect in which two microorganism culture sheets (B) are fixed to the mount. In the present invention, since each microorganism culture sheet (B) is fixed to the mount (A), the microorganism culture sheet (B) that has not been inoculated with the bacterial solution is removed from the mount (A), and the same as in the past. Can be used in the way.
When using the microorganism culture sheet with a mount (100) of the present invention, it is placed in front of the operator who inoculates the bacterial solution, and after injecting the specimen number, date, dilution factor, etc., the bacterial solution is inoculated. At this time, the microbial culture sheet with mount (100) on which the sample information is entered may be rotated 90 ° to inoculate the bacterial solution. Since the peeling direction of the cover sheet (30) of the microorganism culture sheet (B) varies depending on the direction in which the microorganism culture sheet (100) with the mount is placed, the operator can select and use a simple direction.

  Further, in FIG. 7, the microorganism culture sheet (B) on which the dilution factor entry column (40) is displayed is fixed on the mount (A) in four rows, two on each side, for a total of eight, and a color sample distribution is provided. It is a microorganism culture sheet (100) with a mount in which a column table (70) (not shown) is displayed on the open end side of the cover sheet (30). FIG. 7A is a plan view, and FIG. 7B is a cross-sectional view taken along the line XX. The microorganism culture sheet (100) with the mount can be placed in front of the operator in the short direction and the sample number, date, dilution factor, etc. can be entered, and the microorganism culture sheet (B) can be entered without rotating it. ) Cover sheet (30) is peeled from the right side to the left side of the operator, and the bacterial solution can be inoculated.

(3) Microorganism culture sheet The microorganism culture sheet used in the present invention covers at least the base material sheet (10), the dry culture layer (20) formed on one surface of the base material sheet, and the dry culture layer. What is necessary is just to have a cover sheet (30) to do.

  As a base material sheet (10), what has water resistance and mechanical strength suitable for formation of a dry culture layer (20) can be used widely. The base sheet may be a single layer or a laminate of two or more layers. As the base sheet, a polyolefin resin such as polyethylene, polypropylene, and polystyrene, a polyester sheet such as polyethylene terephthalate, and a plastic sheet such as polyamide can be suitably used. These plastic sheets are excellent in transparency, and colonies can be observed through the base sheet. Moreover, the synthetic paper shape | molded like paper by using a paper base material or synthetic resin as a main raw material may be sufficient. The base sheet may be printed with a square print with ink that does not affect the growth of microorganisms.

  A dry culture layer contains a nutrient component and the like in addition to a gelling agent for containing moisture suitable for the growth of microorganisms. The gelling agent is water-absorbing (or water-swelling) and water-insoluble, and can substantially retain its shape when it absorbs water or swells with water. Polyvinyl alcohol (PVA) , Modified PVA, cellulose derivatives, starch and derivatives thereof, cross-linked products of water-soluble polymers such as cellulose derivatives and polysaccharides other than starch, acrylic acid and derivatives thereof, polyethers, and proteins; starch-based water-absorbing polymers; Acrylic polymer water-absorbing polymer; maleic anhydride copolymer; vinyl pyrrolidone copolymer; polyether water-absorbing polymer; acrylic fiber hydrolyzate; and other water-absorbing polymers, guar gum, psyllium seed gum, xanthan gum, hydroxyethyl cellulose Carboxymethyl Loin, polyacrylamide, locust bean gum, and the like algin.

  Examples of nutritional components that can be blended in the dry culture layer include, for example, yeast extract / pepton / glucose mixture, meat extract / pepton mixture, peptone / soybean peptone / glucose mixture, etc. For inspection, peptone, ammonium iron citrate, lactose, etc., for staphylococci, meat extract, peptone, mannitol, egg yolk mixture, peptone, meat extract, yeast extract, sodium pyruvate, etc., for vibrio Yeast extract, peptone, sucrose, sodium thiosulfate, sodium citrate, etc. for enterococci, bovine brain extract, heart extract, peptone, glucose, etc., for fungi, peptone, glucose mixture, yeast extract, A glucose mixture, a potato extract / glucose mixture and the like are used. One or more can be selected from these according to the microorganism to be grown and mixed for use. Furthermore, you may contain other components, such as coloring indicators, selective agents, such as antibiotics, a substrate, and a buffer (pH adjuster). In addition, as a color development indicator, tetrazolim salts such as triphenyltetrazolium chloride (hereinafter referred to as TTC), p-tolyltetrazolium red, tetrazolium violet, petetylyltetrazolium blue, neutral red mixture, phenol red, promthymol blue, thymol blue mixture There are pH indicators such as As the microorganism grows, the color develops or changes color so that the growth of the microorganism becomes easy to see, and the specific microorganism can be detected by adding the color developed or fluorescent enzyme substrate of the enzyme possessed by the particular microorganism.

  As the cover sheet, a cover sheet having flexibility that can be easily contacted with the culture layer, waterproof, and water vapor impermeable can be preferably used. The cover sheet is preferably transparent so that colonies can be observed and counted through the cover sheet after culturing. For example, a plastic sheet such as polyolefin, polyester, or polyamide can be used in the same manner as the base sheet. In addition, when the grid print pattern is not printed on the base material sheet, the grid print pattern may be printed on the cover sheet.

  Such a microorganism culture sheet (B) uses a base sheet on which an identifier such as a dilution rate entry column or a two-dimensional barcode is printed, and is a dry culture containing at least a gelling agent above the base sheet. Layers can be formed and manufactured. For example, an adhesive is applied on the base sheet, and other components such as the above gelling agent, nutritional component, color indicator, selective agent, substrate, buffer (pH adjuster) are adhered in a powder form. Can be prepared. Any adhesive can be used as long as it does not inhibit the growth of microorganisms. Preferably, a water-insoluble adhesive such as an acrylic pressure sensitive adhesive can be used. In addition, a medium solution containing at least a gelling agent and containing at least one of nutritional components, color developing indicators, selection agents, substrates, and buffering agents (pH adjusting agents) is printed, applied, applied, sprayed, and the like. You may form above a material sheet. The medium solution is a solution in which at least an adhesive component is dissolved and a component constituting the dry culture layer is dissolved or dispersed so that the component can be directly fixed on the substrate. In addition, a dry culture layer may be formed in a part of upper surface of a base material sheet, as shown in the said FIG. 4, and may be formed in the recessed part of a base material sheet, as shown in FIG.

  In the present invention, it was found that by using a dry culture layer, microbial colonies can be observed and counted in the same manner as an agar medium, and visibility superior to that when an agar medium is used can be secured. did. The culture layer may be dried as long as the solvent contained in the medium solution can be removed, and can be dried according to a conventionally known method such as temperature and pressure.

(4) Manufacturing method of microorganism culture sheet with mount
Pedestal microbial cultures sheet laminated sheet is obtained by fixing the microorganism culture sheet (B) in the above-mentioned mount (A). The partition line (L), a color sample array table such as a color chart or a gray scale chart, a position recognition marker, a serial number, and the like are formed on the mount (A) by printing or the like. Further, when the cutout portion (b) made of a through hole for fixing the microorganism culture sheet (B) is formed on the mount (A) as shown in FIG. What is necessary is just to form a hole. A photo corner for fixing the microorganism culture sheet (B) may be attached.

On the other hand, when forming the adhesive layer on the mount (A), the adhesive layer is formed after printing the serial number entry column, the color sample array table (70), and the like.
A conventionally well-known adhesive can be used as an adhesive. For example, there are acrylic pressure-sensitive adhesives such as acrylic acid ester type and methacrylic acid ester type. In addition, a rubber-based adhesive such as natural rubber or latex can be used. In addition, a silicone pressure sensitive adhesive can also be used. Such pressure-sensitive adhesives may further include a surfactant, a dehumidifying agent, a thickener, a water-resistant agent, a dispersant, an ultraviolet absorber, an antioxidant, an antistatic agent, an ink fixing agent, and a dye as necessary. Auxiliaries such as fixing agents, fluorescent dyes, colored dyes, lubricants, deodorants, deodorants, and antibacterial agents can also be added.

  The pressure-sensitive adhesive layer used in the present invention is only required to have a fixability to such an extent that the fixed microorganism culture sheet can be re-peeled, and conforms to JIS-Z-0237 of the pressure-sensitive adhesive layer. 180 degree peeling adhesive strength with respect to an aluminum plate under the conditions is 100 g / 25 mm to 600 g / 25 mm. Furthermore, after pasting on the aluminum plate, JIS at the time when 30 days passed under the condition of −10 ° C. to 70 ° C. 180 degreeC peeling adhesive force based on -Z-0237 is 100g / 25mm-600g / 25mm.

  The pressure-sensitive adhesive is preferably formed only at the location where the microorganism culture sheet is fixed. For example, when the microorganism culture sheet is fixed with two linear adhesive layers, four linear adhesive layers are formed at the corresponding positions on the mount, and the microorganism culture sheets are sequentially fixed at predetermined positions. Can be manufactured. Alternatively, a method may be used in which an adhesive layer is continuously formed on a long mount, the microorganism culture sheet (B) is sequentially fixed to the adhesive layer, and the long mount is cut at a predetermined position.

(5) Continuous microorganism culture sheet
The communication設微organism culture sheet, comprising a base sheet and a plurality of drying the culture layer formed on one surface of the base sheet, a cover sheet covering said plurality of drying the culture layer, respectively. Like the above-mentioned microorganism culture sheet with a mount (100), it is easy to maintain the horizontal by continuously arranging a plurality of microorganism culture sheets, and can be carried into the culture apparatus after inoculating the bacterial solution in a short time, Image formation after colony formation is also easy.

A preferred embodiment of the continuous microorganism culture sheet (100 ′) is shown in a plan view of FIG. 8A and a cross-sectional view taken along line XX of FIG. 8B. FIG. 8 shows that a plurality of dry culture layers (20 ′) are formed on a base sheet (10 ′), and a plurality of cover sheets (30 ′) covering the dry culture layers (20 ′) are fixed. The embodiment is shown. If the portion where the base sheet (10 ′) and the cover sheet (30 ′) overlap is defined as the microorganism culture sheet (B ′), the plurality of microorganism culture sheets (B ′) are combined into the same base sheet (10 ′). Since they are formed and connected, they are referred to as “continuous microorganism culture sheets”. The continuous microorganism culture sheet (100 ′) of the present invention may be a case where a plurality of microorganism culture sheets (B ′) are formed using a single substrate sheet (10 ′). A perforation or the like may be formed on the base sheet (10 ′) so that it can be easily separated for each culture sheet (B ′).

As the base material sheet (10 ′) used in the continuous microorganism culture sheet (100 ′), for example, the cover of the microorganism culture sheet (B ′) is the same as the mount (A) of the microorganism culture sheet with mount (100). It is larger than the sheet (30 ′), preferably two or more microorganism culture sheets (A ′) are continuously arranged, more preferably 6 to 10, particularly preferably 6 to 8. As the size of the substrate sheet (10 ′), as in the microorganism culture sheet fixing mount (A), those of A4 size or B4 size can be suitably used. This is because it is easy to form image data with a scanner or the like after colony formation.

In the continuous microorganism culture sheet (100 ′), the same color sample arrangement table (70) and serial number (80) as the above-described substrate (A) for fixing the microorganism culture sheet are displayed on the base sheet (10 ′). May be. When colonies are formed on a plurality of microorganism culture sheets (A ′), image data of the plurality of microorganism culture sheets (B ′) can be formed at a time and the number of colonies can be automatically counted.

The continuous microorganism culture sheet (100 ′) is formed by forming a plurality of dry culture layers (20 ′) on the base sheet (10 ′) and covering the dry culture layers (20 ′) with the cover sheet (30 ′). ') Can be prepared by fixing. According to the continuous microorganism culture sheet (100 ′) of the present invention, since the plurality of dry culture layers (20 ′) are fixed without forming the fixing means to the mount (A), it is lighter and thinner. Can be
(6) Storage tray
Base paper with a microorganism culture sheet (100) and communicating設微organism culture sheet (100 ') can be transferred into the culture device after the inoculated solution is incubated. At this time, if the microorganism culture sheet with a mount (100) and the continuous microorganism culture sheet (100 ′) are stored in a predetermined tray and carried together with the tray into the culture apparatus, the carrying-in operation can be easily performed and a large amount of Since the microorganism culture sheet with the mount and the continuous microorganism culture sheet can be carried into the culture apparatus, the culture operation can be performed efficiently. A perspective view of such a storage tray is shown in FIG. The storage tray (200) shown in FIG. 9 is obtained by covering a shelf step (120) composed of a plurality of shelves with a lid (130).

  A mode in which the lid (130) is removed is shown in FIG. As shown in FIG. 10, the storage tray includes a shelf step portion (120) formed by stacking a plurality of shelves (110) and a lid portion (130) covering the upper portion thereof. The perspective view of the shelf (110) shown in FIGS. 9 and 10 is shown in FIG. 11 (a), the plan view in FIG. 11 (b), the bottom view in FIG. 11 (c), and the side view in FIG. Shown in As shown in FIG. 11, the shelf (110) includes a bottom surface portion (111) and a side wall portion (113) erected on the outer periphery of the bottom surface portion (111), and is arranged at the upper end of the side wall portion (113). A plurality of fitting convex portions (115) are formed, and a plurality of fitting concave portions (117) that can be fitted to the fitting convex portions (115) are formed on the bottom surface of the side wall portion (113). For example, as shown in the perspective view of FIG. 11, such a shelf (110) is fitted into the fitting concave portion (115) of the upper shelf (110) and the fitting convex portion (115) of the lower shelf (110). ) Are connected to form a shelf step (120), and a cover (130) is covered on the uppermost shelf (110). A microorganism culture sheet with a mount (100) or a continuous microorganism culture sheet (100 ') can be placed. Although only two shelves are shown in FIG. 11, in the present invention, a plurality of shelves can be connected and assembled according to the purpose.

The size of the shelf can be appropriately selected according to the size of the microorganism culture sheet with mount (100) and the continuous microorganism culture sheet (100 ′). For example, when the storage tray shown in FIG. 11 stores the A4 size (210 mm long × 297 mm wide) microbial culture sheet with mount (100), the bottom surface of the shelf (110) as shown in FIG. The width (Wl) of the part (111) can be set to exceed 297 mm, and the length (Ll) can be set to exceed 210 mm. When the length (L1) is shorter than 210 mm, for example, 200 mm, 10 mm at the front end of the microorganism culture sheet with mount (100) protrudes from the storage tray, so that the microorganism culture sheet with mount (100) is inserted. And can be taken out easily. The length (L2) of the storage tray (200) is obtained by adding the thickness of the back surface portion (119) to the length (L1) described above, and can be selected as appropriate. Moreover, as shown in FIG.11 (d), the shelf (110) makes the height (h1) of a side wall part (113) 5-50 mm, More preferably, it is 8-30 mm, Most preferably, it is 8-20 mm. In addition, the height (h2) of the fitting convex part (115) is 3-20 mm, More preferably, it is 3-15 mm, Most preferably, it is 5-12 mm. Further, the depth (d1) of the fitting recess (117) is 3 to 20 mm, more preferably 3 to 15 mm, and particularly preferably 5 to 12 mm, similarly to the fitting protrusion. Within this range, the upper and lower shelves can be fitted and stably connected. In the present invention, as shown in FIG. 11, the side wall (113) may be formed with a window (113a). Formation of the window portion (113a) enables circulation of the atmosphere from the side wall portion (113), and the microorganism culture sheet (B) after being carried into the culture apparatus can be quickly heated to the culture temperature. Similarly, the bottom surface part (111) only needs to have a support part to the extent that the microorganism culture sheet with mount (100) or the like is placed. FIG. 11 shows a mode in which two rectangular through portions (111a) are formed on the bottom surface portion (111). Further, the shelf (110) may be further provided with a back surface portion (119) connecting the side wall portion (113) and the side wall portion (113). The back surface portion (119) is configured to be lower than the height (h1) of the side wall portion (113), and enables circulation of the atmosphere from the back surface portion (119).
The shelves (110) may be stacked by being combined with each other. As shown in FIG. 12, the storage tray of the present invention can be assembled if the shelves (110) are fitted together and stacked, and the upper part is covered with a lid (130).

  The shelf (130) used in the invention is not limited to the above. As shown in FIG. 13, the entire surface of the bottom surface portion (111) may be constituted by a support portion. Moreover, a side wall part (113) and a back surface part (119) may be comprised by the same height. The bottom surface portion (111) only needs to be able to support the microorganism culture sheet (100) with a mount, and through holes such as a circle, a rectangle, and a polygon are formed on the entire surface in order to reduce weight and improve the circulation of the atmosphere. Alternatively, a mesh shape may be used.

The storage tray can be manufactured by injection molding using a shelf and a lid, for example, a polyolefin resin such as polyethylene and polypropylene, a polyester resin such as polyethylene terephthalate, and a polyamide resin. .

(7) Method of using microorganism culture sheet with mount and continuous microorganism culture sheet As a method of using the microorganism culture sheet with mount and the continuous microorganism culture sheet of the present invention, eight microorganism cultures are performed on the mount (A) shown in FIG. The case where the sheet is fixed and the storage tray of the present invention is used in combination to store the microbial culture sheet with mount (100) inoculated with the bacterial solution will be described. The same applies to the continuous microorganism culture sheet (100 ′).

  For example, when evaluating the number of general viable bacteria and Escherichia coli for four specimens, spread two microorganism culture sheets with a mount for general viable bacteria and one microorganism culture sheet with a mount for Escherichia coli on the desk, Enter the dilution rate on the microorganism culture sheet (B) to be inoculated in the dilution rate entry field (40) from the top of the cover sheet (30), and enter the sample number in the empty space. In addition, a bacterial solution with different dilution ratios is prepared for each specimen according to a conventional method. A serial number may be entered for each microbial culture sheet with a mount. After entry, two microbial culture sheets with a mount for general viable bacteria and one microbial culture sheet with a mount for Escherichia coli are inoculated with the bacterial solution. It can be previously placed on the lid of the storage tray of the present invention.

  About the sample, the microbial culture sheet with a mount for Escherichia coli filled with the sample information and the E. coli for general living bacteria are placed with the short direction facing the operator, and the cover sheet (30) is placed. For example, it peels from right to left, inoculates two microbial culture sheets (B) for E. coli, and then inoculates two microbial culture sheets (B) for live bacteria at a predetermined dilution ratio. Inoculate the solution, and then inoculate the two microorganism culture sheets (B) with different dilution ratios. This operation is repeated 4 times for each specimen. Thereby, 4 samples can be inoculated on one microorganism culture sheet with a mount for Escherichia coli, and 4 samples can be inoculated on two microorganism culture sheets with a mount for live bacteria. In the microorganism culture sheet with a mount of the present invention, since each microorganism culture sheet (B) is fixed to the mount (A), the turning operation of the cover sheet (30) of the microorganism culture sheet (B) can be stably and easily performed. It can be carried out. After inoculating the dried culture layer (20) of the microorganism culture sheet (B) and coating the cover sheet (30), ensure the level until the bacterial solution infiltrate the dried culture layer (20) for 1-3 minutes. Even if it is necessary to leave it alone, it is easy to ensure horizontality because it is fixed on a mount larger than the microorganism culture sheet (B). After inoculating the bacterial solution, it is placed in the storage tray via the mount (A). Eight microbial culture sheets (B) can be stored together. Next, the storage tray (200) is carried into the culture apparatus and cultured for a predetermined time. At this time, the lid (130) may not be attached.

  Since the storage tray of the present invention can be assembled by fitting a plurality of shelves, for example, five shelf steps (120) composed of ten shelves (110) shown in FIG. Can be divided into two shelves (110), one containing a microorganism culture sheet with a mount for general living bacteria, and the other with a microorganism culture sheet with a mount for Escherichia coli, carried into a culture apparatus, and cultured. it can.

  Further, in the present invention, the microbial culture sheets with a mount may be stored one by one on the shelf of the storage tray, but two microbial culture sheets with a mount for living organisms may be stacked and stored in one shelf. Good.

  After completion of culturing for a predetermined time, the cells are taken out from the culture apparatus, and the colonies are observed by visual observation, a colony counter, or other methods.

(8) Microorganism testing method The microbial culture sheet with mount (100) or the continuous microbial culture sheet (100 ′) of the present invention is obtained by inoculating the microbial culture sheet (B ′, B) with a bacterial solution and cultivating the colony. And forming image data of a microbial culture sheet with a mount and a continuous microorganism culture sheet containing the colonies, and individually using the image data of a plurality of culture layers in which the colonies included in the image data are expressed. The number of colonies in each culture layer can be counted. In that case, the pixel corresponding to the colony contained in each culture layer can be judged as a colony, and the number of colonies can be automatically counted by image analysis.
In the present invention, since a plurality of microorganism culture sheets (B) can be fixed to the mount (A) and image data obtained by inoculating, culturing, and imaging the bacterial solution can be obtained, a plurality of microorganism culture sheets (B) can be obtained by one imaging operation. The feature is that the image data can be acquired. A method of counting the number of colonies from the image data is known, and the number of colonies of each microorganism culture sheet (B) can be counted from the image data corresponding to the dry culture layer (20).
A color chart, gray scale, position recognition marker, and the like can be displayed on the mount (A). When such display is included in the mount (A), the captured image data also includes these displays. Therefore, it is possible to correct the position of the image data using the position recognition marker, perform color correction using a color chart and a gray scale, and then count the number of colonies. The colony count by such image analysis can be performed by inputting image data to a processing apparatus in which a position correction program, a color correction program, and a colony count count program are constructed. Hereinafter, an aspect in the case of performing position correction and color correction will be described. In addition, although demonstrated when the microorganism culture sheet (100) with a mount is used, it is the same for the continuous microorganism culture sheet (100 ′).

(A) Acquisition of image data Image data (Dl) of a culture sheet with microorganisms after mounting can be acquired by a flatbed scanner or a digital camera. The resolution of the image can be arbitrarily selected. Preferably it is 150-600 DPI.
The image data (Dl) is image data of a microorganism culture sheet with a mount, in which a microorganism culture sheet is inoculated with a bacterial solution and cultured to express colonies, and the colonies are included. Accordingly, the image data includes the medium layer of each microorganism culture sheet (B), the colonies expressed in the medium layer, and all the serial numbers and the color sample arrangement table described on the mount.

(B) Position correction For example, the position recognition markers (75a, 75b, 75c, 75d) displayed in the color sample array table (70) included in the image data (Dl) are detected and the inclination of the image data (Dl) The position can be corrected. As shown in FIG. 2, in the case where the position recognition markers (75a, 75b, 75c, 75d) are composed of a circle and a crosshair displayed therein, the brightness of each pixel of the image data increases toward the outside. Is determined under a predetermined condition, and a pixel satisfying this condition is determined to be at the center position (coordinates) of the position recognition markers (75a, 75b, 75c, 75d).

  Specifically, each pixel of the image data (Dl) is converted from the RGB color system to luminance (Y) by a known conversion formula. Here, when the difference in luminance between the two pixels is smaller than a predetermined threshold, it is determined that they have the same luminance. Then, the following determination is made for each pixel of the image data. Here, the luminance value range is set to 0 to 255.

  First, the luminance is examined while moving from the vertical to a plurality of angular directions with the target pixel as the center, the luminance once becomes a different value as it moves, and then becomes the same as the central luminance again, and further the distance (radius) in four directions. ) Is assumed to be a circle obtained from the center and the radius (see FIG. 14A). Furthermore, when adjacent pixels having the same luminance are connected one after another from the target pixel, the circle reaches the circumference in four directions at intervals of 90 ° (see FIG. 14B).

  When the above conditions are satisfied, it is determined that the pixel is the center of the position recognition marker (75a, 75b, 75c, 75d). In the present invention, among the position recognition markers (75a, 75b, 75c, 75d), 75c is displayed in white at the center, and the other position recognition markers (75a, 75b, 75d) are displayed in black at the center. Among those determined as position recognition markers (75a, 75b, 75c, 75d), those whose center pixel has a luminance equal to or higher than a predetermined dark value (for example, an intermediate value of the above range) are displayed in white for the top and bottom recognition reversal marker 75c. Judge.

  When one of the line segments extending at the same luminance is further extended from each position recognition marker (75a, 75b, 75c, 75d), four position recognition markers (75a, 75b, 75c, 75d) are combined into one set. Since a rectangle having the center coordinates (cx, cy) is formed (see FIG. 14C), the coordinates of the center pixels of the position recognition markers (75a, 75b, 75c, 75d) at this time (x1, The inclination θ of this rectangle R is calculated from (yl), (x2, y2), (x3, y3), (x4, y4). The correction coordinate obtained by rotating the inclination θ in the reverse direction is repeated. If the correction coordinates of the coordinates (X1, Yl) of the image data are (X2, Y2), the width of the rectangle R ′ whose inclination is corrected is W, and the height is H, the coordinates of the vertices of the rectangle after the inclination correction are The upper left vertex can be represented by the upper right vertex (W-1, 0), the lower left vertex (0, H-1), and the lower right vertex (W-1, H-1). Conversely, the coordinates (X1, Yl) before correction can be obtained from the coordinates (X2, Y2) in R ′. The corrected image data (D2) obtained by this processing is corrected coordinates (X2, Y2) in the rectangle R ′ in which the pixel coordinates (X1, Yl) in the rectangle R of the original image data (D1) are tilt corrected. This is a collection of pixels converted to. Since this corrected image data (D2) is trimmed into a rectangle having no inclination determined by the width and height of the rectangle R and the center coordinates (cx, cy), each cultured microorganism culture sheet (B) or color chart is trimmed. (71), the grace scale chart (73), and each region such as each color of these charts are specified in advance by the relative position (logical coordinate) in the rectangle R, thereby depending on the resolution of the original image data (Dl). Each region can be relatively positioned from the whole of the corrected image data (D2) without doing so. Further, when the image data (Dl) is upside down, the positional relationship (coordinates) of the inversion marker (75c) in the four position recognition markers (75a, 75b, 75c, 75d) that are the vertices of the rectangle. Since it can be determined that the top and bottom are reversed in the original image data (Dl), the top and bottom can be corrected by rotating 180 degrees.

(C) Image Quality Correction Processing Further, image quality correction processing such as hue and brightness can be performed using the color sample array table (70).
That is, different culture media are used for the microorganism culture sheet (B). For example, a general viable culture medium contains TTC (triphenyltetrazolium chloride), which is reduced to red TPE (triphenyl). Phenylformazan) is generated and observed as a red colony. Staphylococcus aureus decomposes the enzyme substrate X-Phos (5-bromo-4-chloro-3-indolyl β-D-phosphate) with acid phosphatase to give blue color. Indigo, and the E. coli group reacts with the β-galactosidase of the E. coli group as an enzyme substrate in the medium to react with X-Gal (5-bromo-4-chloro-3-indolyl β-galactopyranoside) or Hydrolysis of Magenta-Gal (5-bromo-6-chloro-3-indolyl β-galactopyranoside) To produce purple or purple 5,5-dibromo-6,6-dichloroindigo, and β-glucuronidase specifically possessed by Escherichia coli is an enzyme substrate X-GLUC (5-bromo-4-chloro-3-indolyl). β-glucuronide) is decomposed in the same manner as above to produce blue 5,5-dibromo-4,4-dichloroindigo. For example, when both colonies of E. coli and E. coli are cultured in the same medium and the number of colonies is counted for each bacterial species, it is necessary to distinguish between the red-purple to purple colonies of the coliforms and the blue colonies of E. coli. The colony count for each bacterial species cannot be accurately counted. For this reason, the hue and brightness of the pixels in the image data are corrected. The image data to be corrected is corrected image data (D2) subjected to the position correction described above. A known method can be used for such color correction.

  For example, each pixel data of the image data is represented by an RGB color system, which is converted into an HLS color system expressed by hue (H), lightness (L), and saturation (S). Image quality correction processing can be performed by comparing the values with each other and linearly interpolating to perform image quality correction and then converting it to the RGB color system again. Specifically, each color in the color chart (71) of the color sample arrangement table (70) displayed on the microorganism culture sheet with a mount (100) is numbered from 1 in order of hue, and this is referred to as a reference value (Hl). The measured value (H′1) is correlated, the hue (n) is associated with the reference value (Hn) and the measured value (H′n), the hue (max) is correlated with the reference value (Hmax), and the measured value (H A color comparison table corresponding to 'max) is created. Similarly, each color of the gray scale chart (73) is numbered from 1 in order of brightness, and the reference value (Ll) and the measured value (L′ 1) are associated with each color, and the reference value (Ln) is associated with the brightness (n). The gray scale chart comparison is made by associating the measurement value (L'n) with the reference value (Lmax) corresponding to the lightness (max) and the measurement value (L'max) with the reference value and the measurement value. Create a table.

  The reference value is a value obtained by acquiring data of each color of the color chart (71) and each color of the gray scale chart (73) with a specific scanner as a reference in advance, and converting from the RGB color system to the HLS color system as described above. is there. In addition, the count value is obtained from the RGB color system from each color of the color chart (71) included in the original image data (Dl) and each pixel of the portion corresponding to each color (grayscale) of the grayscale chart (73) from the HLS. The value converted to the color system. The data of each color is a value obtained by averaging RGB elements of a plurality of pixels included in the area. Thereafter, for example, a corrected hue value h is obtained for H obtained by converting each pixel of the image data (D2) into the HLS color system, and similarly, a corrected hue value 1 and a corrected hue value s are obtained for L and S. Is converted into a value of the RGB color system, and image data (D3) in which the position, hue, and brightness are corrected is obtained.

  For example, the hue H obtained by converting each pixel of the image data (D2) into the HLS color system is referred to the color comparison table, and the hue value range is set to 1 to 360, and H ≧ H′max. H0 = Hmax, hl = Hl + 360, h′0 = H′max, h′1 = H′1, and if h′0> h′1, then h′1 = h′1 + 360 To do. When H <H′1, h0 = Hmax−360, hl = H1, h′0 = H′max, h′1 = H′1, and if h′0> h′1, It is assumed that h′0 = h′0-360. In cases other than the above, n satisfying H′n ≦ H′n + 1 is found by incrementing from 1. h0 = Hn, hl = n + 1, h′0 = H′n, h′1 = H′n + 1. Then, the corrected hue value h is obtained as h = h0 + ((hl−h0) × (H−h′0) / (h′−h′0)). Here, if h <0, h = h + 360, and if h> 360, h = h-360.

  Regarding the brightness, the corrected brightness value 1 can be obtained in the same manner as described above with reference to the gray scale chart comparison table. The HLS color system values h, 1, and S (1 = L when saturation correction is not performed) corrected in this way are converted into RGB color system values. Thereby, the image data (D3) in which the position, hue, and brightness are corrected can be obtained.

(D) Counting the number of colonies The number of colonies is automatically counted for this image data (D3). With respect to the dry culture layer (20) specified by the relative coordinates of the image data (D3), the number of colonies contained in the image region of each culture layer is measured while moving in a predetermined order. In addition, regardless of whether or not a microorganism culture sheet is used as the culture layer, a method of imaging the culture medium after the culture and counting the number of colonies from the obtained image data is known. Automatic counting of the number of colonies of each microorganism culture sheet when the mount (A) of the present invention is used can be performed by the following method.

  In the present invention, for example, since eight microbial culture sheets (B) are fixed to one mount, the position-corrected image data corresponds to each culture layer of the microbial culture sheet with mount (100) in advance. The position to do is set as a colony setting location, and the number of colonies in each microorganism culture sheet (B) can be counted. Such a culture position can be appropriately set according to the shape of the corresponding microorganism culture sheet with a mount, the number of fixed microorganism culture sheets, and the like. Furthermore, at each colony count position, the entire culture medium can be used as the colony count measurement position, or only a predetermined location in the culture layer can be set as the colony count measurement position. Furthermore, when the dividing line (L) and the frame (F) are formed in advance on the mount (A), the dividing line (L) and the frame (F) included in the image data are identified and the microorganism culture sheet ( B) and the culture layer can be recognized and set at the colony count measurement position. In particular, when the frame (F) is formed, the size of the mount (A) can be easily recognized, so that the colony number measurement position can be recognized promptly without error.

  At each colony count position included in the image data, measurement can be performed by detecting pixels corresponding to the colonies in the culture layer. When counting the number of colonies in the culture medium for general viable bacteria, TPE (triphenylformazan) reduced with TTC (triphenyltetrazolium chloride) is observed as a red colony. Therefore, from the image data (D3) The pixel of the red colony present at the position corresponding to the culture layer of the microorganism culture sheet is selected and counted.

  More specifically, the color of the red colony due to the general viable bacteria is specified by a numerical value of the RGB color system, and the pixel of the numerical value is detected from the image data (D3) and counted as the number of colonies. At that time, the color of the culture medium is specified by a numerical value of the RGB color system, and if the color of the culture medium at the colony count position is a specified value and is a numerical value of the RGB color system of the red colony, it is counted as a target colony. May be. At this time, as the minimum area of the colony, when the image data (D3) is configured with, for example, 300 DPI, a range of 4 pixels or more may be counted as the colony. The minimum area of such a colony can be set arbitrarily.

  Since the culture layer varies depending on the bacterial solution to be evaluated, the number of colonies in different culture layers can be accurately counted by setting the color of the culture layer and the color of the colony in advance. As for the colony count, a colony counting program such as a colony counting system “Isac / iSac” (registered trademark) provided by Elmex Co., Ltd. is used for the image data (D3). Can be done using.

  When the serial number is described on the mount (A) used in the present invention and the specimen number and the serial number input to the two-dimensional barcode are also described on the microorganism culture sheet (B), these are compared. Thus, a certain microorganism culture sheet (B) and image data (Dl) including the microorganism culture sheet (B) can be easily associated with each other.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

Example 1
224 g of a gelling agent in which 4: 6 parts by mass of guar gum and psyllium seed gum are mixed, 28 g of glycerin as a plasticizer, 70 g of PVP (polyvinylpyrrolidone) as a binder, 26.7 g of tryptone, 3.3 g of beef extract, A coating solution was prepared by mixing 1.0 g of yeast extract, 0.5 g of sorbitol, 3.0 g of disodium hydrogen phosphate, 1.5 g of sodium pyruvate, and 0.05 g of TTC.
0.36 g of this coating solution was applied to a circle having a radius of 25 mm by a dispenser at a length of 90 mm, a width of 70 mm, and a substantially center of the paper substrate on which the dilution factor entry column was printed. This coating layer was dried at 50 ° C. for 15 minutes to form a dry culture layer.
Subsequently, a polyethylene terephthalate film having a thickness of 50 mm was laminated on the base material on which the dry culture layer was formed, the ends were adhered, and sterilized by γ rays to obtain the microorganism culture sheet (B) of the present invention.
An adhesive layer with a length of 280 mm and a width of 10 mm is placed on an A4 size mount with a basis weight of 39 g, a length of 210 mm, and a width of 297 mm from the position of 9 mm from the left end, centering at 15 mm, 80 mm, 105 mm, and 170 mm from the bottom of the mount. Four were formed. Next, in the same manner as in FIG. 3, two microbial culture sheets (B) were fixed on the pressure-sensitive adhesive layer in four rows, and 8 microbial culture sheets (B) were detachably fixed to produce a microbial culture sheet (100) with a mount.

  The cover of the microorganism culture sheet (B) of the microorganism culture sheet (100) with the mount was turned over, 1 ml of the bacterial solution was inoculated, and the operation of covering the cover sheet was performed on eight microorganism culture sheets, and the inoculation time was measured. . In addition, this operation was performed on six microbial culture sheets with a mount, and the inoculation time was measured. The results are shown in Table 1. Eight microbial culture sheets fixed to the microbial culture sheet with the mount could be inoculated with the bacterial solution in about 36 seconds. Therefore, the time required to inoculate 48 microbial culture sheets was only 3.6 minutes.

(Comparative Example 1)
Eight microorganism culture sheets (B) prepared in Example 1 were prepared, each microorganism culture sheet (B) was inoculated with 1 ml of the bacterial solution used in Example 1, and the time required for the eight inoculations was measured. did. This was repeated 6 times. The results are shown in Table 1. An average of 47 seconds was required to inoculate the bacterial solution on the 8 microbial culture sheets, and 4.7 minutes were required to inoculate the 48 microbial culture sheets.

(Comparative Example 2)
Using a microorganism culture sheet in which a culture layer is formed on the entire surface of the base sheet and a culture layer is also formed on the surface of the cover sheet facing the culture layer, the cover sheet is peeled off and 1 ml of the bacterial solution is applied to the culture layer. After inoculating one by one and covering the cover sheet, an operation of forming a circular recess by pressing with a spreader from above the bacterial solution was performed on eight microorganism culture sheets, and the time required was measured. This was repeated 6 times. The results are shown in Table 1. An average of 85 seconds was required to inoculate the bacterial solution on the eight microbial culture sheets, and 8.5 minutes were required to inoculate the 48 microbial culture sheets.

(Comparative Example 3)
A circular culture layer is formed in the center of the base material sheet, an adhesive layer is formed on the outer periphery of the culture layer, and the microorganism culture sheet is used. The cover sheet is peeled off and 1 ml of the bacterial solution is inoculated into the culture layer. The operation | work which coat | covers a cover sheet | seat was performed about eight microorganisms culture sheets, and the time required was measured. This was repeated 6 times. The results are shown in Table 1. It took 124 seconds on average to inoculate the bacterial solution on 8 microorganism culture sheets, and 12.4 minutes to inoculate 48 microorganism culture sheets.

(result)
From Example 1 and Comparative Example 1, when the microbial culture sheet with a mount of the present invention is used, the time is shortened by 23% (= (1- (3.6 / 4.7)) × 100 (%)). I was able to. Further, when Example 1 was compared with Comparative Example 2, it was possible to reduce the time by 58% (= (1− (3.6 / 8.5)) × 100 (%)). Furthermore, when Example 1 and Comparative Example 3 were compared, the bacterial solution could be inoculated in one third of the time.

(Example 2)
The microbial culture sheet with a mount inoculated with the bacterial solution prepared in Example 1 was cultured at 36 ° C. for 48 hours, and after culturing, image data was acquired from the surface of the microbial culture sheet with a mount using a scanner, and the image data acquisition time was measured. did. Image data is shown in FIG. A red colony can be observed in the culture layer by visual observation.

  This image data is sent to a processing apparatus in which a position correction program, a color correction program, and a colony count program are constructed, position correction and color correction are performed, and the red color of TPE (triphenyl formazan) is counted as the number of colonies, The time required for correction, color correction and colony count was measured. The results are shown in Table 2. Note that the Isaac of Elmex Corporation was used for the colony count program. This was carried out on six sheets of microorganism culture sheets with a mount. The results are shown in Table 2.

(Comparative Example 4)
The microorganism culture sheet inoculated with the bacterial solution prepared in Comparative Example 1 was cultured at 36 ° C. for 48 hours. After the culture, image data was acquired from the surface of each microorganism culture sheet with a scanner, and the image data acquisition time was measured.

  This image data is sent to the processing apparatus in which the position correction program, the color correction program, and the colony count program are constructed in the same manner as in the second embodiment, and the image data for eight sheets are subjected to position correction, color correction, and colony count. The time required was measured. This was carried out on 48 microbial culture sheets with a mount. The results are shown in Table 2.

  As shown in Table 2, when the microorganism culture sheet with a mount of the present invention is used, the image data of eight microorganism culture sheets can be obtained by one scanner operation, so that it is possible to easily perform the trouble of image data formation. Image data formation time can be shortened.

  Further, according to the microorganism culture sheet with a mount of the present invention, the position and color correction of eight microorganism culture sheets can be performed by one position correction and color correction, so that the number of colonies of eight microorganism culture sheets can be counted. Can be performed in a short time.

  The microbial culture sheet with a mount and the continuous microbial culture sheet of the present invention have a plurality of microbial culture sheets formed on the mount and the base sheet, so that it is easy to inoculate a plurality of bacterial solutions. Therefore, after inoculating the bacterial solution, the microorganism culture sheet can be maintained horizontally, and can be carried into the culture apparatus immediately after the inoculation.

10, 10 '... substrate sheet,
20, 20 '... dry culture layer,
30, 30 '... cover sheet,
40: Enter the dilution factor,
50 ... Medium identification color bar,
60 ... identifier,
70 ... Sample arrangement table,
80 ... serial number,
100 ... microorganism culture sheet with mount,
100 '... Continuous microorganism culture sheet,
110 ... shelf,
120 ... shelf step,
130 ... Lid 200 ... Storage tray,
A: Mount for fixing microorganism culture sheet,
B ... Microorganism culture sheet,
B ′: Microorganism culture sheet in a continuous microorganism culture sheet,
C ... Adhesive layer F ... Frame

Claims (2)

A microorganism culture sheet fixing mount made of a synthetic resin for fixing a plurality of microorganism culture sheets ,
A substrate for fixing a microorganism culture sheet, wherein the microorganism culture sheet can be detachably fixed, a partition indicating a fixing position of each microorganism culture sheet is formed, and a frame is formed with a low-lightness color. Inoculated with Kin'eki the microorganism culture sheet, and cultured to express colony, to secure the plurality of microbial cultures sheet was expressed the colonies microbial cultures sheet fixing mount according to claim 1,
Forming image data of a mount on which the microorganism culture sheet is fixed ;
A microorganism testing method, wherein the number of colonies for each microorganism culture sheet is counted using the image data of the microorganism culture sheet included in the image data .

Images