JP5875787B2 - Microorganism recovery apparatus, microorganism recovery method, and liquid bacteria collection cartridge - Google Patents

Description

  The present invention relates to a microorganism collection apparatus, a microorganism collection method, and a liquid bacteria collection cartridge that collect microorganisms in a liquid.

  In manufacturing sites, research and development, and quality assurance in pharmaceutical factories, food factories, regenerative medical facilities, etc., there are many opportunities to check for the presence of contamination by microorganisms such as bacteria and the breeding status.

A culture method is known as a method for counting microorganisms. As is well known, the counting of microorganisms by a culture method requires a great deal of time and labor.
On the other hand, an ATP method for indirectly counting microorganisms by quantifying ATP (adenosine triphosphate) extracted from microorganisms is known as a microorganism counting method.

  Patent Document 1 describes an invention for reliably determining the presence or absence of one microorganism by completely removing noise emission points other than microorganisms in the method of detecting the number of microorganisms by the ATP / bioluminescence method using a filtration membrane. ing.

  In this ATP method, an ATP extraction reagent is brought into contact with a collected microorganism to extract ATP present in the microorganism, and the microorganism is counted according to the luminescence intensity when the ATP is reacted with the luminescence reagent. According to this ATP method, for example, in the method of counting microorganisms collected by the number of colonies of microorganisms cultured on a plate medium, it takes several days. The time can be dramatically reduced.

However, since this ATP method counts microorganisms based on weak luminescence intensity, it is necessary to reduce as much as possible the possibility of substances that cause disturbances in the sample to be counted.
Patent Document 2 describes an invention in which a small number of microorganisms contained in a large amount of sample are detected and quantified quickly, with high accuracy, high sensitivity, and simply.

JP-A-11-137293 JP 2010-193835 A

  In the invention described in Patent Document 2, it is possible to filter a sample solution (specimen solution) containing microorganisms with a membrane filter and extract the microorganisms on the membrane filter by the ATP method.

However, some sample solutions may contain a lot of impurities other than microorganisms. For example, fruit juice components in soft drinks containing fruit juice, beverages containing jelly, and the like. These contaminants are substances that cause disturbance in counting microorganisms.
In addition, these sample liquids may have high viscosity, and thus there is a possibility that the filter may not be sufficiently filtered.

  Therefore, an object of the present invention is to provide a microorganism collection apparatus, a microorganism collection method, and a liquid bacteria collection cartridge that can collect microorganisms from a sample solution and detect and count microorganisms with high accuracy.

In order to solve the above problems and achieve the object of the present invention, the present invention is configured as follows.
That is, the microorganism collection apparatus of the present invention is a microorganism collection apparatus that collects microorganisms contained in the sample liquid, and filters out contaminants contained in the sample liquid by the primary filtration means, and by the secondary filtration means, A microorganism recovery apparatus for capturing the microorganisms contained in the sample liquid from which contaminants have been removed by filtration , wherein the diluent is added to the sample liquid and stirred before the primary filtration means, and the viscosity of the sample liquid is Viscosity adjusting means for adjusting the viscosity of the sample liquid, wherein the viscosity adjusting means adds a dilution liquid by rotating a dilution tank that can be rotationally driven until the sample liquid has a predetermined viscosity .
Other means will be described in the embodiment for carrying out the invention.

  According to the present invention, it is possible to provide a microorganism collection apparatus, a microorganism collection method, and a liquid bacteria collection cartridge capable of collecting microorganisms from a sample solution and detecting and counting microorganisms with high accuracy.

It is a figure which shows the structure and operation | movement of the microorganisms detection system in 1st Embodiment. It is a schematic block diagram of the microorganisms collection apparatus in 1st Embodiment. It is a block diagram of the cartridge in 1st Embodiment. It is a block diagram of the tank body of the filling dilution tank in 1st Embodiment. It is a schematic block diagram of the microorganisms counting device in 1st Embodiment. It is a perspective view which shows the mounting part vicinity of the microorganisms counting device in 1st Embodiment. It is a flowchart which shows the microorganisms collection process in 1st Embodiment. It is a figure which shows the sequence of the microorganisms collection process in 1st Embodiment. It is a figure which shows the operation | movement of the microbe collection process in 1st Embodiment. It is a flowchart which shows the microorganisms counting process in 1st Embodiment. It is a block diagram of the cartridge in 2nd Embodiment. It is a block diagram of the tank body of the filling dilution tank in 2nd Embodiment.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings.

(Configuration of the first embodiment)
FIG. 1 is a diagram showing the configuration and operation of the microorganism detection system in the first embodiment.
The microorganism detection system according to the first embodiment includes a sample liquid container 31 that stores a sample liquid that is a specimen liquid, a cartridge 20, a dilution buffer liquid container 41 that stores a sterilized dilution buffer liquid, and a microorganism recovery apparatus 10. And a microbe counting apparatus 100. The cartridge 20 includes a filling dilution tank 21 that is a tank portion that holds a liquid, and a recovery container 25. The cartridge 20 is a liquid bacteria collection cartridge that collects microorganisms contained in the sample liquid.
The filling dilution tank 21 and the collection container 25 of the cartridge 20 are configured to be separable.
Thereby, it can replace | exchange to the unit of the filling dilution tank 21 provided with the primary filter 23 (FIG. 2) from which a specification differs according to the kind of sample liquid. Furthermore, the collection container 25 separated from the filling dilution tank 21 is configured to be installed in the microorganism counting apparatus 100 as it is.
The cartridge 20 is configured to be rotationally symmetric . Thus, the sample liquid and the dilution buffer liquid stored in the filling dilution tank 21 can be stirred by rotating smoothly.

In the microorganism detection system of the first embodiment, in order to prevent the contamination between the sample liquids (experimental contamination) from affecting the analysis result, the parts such as the cartridge 20 and the sample liquid container 31 that are in contact with the sample liquid. Are all disposable products.
The filling dilution tank 21 and the collection container 25 are sterilized with ethylene oxide gas or the like, packaged in individual bags, maintained in a sterilized state, and distributed. The filling dilution tank 21 and the collection container 25 may be connected after being sterilized with ethylene oxide gas or the like, packaged in a single bag, maintained in a sterilized state, and distributed.

  The microorganism collection apparatus 10 includes an opening / closing door 11 provided on the left side of the center, a display unit 12, and an operation unit 13. By opening the door 11, the sample solution container 31, the cartridge 20, and the dilution buffer solution container 41 can be set in the microorganism collection apparatus 10. The microorganism collection device 10 collects microorganisms contained in the sample liquid.

  The cartridge 20 that collects microorganisms is separated into a collection container 25 and a filling dilution tank 21 by the microorganism collection apparatus 10. The collection container 25 is set in the microorganism counting device 100. The microorganism counting apparatus 100 counts the microorganisms collected in the collection container 25 by the ATP method. The microbe counting apparatus 100 is a BioMaiter (registered trademark) marketed by the present applicant.

The operation of the microorganism detection system in the first embodiment will be described with reference to FIG.
When the processing of the microorganism detection system is started, in step S10, the inspector moves the sample liquid container 31 containing the sample liquid, the unused cartridge 20 and the dilution buffer liquid container 41 containing the dilution buffer liquid to the microorganism. Mounted on the collection device 10.

  In step S <b> 11, the microorganism collection device 10 performs a microorganism collection process for collecting microorganisms from the sample solution stored in the sample solution container 31. The processing in step S11 will be described in detail with reference to FIG.

  In step S <b> 12, the inspector takes out the collection container 25 of the cartridge 20 from which the microorganism has been collected from the microorganism collection apparatus 10 and mounts it on the microorganism counting apparatus 100.

In step S <b> 13, the microorganism counting device 100 performs a counting process of microorganisms in the housing 26 of the collection container 25. The process of step S13 will be described in detail with reference to FIG.
When the process of step S13 ends, the process of FIG. 1 ends.

FIG. 2 is a schematic configuration diagram of the microorganism collection apparatus according to the first embodiment.
The microorganism collection apparatus 10 includes a processing stage 14, a sample collection unit 30, a dilution buffer supply unit 40, a cartridge motor 50, a rotation shaft 51, a rotary dilution tank holder 53, a collection container holder rotation unit 54, A collection container holder 55, a collection container heater 56, a suction unit 60, a suction head drive unit 70, and a control unit 80 are included. The sample liquid container 31, the cartridge 20, and the dilution buffer liquid container 41 are installed on the processing stage 14 by the inspector.

The sample recovery unit 30 is configured so that a sample liquid supply pipe 32 can be set in the liquid feed pump 33 in order to collect a predetermined amount of sample liquid from the sample liquid container 31.
The sample liquid container 31 stores a sample liquid that is a specimen liquid that is a target for counting microorganisms. The inspector collects the sample solution in the sample solution container 31 and sets it in the microorganism collection apparatus 10. However, the present invention is not limited to this. For example, when inspecting an injection, the sample collection unit 30 may be configured so that a vial (product container) can be used as it is as a sample solution container.

  The sample liquid supply pipe 32 is a resin tube and serves as a path for sending the sample liquid from the sample liquid container 31 to the filling dilution tank 21. The sample liquid container 31 and the sample liquid supply pipe 32 are disposable products in order to suppress contamination between the sample liquids. The sample collection unit 30 is configured such that the sample solution supply pipe 32 has the shortest length in order to improve the sample collection rate and suppress contamination.

  The liquid feed pump 33 is a pump that feeds the sample liquid supply pipe 32 without contact with the sample liquid, and is controlled by a flow rate control unit 81 of the control unit 80 described later. The liquid feeding amount and driving torque of the liquid feeding pump 33 are notified to the flow rate control unit 81 of the control unit 80.

  The dilution buffer supply unit 40 is configured such that a dilution buffer liquid supply pipe 42 can be set to the liquid feed pump 43 and the buffer heater 44 in order to supply a predetermined amount of the dilution buffer liquid from the dilution buffer liquid container 41.

  The dilution buffer solution container 41 stores a sterilized dilution buffer solution. This diluted buffer solution is a buffer solution that controls the viscosity by diluting the sample solution. The inspector sets the dilution buffer solution container 41 in which the dilution buffer solution is stored in the microorganism collection apparatus 10.

  The dilution buffer solution supply pipe 42 is a resin tube similar to the sample solution supply pipe 32, and sends the dilution buffer solution from the dilution buffer solution container 41 to the filling dilution tank 21 of the cartridge 20. The dilution buffer solution supply pipe 42 is a disposable product in order to suppress contamination.

  Similarly to the liquid feed pump 33, the liquid feed pump 43 is a pump that feeds the diluted buffer liquid supply pipe 42 in a non-contact manner with the diluted buffer liquid, and is controlled by a flow rate control unit 81 of the control unit 80 described later. Be controlled. The liquid feeding amount and driving torque of the liquid feeding pump 43 are notified to the flow rate control unit 81 of the control unit 80.

  The buffer heater 44 is a heater that heats the diluted buffer solution through the diluted buffer solution supply pipe 42 in a non-contact state with the sterilized diluted buffer solution. This makes it possible to control the viscosity of a sample liquid whose viscosity changes with temperature, such as a jelly beverage. The temperature range in which the buffer heater 44 heats is a temperature range in which the microorganism to be measured can survive. The buffer heater 44 is controlled by a heater control unit 82 of the control unit 80 described later.

The cartridge 20 includes a filling dilution tank 21 (FIG. 1) provided in the upper part and a recovery container 25 (FIG. 1) provided in the lower part.
The filling dilution tank 21 (FIG. 1) includes a tank body 22, a primary filter 23, and a primary filter fixing ring 24. The collection container 25 (FIG. 1) includes a housing 26, a secondary filter 27, and a secondary filter fixing ring 28. The detailed structure of these cartridges 20 will be described in detail with reference to FIG.

  The cartridge motor 50 is connected to the rotating shaft 51 and rotates the rotating shaft 51. A belt 52 is stretched between the rotary shaft 51 and the rotary dilution tank holder 53. The cartridge motor 50 is controlled by a motor control unit 83 of the control unit 80 described later.

  The collection container holder 55 is fixed to the processing stage 14, for example. The collection container holder 55 is in contact with the collection container holder rotating portion 54 by a ball bearing or the like, and supports the collection container holder rotating portion 54 so as to be rotatable. The rotary dilution tank holder 53 is installed in the collection container holder rotating unit 54. As a result, the cartridge motor 50 rotates the rotary dilution tank holder 53 and the collection container holder rotating portion 54 via the rotating shaft 51 and the belt 52, thereby rotating the cartridge 20.

  The cartridge motor 50 rotates the rotating shaft 51. The belt 52 transmits the rotational force of the rotary shaft 51 to the rotary dilution tank holder 53. The rotary dilution tank holder 53 holds and rotates the filling dilution tank 21 of the cartridge 20. As the filling dilution tank 21 of the cartridge 20 rotates, the recovery container 25 of the cartridge 20 rotates on the recovery container holder rotating unit 54.

  The collection container heater 56 is a heater that heats the collection container 25. The recovery container heater 56 controls the viscosity of the sample liquid in the recovery container 25 by heating. The recovery container heater 56 is configured so as not to contact the sample liquid.

  The suction unit 60 includes a suction head 61, an aspirator pump 62, and a suction pipe 63. The suction head 61 is driven up and down by a suction head drive unit 70 to be described later, and is pressure-bonded to the lower portion of the collection container 25 and sucked with negative pressure. The suction pipe 63 connects the suction head 61 and the aspirator pump 62 so that the air in the suction head 61 can be sucked out by the aspirator pump 62. The aspirator pump 62 is a suction pump that sucks the sample liquid or the sample liquid whose viscosity is adjusted by the venturi effect through the primary filter 23 and the secondary filter 27 with a predetermined negative pressure. The aspirator pump 62 filters and removes contaminants contained in the sample liquid by the primary filter 23, and captures and collects microorganisms contained in the sample liquid from which the contaminants are filtered and removed by the secondary filter 27.

The suction head drive unit 70 includes a suction head motor 71, a nut 72, and a ball screw shaft 73.
The suction head motor 71 rotationally drives the nut 72 to move the ball screw shaft 73 up and down. The suction head 61 described above is fixed to the upper portion of the ball screw shaft 73. As the ball screw shaft 73 moves up and down, the suction head 61 also moves up and down. The suction head motor 71 is a stepping motor, for example, and can be driven to rotate by a rotation amount corresponding to the drive pulse. The suction head motor 71 is controlled by a motor control unit 83 described later.
When the cartridge motor 50 is rotated, the suction head 61 is controlled so as to be positioned below, so that free rotation of the cartridge motor 50 is not hindered.

  The control unit 80 includes a flow rate control unit 81, a heater control unit 82, a motor control unit 83, and a pump control unit 84, and includes a display unit 12 (FIG. 1) and an operation unit 13 (FIG. 1). It is connected.

  The flow rate controller 81 is connected to the liquid feed pump 33 and the liquid feed pump 43. The flow rate control unit 81 can drive the liquid feed pump 33 and the liquid feed pump 43 and can measure the drive torque thereof. The flow control unit 81 drives the liquid feed pump 33 and the liquid feed pump 43 in a predetermined sequence in accordance with instructions from the control unit 80.

  The heater control unit 82 is connected to the buffer heater 44 and the collection container heater 56. The heater control unit 82 turns on the buffer heater 44 and the collection container heater 56 in a predetermined sequence in accordance with an instruction from the control unit 80.

  The motor control unit 83 is connected to the cartridge motor 50 and the suction head motor 71. The motor control unit 83 can further measure the drive torque of the cartridge motor 50. The motor control unit 83 drives the cartridge motor 50 so that the drive torque of the cartridge motor 50 becomes a predetermined value. The motor control unit 83 further rotates the suction head motor 71 by a predetermined amount so that the suction head 61 is at a desired position.

  The pump control unit 84 is connected to the aspirator pump 62. The pump control unit 84 drives the aspirator pump 62 in a predetermined sequence in accordance with an instruction from the control unit 80.

The control unit 80 serving as a viscosity adjusting unit adds a dilution buffer solution to the sample solution according to an instruction from the flow rate control unit 81. Further, according to an instruction from the motor control unit 83, the filling dilution tank 21 is rotated to a predetermined value to stir the sample liquid, and the torque of the cartridge motor 50 is measured to measure the viscosity of the sample liquid. The dilution buffer solution is adjusted by dropping it into the filling dilution tank 21 so as to be as follows. The torque of the cartridge motor 50 depends on the mass of the cartridge 20, the rotary dilution tank holder 53 and the collection container holder rotating portion 54, the amount of sample liquid stored in the filling dilution tank 21, and the like. The control unit 80 calculates and measures the viscosity of the sample liquid stored in the filling dilution tank 21 by adding such information to the torque information of the cartridge motor 50.
The control unit 80 includes a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), various interfaces, an electronic circuit, and the like. The control unit 80, the flow rate control unit 81, the heater control unit 82, the motor control unit 83, and the pump control unit 84 read software programs and data recorded in the ROM onto the RAM, and are executed by the CPU. It is embodied by being executed.

FIG. 3 is a configuration diagram of the cartridge according to the first embodiment. FIG. 3 is an exploded perspective view when the cartridge 20 is looked down obliquely from above.
The cartridge 20 according to this embodiment is arranged and used in the microorganism recovery apparatus 10 shown in FIG. 2 described above when collecting microorganisms that are detection objects in a sample liquid that is a specimen liquid. When counting the collected and collected microorganisms, only the collection container 25 provided in the lower part is mounted on the microorganism counting apparatus 100 and used.
The cartridge 20 includes a filling dilution tank 21 provided in the upper part and a recovery container 25 provided in the lower part.
The filling dilution tank 21 further includes a tank body 22, a primary filter 23, and a primary filter fixing ring 24. The filling dilution tank 21 adjusts the viscosity by mixing the sample solution and the dilution buffer solution.
Tank body 22 forms a peripheral portion of the filling dilution tank 21, and an upper tape path portion 22a, and a lower cylindrical portion 22b is formed. Inside the upper tape path portion 22a, stirred ribs 22d are formed four. An engaging claw 22c is formed in the lower cylindrical portion 22b. The engaging claw 22c engages with the L-shaped groove 26c of the housing 26 of the recovery container 25, and detachably connects the filling dilution tank 21 and the recovery container 25. The structure of the tank body 22 will be described in more detail with reference to FIG. The tank body 22 is a tank unit that holds a liquid.

The primary filter 23 is installed between the inside of the cylindrical portion 22 b of the tank body 22 and the primary filter fixing ring 24. The diameter of the primary filter 23 is formed larger than the inner diameter of the cylindrical portion 22 b of the tank body 22. The primary filter 23 is configured to have a larger filtration area than a secondary filter 27 described later. The primary filter 23 captures and removes large-sized particles from the sample solution dropped into the filling dilution tank 21. However, the present invention is not limited to this, and a specific substance or dye that inhibits detection by the ATP method may be removed specifically or non-specifically. The primary filter 23 is primary filtering means for filtering out impurities contained in the sample liquid. The primary filter 23 does not filter the sample solution and the dilution buffer solution under normal pressure (under atmospheric pressure), and removes impurities by filtering the sample solution and the dilution buffer solution by suctioning at a predetermined negative pressure.
Supplementally, the primary filter 23 has a larger opening than the secondary filter 27 described later, and is entirely hydrophobic. The primary filter 23 is configured not to pass liquid unless it is sucked by the aspirator pump 62.

The primary filter fixing ring 24 has a hollow cylindrical shape having the same diameter as the inner diameter of the cylindrical portion 22b of the tank body 22, and includes a rib portion 24b on the upper portion. The rib portion 24b includes a rib extending radially from the central portion, and a rib formed in a circular shape from the central portion and intersecting the radial rib described above. The rib portion 24 b supports the primary filter 23 from below when the primary filter fixing ring 24 and the primary filter 23 are connected to the tank body 22. The primary filter fixing ring 24 is hollow except for the portion of the rib portion 24b, and allows liquid or gas to pass therethrough.
Three engaging claws 24 a are formed on the cylindrical outer peripheral side surface of the primary filter fixing ring 24. The primary filter fixing ring 24 is engaged with three L-shaped grooves (not shown) provided at the lower part of the tank body 22 by the three engagement claws 24 a and is connected to the tank body 22. Since the outer diameter of the primary filter 23 is larger than the inner diameter of the cylindrical portion 22b of the tank body 22 and the outer diameter of the primary filter fixing ring 24, the end portion of the primary filter 23 is connected to the cylindrical portion 22b and the primary filter fixing ring 24. It is clamped between and fixed.

The collection container 25 further includes a housing 26, a secondary filter 27, and a secondary filter fixing ring 28.
The housing 26 has an upper cylindrical portion having an inner diameter substantially the same as the outer diameter of the cylindrical portion 22b of the tank body 22, and a lower side having an inner diameter that is further reduced in diameter than the inner diameter of the upper cylindrical portion. A cylindrical part, an inverted conical lottery part having an inner diameter gradually reduced from the inner diameter of the lower cylindrical part, and a filter mounting part provided around the outlet of the discharge opening formed at the lowermost part of the lottery part Are integrated in this order. The housing 26 has four engaging claws 26a formed on the outer peripheral side surface of the lower cylindrical portion, and three L-shaped grooves 26c formed on the inner peripheral side surface of the upper cylindrical portion. Lot of the inverted cone shape of the housing 26 to capture efficiently bacteria in the secondary filter 27, is formed in a tape path shape.

On the inner peripheral surface of the upper cylindrical portion, three L-shaped grooves 26c into which the engaging claws 22c of the tank body 22 are fitted are formed along the inner circumference at positions corresponding to the engaging claws 22c.
The lower cylindrical portion is connected to the upper cylindrical portion via a shelf portion. Four engaging claws 26a are formed on the outer peripheral surface of the lower cylindrical portion. These engaging claws 26a are formed at equal intervals along the circumferential direction so as to extend outward in the radial direction of the lower cylindrical portion. The four engaging claws 26a engage with four L-shaped grooves (not shown) of the collection container holder rotating portion 54 (FIG. 2) of the microorganism collection apparatus 10, and rotate the collection container 25 (housing 26) with the collection container holder. The unit 54 is detachably installed.
The inner diameter of the lotter part gradually decreases toward the bottom, so that the contents easily flow down toward the lowermost discharge opening.
The filter mounting portion supports a filter housing portion (not shown) that forms a thin disk-like space and a secondary filter fixing ring 28 in accordance with the shape of the secondary filter 27 arranged so as to close the outlet that is the discharge opening. The ring support part (not shown) is integrally formed.
Four L-shaped grooves (not shown) are formed on the inner peripheral surface of the ring support portion. These four L-shaped grooves are formed at equal intervals along the circumferential direction. These four L-shaped grooves engage and engage with engaging claws 28a formed on a secondary filter fixing ring 28, which will be described later, and fix the secondary filter fixing ring 28 in a detachable manner.

The housing 26 is engaged with four L-shaped grooves (not shown) of the collection container holder rotating portion 54 (FIG. 2) by four engagement claws 26a formed on the outer peripheral side surface of the lower cylindrical portion, and the microorganisms are collected. It is detachably installed on the processing stage 14 (FIG. 2) of the apparatus 10. The housing 26 is further engaged with four L-shaped grooves (not shown) provided in the microorganism counting device 100 by four engaging claws 26 a formed on the outer peripheral side surface of the lower cylindrical portion, and the microorganism counting device 100. Is detachably installed.
The housing 26 is engaged with the three engaging claws 22c of the tank body 22 by the three L-shaped grooves 26c formed on the inner peripheral side surface of the upper cylindrical portion, and the filling dilution tank 21 is airtight. Removably connected in a state where is maintained. Thereby, the sample liquid stored in the primary filter 23 of the filling dilution tank 21 is sucked by a negative pressure from the discharge opening formed in the lower part of the collection container 25, and microorganisms are put on the secondary filter 27. Can be recovered and captured. The housing 26 is a liquid holding unit that holds a liquid such as a sample liquid.

The secondary filter 27 is a single filter that captures microorganisms, or a two-filter configured by superposing a hydrophilic filter that captures bacteria and a hydrophobic filter that retains reagents. In order to perform counting with the microorganism counting apparatus 100 easily and with high sensitivity, the filtration area is smaller than that of the primary filter 23 and has a narrow opening. The secondary filter 27 is a secondary filtration means for capturing microorganisms contained in the sample liquid from which impurities have been removed by filtration.
Commercially available products can be used for the hydrophilic filter and the hydrophobic filter constituting the secondary filter 27. Examples of the hydrophilic filter include MF-Millipore (Nippon Millipore), Durapore (Nippon Millipore), Isopore (Nippon Millipore).
Examples of the hydrophobic filter include Mytex (manufactured by Nihon Millipore) and polypropylene prefilter (manufactured by Nihon Millipore).
The outer diameter of the secondary filter 27 is formed larger than the inner diameter of the discharge opening.
Since the primary filter 23 needs to store the sample solution and the dilution buffer solution on the primary filter 23, it is possible to use a material similar to the secondary filter 27 and having a large opening. is there. The primary filter 23 does not need a function of capturing bacteria.

The secondary filter fixing ring 28 fixes the secondary filter 27 to the housing 26 (lotter part). The secondary filter fixing ring 28 has a through hole at a position where it communicates with the discharge opening of the lottery portion of the housing 26 via the secondary filter 27. The secondary filter fixing ring 28 is formed in a ring body having an outer diameter substantially the same as the inner diameter of the ring support portion of the filter mounting portion of the housing 26 and is formed below the ring body, and has a diameter larger than the outer diameter of the ring body. The flange portion is formed.
The secondary filter fixing ring 28 is integrally formed on the upper surface of the ring body, and is erected around a fitting portion that fits inside the filter housing portion of the housing 26 and the opening of the through hole at the fitting portion. And an annular rib. The secondary filter 27 is pressed around the outlet of the discharge opening by the annular rib.
On the peripheral surface of the secondary filter fixing ring 28 ring body, four engaging claws 28a are formed at equal intervals along the circumferential direction so as to extend outward in the radial direction. These engaging claws 28a are formed at positions corresponding to L-shaped grooves (not shown) of the ring support portion, and are fitted into these L-shaped grooves, whereby the secondary filter 27, the secondary filter fixing ring 28, Are detachably connected.

The cartridge 20 has a lower discharge opening at normal pressure, and when sample liquid or dilution buffer liquid is dropped into the filling dilution tank 21, the liquid is retained and diluted by rotation to be stirred.
When a negative pressure is applied to the lower discharge opening of the cartridge 20 and the diluted sample liquid is stored in the filling dilution tank 21, the liquid is filtered by the primary filter 23 and the secondary filter 27, and microorganisms Is collected on the surface of the secondary filter 27.

The cartridge 20 that has collected microorganisms separates the filling dilution tank 21 and the collection container 25, and only the collection container 25 is installed in the microorganism counting device 100, and the collected microorganisms are counted.
The cartridge 20 as described above can be formed of a moldable resin except for the primary filter 23 and the secondary filter 27. Of these, polypropylene is preferable.

  FIGS. 4A and 4B are configuration diagrams of the tank body of the filling dilution tank in the first embodiment.

4A is a top view of the tank body 22 of the filling dilution tank 21. FIG.
The tank body 22 has a columnar cross rib 22g inside, and supports the stirring rib 22d described above from below. The tank body 22 is hollow except for the portions of the cross ribs 22g and the stirring ribs 22d, and is configured to allow liquid or gas to pass in the vertical direction.

4B is a cross-sectional view of the tank body 22 of the filling dilution tank 21 taken along the line K0-K1.
Tank body 22 has an upper tape path portion 22a, the cross rib 22g is formed in a plane that separates the lower side of the cylindrical portion 22b. Plate-shaped stirring ribs 22d are respectively formed on both upper sides of the cross rib 22g. The cross rib 22g and the stirring rib 22d are configured to be rotationally symmetric . And the tank body 22 is also comprised by the rotational symmetry .
The primary filter 23 shown in FIG. 3 is sandwiched and fixed by the cross rib 22g and the primary filter fixing ring 24 (FIG. 3).

FIG. 5 is a schematic configuration diagram of the microorganism counting apparatus according to the first embodiment.
The microorganism counting apparatus 100 is an apparatus that counts microorganisms contained in a sample based on the ATP method.
In the housing 101, a mounting unit 102 for mounting the collection container 25, a hot water supply unit 103, a suction unit 104, a reagent cartridge 110 for arranging a plurality of reagents R, a buffer tank 105, a dispensing unit 106, The light emission intensity measuring unit 107 and the control unit 108 are provided.
In FIG. 5, the casing 101 and the reagent cartridge 110 are indicated by alternate long and short dash lines for convenience of drawing, and the collection container 25 is not shown. The setting of the collection container 25 to the microorganism counting device 100 will be described in detail with reference to FIG.

  The hot water supply unit 103 is for injecting hot water into the internal space of the housing 26 (FIG. 3). Although not shown, the hot water from the cartridge heater is passed through a nozzle formed of a flexible tube or the like with a peristaltic pump, for example. To be discharged. This nozzle is inserted into the internal space of the housing 26 in advance when the collection container 25 is mounted on the mounting portion 102. The hot water supply unit 103 can be omitted in the microorganism counting process of the collection container 25 as in the present embodiment.

  The suction unit 104 sucks hot water (injection may be omitted) injected into the internal space of the housing 26 (FIG. 3), a reagent R, a buffer solution, and the like, which will be described later, through the secondary filter 27. To be discharged. For example, the suction unit 104 includes a suction head (not shown), a suction pump (not shown) connected to the suction head via a predetermined pipe, and a waste liquid tank (not shown). The configuration of the suction unit 104 is the same as the configuration of the suction unit 60 of the microorganism collection device 10.

  The suction unit 104 in the present embodiment moves the suction head up and down so that the suction head (not shown) can be connected to and separated from the housing 26 (see FIG. 3) supported by the mounting portion 102. It further includes a lifting device that does not. The configuration of the lifting device is the same as the configuration of the suction head drive unit 70 of the microorganism collection device 10.

  The reagent cartridge 110 has a plurality of reagents R required for the ATP method arranged together. The reagent cartridge 110 is disposed at a predetermined position in the vicinity of the mounting portion 102. In this reagent cartridge 110, a dispensing nozzle 106a of a dispensing unit 106, which will be described later, measures the reagent R in a predetermined order in the housing 26 (FIG. 3) of the recovery container 25 (FIG. 3) and the emission intensity measurement. The reagent R is positioned so that it can be dispensed into the light emission tube 107a of the unit 107. That is, the position (coordinates) of the reagent R is stored in the control unit 108 that controls the dispensing unit 106, as will be described later.

The reagent R necessary for the ATP method includes, for example, an ATP elimination reagent that erases ATP present outside the collected microorganism, an ATP extraction reagent that extracts ATP contained in the microorganism, and ATP extracted from the microorganism. Examples thereof include an ATP luminescent reagent that emits light and an ATP standard reagent for obtaining a reference luminescence amount for converting the ATP amount from the luminescence amount.
Examples of the ATP elimination reagent include ATP degrading enzyme.
Examples of the ATP extraction reagent include benzalkonium chloride, trichloroacetic acid, Tris buffer, and the like.
Examples of the ATP luminescent reagent include a luciferase / luciferin reagent.
Note that the reagent R can include a correction reagent of the luminescence intensity measurement unit 107, sterilized pure water, and the like.
Examples of the ATP luminescent reagent include those obtained by dissolving ATP quantified by HPLC (High Performance Liquid Chromatography) or the like in a Tris buffer.

  As will be described later, the buffer tank 105 stores a buffer solution such as a phosphoric acid aqueous solution for preparing a sample solution containing microorganisms in addition to the microorganisms separated and retained by the filter.

  The dispensing unit 106 dispenses the buffer solution and the reagent R described above into the housing 26 (FIG. 3) of the recovery container 25 (FIG. 3). The dispensing unit 106 dispenses the reagent R and the ATP extract described later in the housing 26 (see FIG. 3) into the light emission tube 107 a of the light emission intensity measurement unit 107.

  The dispensing unit 106 includes a dispensing nozzle 106a formed of a thin tube, an actuator 106b for moving the dispensing nozzle 106a in three axial directions, and a syringe pump 106c connected to the dispensing nozzle 106a by a predetermined flexible pipe. A pipe (not shown) for supplying the buffer liquid from the buffer tank 105 to the dispensing nozzle 106a via the syringe pump 106c is provided.

  The emission intensity measuring unit 107 receives a later-described ATP extract dispensed from the housing 26 (FIG. 3), emits ATP, and emits ATP. The photomultiplier tube detects the emission intensity of the ATP. And a light detection unit main body 107b.

  The control unit 108 controls the microorganism counting apparatus 100 as a whole, and after mounting the collection container 25 (FIG. 3) on the mounting unit 102, the hot water supply unit 103, the suction unit 104, the dispensing unit 106, The emission intensity measuring unit 107 is controlled by the procedure described below. The control unit 108 includes a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), various interfaces, an electronic circuit, and the like.

FIG. 6 is a perspective view showing the vicinity of the mounting portion of the microorganism counting device according to the first embodiment.
The reagent cartridge 110 is locked at a predetermined position of the apparatus main body 100a by a locking portion (not shown).

The mounting portion 102 has a recess 102a for accommodating the housing 26 of the collection container 25, and an engagement ring 102b is further disposed around the opening of the recess 102a.
The engagement ring 102 b is configured to support the housing 26 on the sample mounting portion 102 by engaging with an engagement claw 26 a provided on the housing 26 of the collection container 25. The engagement ring 102b has a notch having a planar shape so that the engagement claw 26a of the housing 26 can be accommodated, and a gap with a thickness capable of receiving the engagement claw 26a between the engagement ring 102b and the apparatus main body 100a in which the recess 102a is formed. G is formed.

  When the housing 26 is fitted into the recess 102a, the engaging claw 26a is inserted into the recess 102a through the notch, and the housing 26 is rotated to slide the engaging claw 26a into the gap G. 26 engages with the engagement ring 102b. Thereby, the collection container 25 is set in the microorganism counting apparatus 100, and the collected microorganisms can be counted.

(Operation of the first embodiment)

FIG. 7 is a flowchart showing the microorganism recovery process in the first embodiment.
In step S20, the inspector collects the sample solution in the sample solution container 31, closes the open / close door 11, and then turns on a switch (not shown) of the operation unit 13 of the microorganism collection device 10 to collect the microorganism. To start.
In step S21, the control unit 80 of the microorganism collection apparatus 10 rotates the filling dilution tank 21 (cartridge 20) in a predetermined pattern. The predetermined pattern is, for example, a pattern in which high-speed rotation and low-speed rotation are repeated, a pattern in which the rotation speed is changed into a rectangular wave shape, a pattern in which the rotation speed is changed in a sine wave shape centered on the predetermined rotation speed, and the like. .

In step S22, the control unit 80 starts feeding the dilution buffer solution feeding pump 43 and the sample solution feeding pump 33 at the same time, and adds the diluted buffer solution by the buffer heater 44 as necessary. while heated, it is added dropwise to the tape path portion 22a of the filling dilution tank 21. Since the filling dilution tank 21 (cartridge 20) is rotated in a predetermined pattern in step S21, the control unit 80 can efficiently stir the sample solution and the dilution buffer solution by the stirring rib 22e.
In step S23, the control unit 80 determines whether or not a predetermined amount of the sample liquid has been dropped. This predetermined amount is a predetermined value. However, the value is not limited to this, and may be a value input by the inspector through the operation unit 13.
In step S24, the control unit 80 stops the sample solution feeding pump 33.
In step S25, the control unit 80 measures the driving torque (power consumption) of the cartridge motor 50 that drives the filling dilution tank 21. This measurement is periodically performed, and the control unit 80 estimates the viscosity of the liquid based on the measurement value.

  In step S26, the control unit 80 determines whether or not the driving torque of the cartridge motor 50 that drives the filling dilution tank 21 is within a predetermined value. This driving torque is measured, for example, with the rotational speed of the cartridge motor 50 being constant for a predetermined time. If the drive torque of the cartridge motor 50 is within the predetermined value (Yes), the process of step S27 is performed. If the drive torque of the cartridge motor 50 is not within the predetermined value (No), the process returns to step S25. However, the present invention is not limited to this, and the torque when the rotational speed is changed may be measured. The viscosity of the liquid is estimated based on the measured torque, and it can be determined whether or not proper filtration is possible.

In step S27, the control unit 80 stops the operation of the buffer liquid feed pump 43, the buffer heater 44, and the rotation operation of the filling dilution tank 21, and waits until the rotation of the filling dilution tank 21 is stopped. .
In step S28, the control unit 80 raises the suction head 61 by giving a predetermined amount of driving pulses to the suction head motor 71, and turns on the collection container heater 56 as necessary.

In step S29, the control unit 80 drives the aspirator pump 62 for a predetermined time, and collects the diluted sample liquid by filtration. This predetermined time is a value obtained by dividing the amount of liquid stored in the filling dilution tank 21 by the filtration amount per hour of the secondary filter 27 at a predetermined negative pressure. The diluted sample liquid stored in the filling dilution tank 21 is filtered through the primary filter 23 and dropped into the housing 26 by applying a negative pressure through the primary filter 23 and the secondary filter 27. The sample liquid is filtered through the secondary filter 27 and sucked into the suction head 61.
In step S <b> 30, the controller 80 lowers the suction head 61 by applying a predetermined amount of drive pulses to the suction head motor 71 and turns off the collection container heater 56.

  In step S31, the control unit 80 determines whether reprocessing has been instructed. If re-processing is instructed (Yes), the process returns to step S21. If the reprocessing is not instructed (No), the processing in FIG. Thereby, the inspector can operate the operation unit 13 and instruct reprocessing when the amount of bacteria in the sample seems to be small.

  With the processing in FIG. 7, the control unit 80 of the microorganism collection apparatus 10 controls the sample liquid to be equal to or lower than a predetermined viscosity, and captures and removes large-sized particles by the primary filter 23. Further, microorganisms are captured and collected by the secondary filter 27 from the sample liquid filtered by the primary filter 23. As a result, the contaminants other than the microorganisms contained in the sample solution are removed, and then the microorganisms contained in this sample solution are captured. Therefore, the substances that cause disturbance in counting the microorganisms are removed. Later, it is possible to recover the microorganisms.

  FIGS. 8A to 8I are diagrams showing a sequence of the microorganism recovery process in the first embodiment.

  The vertical axis | shaft of Fig.8 (a) has shown the liquid quantity of the filling dilution tank 21. FIG. The vertical axis in FIG. 8B indicates the amount of liquid in the housing 26. The vertical axis in FIG. 8C indicates on / off of the cartridge motor 50. The vertical axis in FIG. 8D indicates ON / OFF of the sample liquid feeding pump 33. The vertical axis in FIG. 8 (e) indicates ON / OFF of the solution pump 43 for the diluted buffer solution. The vertical axis in FIG. 8F indicates on / off of the buffer heater 44. The vertical axis in FIG. 8G indicates the position of the suction head 61. The vertical axis in FIG. 8H indicates on / off of the recovery container heater 56. The vertical axis in FIG. 8 (i) indicates on / off of the aspirator pump 62. The on / off shown in FIG. 8 is a schematic. For example, the buffer heater 44 and the collection container heater 56 are turned off when the temperature is too high, even if they are turned on, and excessive heating is suppressed.

When the microorganism collection process is started, the cartridge motor 50, the sample solution feeding pump 33, the buffer solution feeding pump 43, and the buffer heater 44 are turned on at time t0. Thereafter, the amount of liquid in the filling and dilution tank 21 gradually increases.
At a time t1 when a predetermined amount of the sample liquid is dropped, the sample liquid feeding pump 33 is stopped (turned off). After that, the increasing speed of the liquid amount in the filling dilution tank 21 is slightly decreased as compared with the times t0 to t1.

At the time t2 when the drive torque of the cartridge motor 50 falls within a predetermined value, the cartridge motor 50, the buffer solution feed pump 43, and the buffer heater 44 are stopped (turned off). After that, the amount of liquid in the filling dilution tank 21 remains constant.
At the time t3 when the rotation of the cartridge 20 is stopped after a predetermined time has elapsed, the position of the suction head 61 starts to rise due to the driving of the suction head motor 71.
When the suction head 61 reaches a predetermined position, at time t4, the suction head motor 71 stops driving, the suction head 61 stops, and at the same time, the collection container heater 56 is turned on.

At a time t5 when a predetermined time has elapsed, the aspirator pump 62 is turned on. Thereby, the inside of the suction head 61 becomes a negative pressure, and the sample liquid stored in the filling dilution tank 21 is filtered by the primary filter 23 and the secondary filter 27. After that, the liquid amount in the filling dilution tank 21 gradually decreases, and the liquid amount in the housing 26 gradually increases.
At the time t6 when the liquid volume in the filling dilution tank 21 reaches zero, the liquid volume in the housing 26 becomes maximum. After that, the liquid amount in the housing 26 starts to decrease gradually. Since the sample liquid dropped on the housing 26 is filtered through the secondary filter 27 at the same time, the maximum liquid amount in the housing 26 is smaller than the maximum liquid amount in the filling dilution tank 21.

At time t7 when the liquid amount in the housing 26 reaches zero, the aspirator pump 62 is stopped (turned off). Thereafter, the suction head motor 71 is driven to lower the position of the suction head 61. The driving time of the aspirator pump 62 (from time t5 to time t7) is a value obtained by dividing the liquid amount in the filling dilution tank 21 by the filtration amount per hour of the secondary filter 27 at a predetermined negative pressure.
At time t8 when the position of the suction head 61 reaches the lowest end, the suction head motor 71 is stopped and the recovery container heater 56 is stopped (off).
If all the sample liquid has not been dropped at time t8, the processing at times t0 to t8 is repeated again. If all the sample liquid has been dripped, the process of FIG. 8 is terminated.

FIGS. 9A1 to 9B5 are diagrams showing the operation of the microorganism recovery process in the first embodiment.
FIG. 9A1 is a diagram showing the state of the cartridge 20 at time t0 to t1 in FIG. A sample solution and a buffer solution are dropped into the filling dilution tank 21.

  FIG. 9 (b1) is a diagram showing the state of the sample liquid in the filling dilution tank 21 in FIG. 9 (a1). The sample liquid contains the microorganism 200 and the impurities 210 at a slightly high concentration.

  FIG. 9A2 is a diagram showing the state of the cartridge 20 at times t2 to t5 in FIG. In the filling dilution tank 21, a sample solution diluted with a buffer solution and having a predetermined viscosity or less is stored.

  FIG. 9 (b2) is a diagram showing the state of the sample liquid in the filling dilution tank 21 in FIG. 9 (a2). The diluted sample liquid contains the microorganism 200 and the contaminants 210 at a slightly lower concentration. Thereby, the impurities 210 can be efficiently removed by the primary filter 23.

  FIG. 9A3 is a diagram showing the state of the cartridge 20 at times t5 to t6 in FIG. In the filling dilution tank 21, a sample solution diluted with a buffer solution and having a predetermined viscosity or less is stored. Stored in the housing 26 is a sample solution from which impurities 210 have been filtered out by the primary filter 23. The sample liquid dropped into the housing 26 is simultaneously filtered through the secondary filter 27 and sucked into the suction head 61.

  FIG. 9 (b3) is a diagram showing the state of the sample liquid in the housing 26 in FIG. 9 (a3). The sample liquid filtered by the primary filter 23 shows that only the microorganism 200 is included, and the contaminant 210 is not included.

FIG. 9A4 is a diagram showing the state of the cartridge 20 at time t8 in FIG. The sample liquid is not stored in the filling dilution tank 21 and the housing 26.
FIG. 9 (b4) is a diagram showing a state on the primary filter 23 in FIG. 9 (a4). On the primary filter 23, the impurities 210 remain without being filtered.

  FIG. 9 (b5) is a diagram showing a state on the secondary filter 27 in FIG. 9 (a4). On the secondary filter 27, the microorganism 200 remains without being filtered. By counting the microorganisms 200 on the secondary filter 27, the microorganisms 200 contained in the sample liquid can be counted.

FIG. 10 is a flowchart showing the microorganism counting process in the first embodiment.
The operation of the microorganism counting device 100 and the principle of counting microorganisms will be described while explaining the procedure executed by the control unit 108 (FIG. 5) of the microorganism counting device 100 (FIG. 5).

  In the microbe counting apparatus 100 (FIG. 5), after the collection container 25 (FIG. 3) is mounted on the mounting unit 102 (FIG. 5), the control unit 108 executes the following procedure by turning on a start switch (not shown). To do.

  In step S40, the control unit 108 (FIG. 5) outputs a command to the dispensing unit 106 (FIG. 5), and the buffer liquid stored in the buffer tank 105 (FIG. 5) is stored in the housing 26 (FIG. 3). Dispense inside. As a result, the microorganisms held in the secondary filter 27 (FIG. 3) move into the buffer solution, and the buffer solution containing the collected microorganisms stays in the housing 26 (FIG. 3).

  In step S41, the control unit 108 outputs a command to the dispensing unit 106 (FIG. 5) to dispense the ATP erasing reagent of the reagent cartridge 110 into the housing 26 (FIG. 3). As a result, the ATP present outside the cells of the microorganism is eliminated from the buffer solution containing the microorganism.

  In step S42, the control unit 108 (FIG. 5) outputs a command to the suction unit 104 (FIG. 5) for suction, and filters the contents of the housing 26 (FIG. 3). As a result, the microorganisms are separated and held by the secondary filter 27 (FIG. 3), and the ATP elimination reagent and the buffer solution are filtered and discharged out of the housing 26.

  In step S43, the control unit 108 (FIG. 5) outputs a command to the dispensing unit 106 (FIG. 5), and the buffer liquid stored in the buffer tank 105 is again stored in the housing 26 (see FIG. 3). At the same time, a command is output to the suction unit 104 (FIG. 5) and suction is performed, and the contents of the housing 26 (FIG. 3) are filtered and washed. As a result, the microorganisms are separated and held by the secondary filter 27 (FIG. 3), and the buffer solution washed in the housing 26 is filtered and discharged out of the housing 26.

In step S44, the control unit 108 outputs a command to the dispensing unit 106 (FIG. 5), and again dispenses the buffer liquid stored in the buffer tank 105 into the housing 26 (FIG. 3). As a result, the microorganisms held in the secondary filter 27 are transferred into the buffer solution, and a sample solution containing the collected microorganisms is prepared in the housing 26.
In step S45, the control unit 108 outputs a command to the light emission intensity measurement unit 107 (FIG. 5) to turn on the light detection unit main body 107b (FIG. 5).

  In step S46, the control unit 108 outputs a command to the dispensing unit 106 (FIG. 5), and causes the ATP luminescent reagent of the reagent cartridge 110 to be dispensed into the luminescent tube 107a (FIG. 5). As a result, the light detection unit main body 107b of the luminescence intensity measurement unit 107 performs background measurement of the ATP luminescence reagent in the luminescence tube 107a.

In step S47, the control unit 108 outputs a command to the dispensing unit 106 (FIG. 5), and causes the ATP standard reagent in the reagent cartridge 110 to be dispensed into the luminescent tube 107a (FIG. 5). As a result, the light detection unit main body 107b (FIG. 5) of the luminescence intensity measurement unit 107 measures the calibration of the ATP luminescent reagent in the luminescence tube 107a (FIG. 5).

  In step S48, the control unit 108 outputs a command to the dispensing unit 106 (FIG. 5) to dispense the ATP extraction reagent of the reagent cartridge 110 (FIG. 5) into the housing 26 (FIG. 3). As a result, ATP present in the microorganism is extracted into the sample solution, whereby an ATP extract is prepared in the housing 26 (FIG. 3).

  In step S49, the control unit 108 outputs a command to the dispensing unit 106 (FIG. 5), and dispenses the ATP extract in the housing 26 to the luminescent tube 107a that has performed background measurement. As a result, in the light emission tube 107a, light is emitted by the reaction between the ATP extract and the ATP luminescent reagent previously dispensed in step S47.

  In step S50, the control unit 108 digitally processes the signal output by the light detection unit main body 107b (FIG. 5) of the light emission intensity measurement unit 107 (FIG. 5) by detecting the ATP emission, and performs single photon counting. The emission intensity is measured based on the method.

In step S51, the control unit 108 dispenses the ATP dispensed into the light emission tube 107a based on the conversion coefficient between the ATP amount (amol) and the light emission intensity (CPS) obtained by the calibration measurement in step S47. The amount of ATP (amol) contained in the extract is calculated, and based on the amount of ATP (amol) and the amount of buffer in the sample solution prepared in step S48, the amount of ATP equivalent to the microorganism contained in the sample solution The microorganism is counted as a conversion value.
The counting time using the microorganism counting apparatus 100 is about one hour.

(Effects of the first embodiment)
The first embodiment described above has the following effects (A) to (E).

(A) The primary filter 23 removes the contaminants 210 that are unnecessary for detecting microorganisms and that are an inhibiting factor. Thereby, it is possible to count highly sensitive microorganisms that are not affected by the contaminants 210.

(B) The filling dilution tank 21 provided with the primary filter 23 can be a detachable unit, and can be replaced with the unit of the filling dilution tank 21 provided with the primary filter 23 having different specifications depending on the object. Thereby, it is possible to recover microorganisms contained in a wide variety of sample solutions.

(C) The sample solution is diluted with a dilution buffer solution. Thereby, filtration of a highly viscous sample liquid and collection | recovery of microorganisms are possible.

(D) The control unit 80, which is a viscosity adjusting unit, dilutes the sample solution with a dilution buffer solution so as to be equal to or lower than a predetermined viscosity, and agitates. Thereby, the microorganisms contained in the wide viscous sample liquid can be recovered by the same microorganism recovery apparatus 10.

(E) The dilution buffer solution is heated to dilute the sample solution. Thereby, it is possible to control the liquid whose viscosity changes depending on the temperature so as to be equal to or lower than a predetermined viscosity.

(Configuration of Second Embodiment)
FIG. 11 is a configuration diagram of a cartridge according to the second embodiment. The same elements as those of the cartridge 20 of the first embodiment shown in FIG.

  The cartridge 20F of the second embodiment has the same configuration as the cartridge 20 of the first embodiment, except that it has a filling dilution tank 21F different from the filling dilution tank 21 of the cartridge 20 of the first embodiment. have.

  The filling dilution tank 21F of the second embodiment has a tank body 22F different from the tank body 22 of the filling dilution tank 21 of the first embodiment, except that the filling dilution tank 21 of the first embodiment. It has the same composition as.

  Unlike the tank body 22 of the first embodiment, the tank body 22F of the second embodiment does not have the stirring rib 22d of the inner peripheral portion, and instead has the stirring rib 22e in the center portion. Other than that, it has the same configuration as the tank body 22 of the first embodiment. The stirring rib 22e has a shape in which flat plates are combined in a cross shape, and the center position of the cross shape of the stirring rib 22e coincides with the rotation center of the tank body 22F. The method for fixing the stirring rib 22e will be described in detail with reference to FIG.

  FIGS. 12A and 12B are configuration diagrams of a tank body of a filling dilution tank in the second embodiment. Elements identical to those of the tank body 22 of the first embodiment shown in FIG.

Fig.12 (a) is a top view of the tank body 22F of the filling dilution tank 21F.
The tank body 22F has a columnar cross rib 22g inside, and supports the stirring rib 22e described above from below. The tank body 22F is hollow except for the cross ribs 22g and the stirring ribs 22e, and is configured to allow liquid or gas to pass in the vertical direction.

FIG.12 (b) is K2-K3 sectional view taken on the line of the tank body 22F of the filling dilution tank 21F.
Tank body 22F includes an upper tape path portion 22a, the cross rib 22g is formed in a plane that separates the lower side of the cylindrical portion 22b. A flat stirring rib 22e different from the stirring rib 22d of the first embodiment is formed at the center of the cross rib 22g. The stirring rib 22e is formed in a shape in which flat plates are combined in a cross shape. That is, the stirring rib 22e is configured such that the ribs for stirring the sample liquid are radially formed.
The cross rib 22g and the stirring rib 22e are configured to be rotationally symmetric . The tank body 22F is also configured rotationally symmetrical .
The primary filter 23 shown in FIG. 11 is sandwiched and fixed by the cross rib 22g and the primary filter fixing ring 24 shown in FIG.

(Operation of Second Embodiment)
The operation of the microorganism collection apparatus 10 of the second embodiment is the same as the operation of the microorganism collection apparatus 10 of the first embodiment. The filling dilution tank 21F can agitate and dilute the dilution buffer solution and the sample solution by the agitation rib 22e provided at the center more efficiently than the agitation rib 22e of the first embodiment.
The operation of the microorganism counting apparatus 100 of the second embodiment is the same as the operation of the microorganism counting apparatus 100 of the first embodiment.

(Effect of 2nd Embodiment)
The second embodiment described above has the following effect (F).

(F) A stirring rib 22e having a shape in which flat plates are combined in a cross shape is provided at the center of the filling dilution tank 21F. This makes it possible to stir and dilute the diluted buffer solution and the sample solution more efficiently than providing the stirring rib 22d on the inner peripheral portion.

(Modification)
The present invention is not limited to the above embodiment, and can be modified without departing from the spirit of the present invention. For example, the following forms (a) to (i) are used as the usage forms and modifications.

(A) The filling dilution tank 21 of the first embodiment is provided with four stirring ribs 22d on the inner periphery. However, the present invention is not limited to this, and any number of stirring ribs 22d may be provided on the inner peripheral portion as long as they are formed rotationally symmetrical .

(B) The filling dilution tank 21F of the second embodiment has four stirring ribs 22e at the center. However, the present invention is not limited to this, and any number of stirring ribs 22e may be provided in the central portion as long as they are formed rotationally symmetrical .

(C) In the first embodiment, the sample liquid and the dilution buffer liquid are dropped into the filling dilution tank 21 to control the viscosity. However, the present invention is not limited to this. When the viscosity of the sample liquid is sufficiently low, only the sample liquid is injected into the filling dilution tank 21 and filtered through the primary filter 23 and the secondary filter 27 to collect microorganisms. Good.

(D) In the first embodiment, the sample solution and the dilution buffer solution are dropped and injected into the filling dilution tank 21. However, the present invention is not limited to this, and when the viscosity of the sample liquid is sufficiently low, only the sample liquid is decanted and injected into the filling dilution tank 21, and the viscosity may be controlled by dropping only the dilution buffer liquid. . Thereby, it is possible to suppress contamination by the sample liquid container 31 and the sample liquid supply pipe 32, and to remove impurities with a large particle diameter by decantation.

(E) In the first embodiment, a fixed amount of sample solution and dilution buffer solution are dropped and injected into the filling dilution tank 21. However, the present invention is not limited to this, and the viscosity of the sample liquid is calculated by measuring the driving torque and the liquid supply amount of the liquid feed pump 33, and the sample liquid and the dilution buffer liquid corresponding to the viscosity are dropped. It may be injected. Thereby, when the viscosity of the sample liquid is high, a small amount is dropped, and thus it is possible to always dilute by stirring so that the viscosity is not more than a predetermined viscosity.

(F) In the first embodiment and the second embodiment, it is assumed that the microorganisms collected in the collection container 25 are counted by the microorganism counting device 100. However, the present invention is not limited to this, and the reagent R may be manually dispensed into the housing 26 and the microorganisms may be counted by the ATP method.

(G) In the first embodiment and the second embodiment, the microorganisms are counted by the ATP method. However, the present invention is not limited to this. Microorganisms are based on fluorescence generated by irradiating in vivo substances such as DNA (DeoxyriboNucleic Acid), RNA (Ribo Nucleic Acid), and NAD (Nicotinamide Adenine Dinucleotide) extracted from microorganisms. May be counted.

(H) When the cartridge 20 collects Gram-negative bacilli contained in the sample liquid and counts it, the endotoxin contained in the cell membrane is used as an index, and the luminescence intensity when the endotoxin is reacted with limulus is used. Based on this, the number of microorganisms may be counted.

(I) In the first embodiment and the second embodiment, the aspirator pump 62 is equal to the time obtained by dividing the liquid amount in the filling dilution tank 21 by the filtration amount per hour of the secondary filter 27 at a predetermined negative pressure. Is driving. However, the present invention is not limited to this, and the end of filtration of the secondary filter 27 may be detected by detecting that the torque of the aspirator pump 62 has become a predetermined value or less.

DESCRIPTION OF SYMBOLS 10 Microorganism collection | recovery apparatus 11 Opening / closing door 12 Display part 13 Operation part 14 Processing stage 20, 20F Cartridge (liquid bacteria collection cartridge)
21 Filling dilution tank 21F Filling dilution tank 22, 22F Tank (tank part)
22d stirring rib 22e stirring rib 23 primary filter (primary filtration means)
24 Primary filter fixing ring 25 Collection container 26 Housing (liquid holding part)
27 Secondary filter (secondary filtration means)
28 Secondary filter fixing ring 25 Recovery container 30 Sample recovery part 31 Sample liquid container 33 Liquid feed pump 40 Dilution buffer supply part 41 Dilution buffer liquid container 43 Liquid supply pump 44 Buffer heater 50 Cartridge motor 51 Rotating shaft 52 Belt 53 Rotational dilution Tank holder 54 Recovery container holder rotating part 55 Recovery container holder 56 Recovery container heater 60 Suction part 61 Suction head 62 Aspirator pump 63 Suction pipe 70 Suction head drive part 71 Suction head motor 80 Control part (viscosity adjusting means)
81 Flow Control Unit 82 Heater Control Unit 83 Motor Control Unit 84 Pump Control Unit 100 Microorganism Counting Device 101 Case 102 Mounting Unit 103 Hot Water Supply Unit 104 Suction Unit 105 Buffer Tank 106 Dispensing Unit 107 Luminous Intensity Measurement Unit 108 Control Unit 110 Reagent cartridge

Claims (13)

A microorganism collection apparatus for collecting microorganisms contained in a sample liquid,
The primary filtration means removes impurities contained in the sample liquid by filtration,
A microorganism collecting apparatus for capturing the microorganisms contained in the sample liquid obtained by filtering and removing the contaminants by secondary filtration means,
Prior to the primary filtration means, a diluent is added to the sample liquid and stirred to provide a viscosity adjusting means for adjusting the viscosity of the sample liquid;
The viscosity adjusting means includes
A microorganism collecting apparatus, wherein the dilution liquid is added by rotating a dilution tank that can be driven to rotate until the sample liquid has a predetermined viscosity. The microorganism dilution apparatus according to claim 1, wherein the dilution tank has a tapered shape. The microorganism collection apparatus according to claim 1 or 2, further comprising:
Equipped with a suction pump,
The suction pump sucks the sample liquid or the adjusted sample liquid through the primary filtration means and the secondary filtration means at a predetermined negative pressure,
While filtering and removing the contaminants contained in the sample liquid by the primary filtration means,
The microorganism recovery apparatus characterized in that the microorganisms contained in the sample liquid obtained by removing the contaminants by the secondary filtration means are captured. The primary filtering means is
Under normal pressure, the sample solution and the diluted solution are not filtered,
The microorganism collection apparatus according to any one of claims 1 to 3, wherein the sample liquid and the diluted liquid are filtered by aspiration with a predetermined negative pressure to remove the contaminants. The microorganism collecting apparatus according to any one of claims 1 to 4, wherein a flow path between the primary filtration unit and the secondary filtration unit has a tapered shape. A microorganism recovery method for recovering microorganisms contained in a sample liquid,
A viscosity adjusting step of adjusting the viscosity of the sample liquid by adding a diluent to the sample liquid;
A primary filtration step of removing contaminants contained in the adjusted sample liquid using a primary filter;
A secondary filtration step of capturing the microorganisms contained in the filtered sample liquid on a secondary filter;
And
The viscosity adjusting step includes
A method for recovering microorganisms, comprising: rotating a dilution tank until the sample liquid has a predetermined viscosity and adding the dilution liquid. A liquid bacteria collection cartridge for collecting microorganisms contained in a sample liquid,
A tank for holding a liquid;
A primary filter that is provided at a lower portion of the tank and filters and removes impurities contained in the sample liquid;
A liquid holding unit for holding liquid;
A secondary filter that is provided at a lower portion of the liquid holding unit and captures the microorganisms from the sample liquid obtained by filtering out the contaminants;
With
Provided inside the tank portion is a rib for stirring the sample liquid,
Rotating, adding a dilute solution to the sample solution and stirring the mixture, adjusting the viscosity of the sample solution , and capable of being driven to rotate until the sample solution has a predetermined viscosity Collection cartridge. The liquid bacteria collection cartridge according to claim 7, wherein the tank part and the primary filter, and the liquid holding part and the secondary filter are configured to be separable. 9. The liquid bacteria collection cartridge according to claim 7, wherein the tank part and the liquid holding part are both tapered. The liquid bacteria collection cartridge according to any one of claims 7 to 9, wherein the liquid bacteria collection cartridge is configured to be rotationally symmetric. The liquid bacteria collection cartridge according to any one of claims 7 to 10, wherein ribs for stirring the sample liquid are rotationally symmetrical inside the tank portion. The liquid bacteria collection cartridge according to any one of claims 7 to 10, wherein ribs for stirring the sample liquid are radially formed in a central portion of the tank portion. The liquid bacteria collection cartridge according to claim 7, wherein the liquid holding unit can be set as it is in a microorganism counting device that counts microorganisms.

Images