Patent Publication Number: US-2023160796-A1

Title: Filtration cartridge and microbial test method

Description:
TECHNICAL FIELD 
     The present disclosure relates to a filtration cartridge and a microbial test method. 
     BACKGROUND ART 
     In the field of recent regenerative medicine products including cell therapeutic agents and gene therapeutic agents, the Act on Securing Quality, Efficacy, and Safety of Products Including Pharmaceuticals and Medical Devices (pharmaceuticals and medical device law) and the Act on Securing Safety of Regenerative Medicine are implemented. In the pharmaceuticals and medical device law, a ministry ordinance as a standard for production management and quality management of regenerative medicine products (GCTP ministry ordinance: Good Gene, Cellular, and Tissue-based Products Manufacturing Practice) is implemented, and a “sterile operation and like zone” and a “cleanliness control zone” are defined as environmental zones. In the ministry ordinance, the “sterile operation and the like zone” is defined as a location where a preparation operation of products and the like that need to be handled by a sterile operation is performed, a location where sterilized containers and the like are exposed to air in an operation place, and a location where the sterile operation such as a “sterility test”is performed in the operation place. Specifically, the locations defined in the ministry ordnance are classified into an important zone: grade A (in safety cabinet, in clean bench, in isolator, or the like), a direct support zone: grade B, and other support zones: grade C or D. In the sterility test, a sterile operator, a sterilized material, and the like are present in grade B, and a sterile dispensing operation of test samples and reagents is performed in grade A. Grade C or D is an area where a pre-sterilized material is handled as a zone less affected product contamination. 
     Hitherto, a culturing method has been used in a microbial test and a sterility test for guaranteeing cleanliness of a production facility and sterility of water (pharmaceutical water) and a product itself used for production. In particular, a membrane filter method (MF method) is generally employed. The MF method is a method of filtering a collected sample through a membrane filter, then placing on a membrane filter obtained by filtering the sample on an agar medium, culturing the sample in a thermostat for up to 14 days, and visually counting the number of colonies of grown bacteria. However, as research on a wide variety of microorganisms has advanced, it has become clear that, since bacteria that grow relatively quickly until the colonies are visually detected require a period of at least several days and a culture medium of the related art has low colony-forming ability, there are bacteria that are difficult to be detected and quantified only by the culturing method. Rapid microbial test techniques using various measurement principles that do not necessarily require culture, which are different from culturing methods of the related art such as the MF method, have been expected as means for solving these problems. 
     The rapid microbial test methods are roughly classified into direct detection method and an indirect detection method. The former includes solid phase cytometry, flow cytometry, and the like. The latter includes immunologic methods, nucleic acid amplification methods, bioluminescence, fluorescence (staining) methods, and the like. Although performance to be prioritized most in performance required for speeding up is sensitivity, improvement in sensitivity usually leads to a decrease in specificity. For example, Adenosine triphosphate (ATP) bioluminescence method (hereinafter, referred to as an ATP method) is a method of converting ATP present in a body cell into light and measuring the ATP by using a luminescence reaction of fireflies. In the ATP method, since presence of ATP molecules contained per cell can be detected as tens of thousands of photons, light corresponding to one cell is detected with high sensitivity by photon counting light measurement. 
     In the ATP method, in order to detect an extremely small amount (several bacteria level) of bacteria mixed in a specimen with high sensitivity and high accuracy, performance of a light emission measurement device must be highly sensitive as well as it is necessary to suppress a false light emission factor due to contamination such as ATP and bacteria present in an environment, microorganisms possessed by humans, and dust adsorbing ATP. In general, since ATP and free ATP derived from dead bacteria are mixed in the specimen, it is difficult to accurately estimate the number of viable bacteria when the specimen is used as it is for luminescence measurement. 
     PTL 1 describes that a specimen liquid is filtered and microorganisms and dead bacteria are decomposed in advance by using an ATP-degrading enzyme such as apyrase in order to remove the free ATP in the specimen. 
     CITATION LIST 
     Patent Literature 
     PTL 1: WO 2016/147313A 
     SUMMARY OF INVENTION 
     Technical Problem 
     Attempts have been made to achieve both sensitivity and high specificity not only in the ATP method but in all the rapid microbial test methods. A special pre-treatment step may be added to eliminate a substance as a false positive factor from contaminants included in a test target. Thus, the number of steps of complicated operations such as reaction with a plurality of reagents, separation and cleaning of a substance using filtration, centrifugation, and the like increases. Thus, there is a problem that contamination from the environment (floating, adhesion, and human-derived) occurs, false positives occur, and the specificity also decreases. 
     Therefore, when the rapid test method is applied, it is often necessary to bring a device such as a centrifuge or an incubator into an area having a level of grade A represented by the sterile operation zone such as the safety cabinet or the isolator. As a result, because a risk of environmental break of grade A increases due to bringing the device, a validity test (validation) is required for proving that the level of grade A is guaranteed. As a result, an operator requires a large amount of labor to introduce a new technique, and may give up introduction while understanding an advantage of the rapid test. 
     Thus, it is desired to realize a pre-treatment kit for the microbial test, a dedicated reaction container or a cartridge for pre-treatment, and automation based on a completely closed system, which are excellent in operability and capable of suppressing intrusion of contaminants from the environment. 
     Therefore, the present disclosure provides a technique for suppressing false positives due to environmental contamination and easily performing a highly sensitive and highly accurate microbial test. 
     Solution to Problem 
     A filtration cartridge according to the present disclosure include a filter cup with a filter capturing microorganisms, a first filter cup attachment that covers a lower portion of the filter cup and has at least one through-hole in a bottom surface portion, and a second filter cup attachment, being connected to the first filter cup attachment, that covers an upper portion of the filter cup, and that has a lid installed at a position away from an opening surface of the filter cup. 
     Further features related to the present disclosure will become apparent from the description of the present specification and the accompanying drawings. The aspects of the present disclosure are achieved and realized by elements, combinations of various elements, the following detailed description, and aspects of the appended claims. 
     The description of the present specification is merely a typical example, and does not limit the scope of claims or application examples of the present disclosure in any sense. 
     Advantageous Effects of Invention 
     According to the filtration cartridge of the present disclosure, it is possible to suppress false positives due to environmental contamination and to easily perform a highly sensitive and highly accurate microbial test. 
     Other objects, configurations, and effects will be made apparent in the descriptions of the following embodiments. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1    is a schematic cross-sectional view illustrating a filtration cartridge according to a first embodiment. 
         FIG.  2 A  is a side view of a filter cup according to the first embodiment. 
         FIG.  2 B  is a bottom view of the filter cup according to the first embodiment. 
         FIG.  3 A  is a schematic cross-sectional view illustrating a configuration example of a first filter cup attachment according to the first embodiment. 
         FIG.  3 B  is a schematic cross-sectional view illustrating another configuration example of the first filter cup attachment according to the first embodiment. 
         FIG.  3 C  is a schematic cross-sectional view illustrating another configuration example of the first filter cup attachment according to the first embodiment. 
         FIG.  3 D  is a schematic cross-sectional view illustrating another configuration example of the first filter cup attachment according to the first embodiment. 
         FIG.  4 A  is a schematic front view of a second filter cup attachment according to the first embodiment. 
         FIG.  4 B  is a schematic side view of the second filter cup attachment according to the first embodiment. 
         FIG.  4 C  is a schematic front view illustrating another configuration example of the second filter cup attachment according to the first embodiment. 
         FIG.  5    is a schematic diagram illustrating a state where a suction filtration attachment and the filtration cartridge are connected. 
         FIG.  6 A  is a schematic diagram illustrating a state where a first centrifugal filtration attachment and the filtration cartridge are connected. 
         FIG.  6 B  is a schematic diagram illustrating a state where a second centrifugal filtration attachment and the filtration cartridge are connected. 
         FIG.  6 C  is a schematic diagram illustrating a modification example in which the first centrifugal filtration attachment also serves as a body portion. 
         FIG.  6 D  is a schematic diagram illustrating a modification example in which the second centrifugal filtration attachment also serves as a body portion. 
         FIG.  7    is a schematic diagram illustrating a biological clean room. 
         FIG.  8    is a flowchart illustrating a rapid microbial test method. 
         FIG.  9    a diagram illustrating a photon counting time course using the filtration cartridge and the suction filtration attachment. 
         FIG.  10    is a flowchart illustrating a rapid microbial test method using the filtration cartridge and the centrifugal filtration attachment. 
         FIG.  11    is a flowchart illustrating a rapid microbial test method using the filtration cartridge, the suction filtration attachment, and the centrifugal filtration attachment. 
         FIG.  12    is a schematic diagram illustrating a microbial test apparatus according to a fifth embodiment. 
         FIG.  13    is a schematic diagram illustrating a microbial test apparatus according to a sixth embodiment. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. However, each embodiment is merely an example for realizing the technique of the present disclosure, and does not limit the technology of the present disclosure. In the drawings, common components are denoted by the same reference signs. 
     First Embodiment 
     Overall Configuration Example of Filtration Cartridge 
       FIG.  1    is a schematic cross-sectional view illustrating a filtration cartridge  1  used for a microbial test according to a first embodiment. As illustrated in  FIG.  1   , the filtration cartridge  1  includes a filter cup  2 , a first filter cup attachment  3 , and a second filter cup attachment  4 . 
     The filter cup  2  is provided, at its bottom portion, with a filter  21  having a pore diameter that basically does not allow passage of microorganisms such as bacteria, fungi, and archaea. The test sample is filtered to capture bacteria on the filter  21  by adding a liquid test sample to the filter cup  2  and causing the test sample to pass through the filter and the test sample can be concentrated by removing filtrate. 
     A flange  22  is provided on an upper portion of the filter cup  2 . An opening portion of the flange  22  serves as an inlet port for introducing the test sample and reagents into the filter cup  2 . 
     The first filter cup attachment  3  covers a lower portion of the filter cup  2  and prevents contamination of an outer wall of the filter cup  2  and the filler  21 . The first filler cup attachment  3  functions as a contamination protection cover for preventing an operator&#39;s finger or a surface such as a bench top, a ceiling, a side wall, or a glass window within an operation area from directly coming into contact with the filter cup  2 . 
     A filtration nozzle  31  (through-hole) is provided at a bottom portion of the first filter cup attachment  3 . As a result, a liquid sample such as the test sample or the reagent added to the filter cup  2  passes through the filter  21 , and thus it becomes possible to forcibly pass the liquid sample from an upper portion to a lower portion of the filter  21 . 
     The second filter cup attachment  4  is connected to the first filter cup attachment  3  and covers the upper portion of the filter cup  2 , in particular the flange  22 , and a lid  41  is provided at a position away from an opening surface of the filter cup  2  by a certain distance. As a result, it is possible to prevent dust from entering the filter cup  2  and to prevent the operator&#39;s hand or arm from touching the filter cup  2 . Generally, an operator who uses the filtration cartridge  1  operates the filtration cartridge in a state where the lid  41  is closed in order to avoid a risk of entering of dust and contamination from the operator or the inside of the operation area, and temporarily opens the lid  41  for use in an operation of dispensing a sample to be tested and reagents for test into the filter cup  2 . The lid  41  is configured to be movable parallel to the opening surface of the filter cup  2 . 
     The filtration cartridge can be provided to a user as a microbial test kit together with a suction filtration attachment and a centrifugal filtration attachment to be described later. The filtration cartridge  1  can be disposable. 
     Configuration Example of Filter Cup 
       FIG.  2 A  is a side view of the filter cup  2 . As illustrated in  FIG.  2 A , the filter cup  2  includes the filter  21 , the flange  22 , a container portion  23 , and a support plate  24 . The filter  21  is provided at a bottom portion of the container portion  23  and is supported by the support plate  24 . 
     The container portion  23  and the support plate  24  can be made of, for example, a resin such as polypropylene, polystyrene, or polyethylene, and can be produced by injection molding, machining, or the like. The filter  21 , the container portion  23 , and the support plate  24  can be fixed by, for example, adhesion using ultrasonic welding. From the viewpoint of a fluid, a shape of the container portion  23  can be a cylindrical shape, but can also be a square tubular shape or a polygonal tubular shape, each having a corner depending on the application. 
     The container portion  23  is provided, on its upper portion, with the flange  22  for supporting the filter cup  2  by the second filter cup attachment  4 . When the filter cup  2  is inserted into the second filter cup attachment  4 , the flange  22  serves as a stopper, and thus the filter cup  2  is fixed at a predetermined position. 
     The filter  21  may be any filter capable of capturing a microbial to be tested, and for example, a membrane filter can be used. For example, cellulose mixed ester, PTFE, polyvinylidene fluoride (PVDF), polycarbonate, and the like can be used as a material of the filter  21 . 
     A pore size of the filter  21  can be appropriately selected in accordance with a size of the microorganism to be tested and various conditions of the microbial test. The pore size can be equal to or greater than, for example, 0.20 μm. Specifically, membrane filters having pore sizes of 0.22 μm, 0.45 μm, and the like can be enumerated as an example of the filter  21 . 
     It is possible to minimize liquid remaining after filtration as much as possible by setting a size (diameter) of the filter  21  and a diameter of the container portion  23  to the same diameter and by straightening both an inner wall of the container portion  23  and a side surface of the filter  21 . Alternatively, it is possible to minimize the liquid remaining after filtration as much as possible by setting to be the same a shape of the side surface of the filter  21  and a shape of the bottom portion of the container portion  23 . Of course, the container portion  23  may be formed in a tapered shape that becomes thinner from the upper portion toward the bottom portion. Depending on the application, although the container portion  23  may have a tapered shape that becomes thicker from the upper portion toward the bottom portion, since a large distribution may occur in the amount of liquid per unit time passing through a filtration surface of the filter  21  during filtration by suction or centrifugation, clogging is caused. Although the container portion  23  may have a structure bent in the middle depending on the application, uniformity of the amount of liquid passing through the filtration surface may be lost, and a large distribution may occur. Thus, clogging is caused. 
     The diameter of the filter  21  can be, for example, several mm to 47 mm. A diameter of the bottom portion of the container portion  23  is substantially equal to the diameter of the filter  21 . A height of the container portion  23  is designed in accordance with the amount of liquid of the test sample, and can be designed such that several μL to several 100 mL can be accommodated, for example. 
     However, a case where an area and a volume for capturing microorganisms are reduced by decreasing the diameter of the filter  21  as much as possible is useful for improving sensitivity since a sample concentration (per unit area or per unit liter) used for measurement is increased. When the area of the filter  21  is reduced, a large volume of test sample can be filtered by attaching a funnel to the opening portion of the filter cup  2 . 
       FIG.  2 B  is a bottom view of the filter cup  2 . As illustrated in the left diagram of  FIG.  2 B , the support plate  24  can be formed by a plurality of support rods radially extending from a center, represented by a cross shape. As illustrated in the center diagram of  FIG.  2 B , the support plate  24  can support the filter  21  by a mesh structure having a plurality of through-holes of levels from several mm to several cm. As illustrated in the right diagram of  FIG.  2 B , the support plate  24  may have a shape of supporting only an end portion of the filter  21 . As described above, the support plate  24  has the through-hole capable of passing through the liquid having passed through the filter  21 . 
     Configuration Example of First Filter Cup Attachment 
       FIG.  3 A  is a schematic cross-sectional view illustrating a configuration example of the first filter cup attachment  3 . As described above, the first filter cup attachment  3  prevents contamination of the outer wall of the filter cup  2  and the filter  21 . The first filter cup attachment  3  can be made of, for example, a resin such as polypropylene, polystyrene, or polyethylene and can be produced by injection molding, machining, or the like. 
     As illustrated in  FIG.  3 A , the first filter cup attachment  3  has a body portion  32  in which the filtration nozzle  31 , an attachment joint hole  33 , and a filter cup insertion portion  34  are formed. A sealing material  35  is provided on a lower surface of the body portion  32 . 
     The attachment joint hole  33  and the filter cup insertion portion  34  correspond to recesses fitted to the outer shape of the container portion  23  of the filter cup  2 , and the filter cup  2  is inserted into these recesses. The filter cup insertion portion  34  is coupled to the filtration nozzle  31 . The filter cup  2  and the first filter cup attachment  3  are fixed by, for example, press-bonding by pressing. A part of the body portion of the second filter cup attachment  4  to be described later is also included in the first filter cup attachment  3 . 
     Since the operator may remove the filter cup  2  from the first filter cup attachment  3  during a step of the microbial test in which the filtration cartridge  1  is used, an inner diameter of the first filter cup attachment  3  is formed to be larger than an outer diameter of the container portion  23  of the filter cup  2 . 
     A connector (claw-like connector or tubular connector to be described later) of the second filter cup attachment  4  is fitted into the attachment joint hole  33 , and thus, the first filter cup attachment  3  is held in state of being close contact with the filter cup  2 . As a result, the filter cup  2  is more stably stored in the first filter cup attachment  3  and the second filter cup attachment  4 . The attachment joint hole  33  illustrated in  FIG.  3 A  is a form suitable for press-bonding by pressing or the second filter cup attachment  4  including a tubular connector. When the tubular connector is a male screw, a wall surface of the attachment joint hole  33  can have a female tap shape. 
     The first filter cup attachment  3  is used in connection with a suction filtration attachment, a first centrifugal filtration attachment, and a second centrifugal filtration attachment to be described later. Thus, the bottom portion of the body portion  32  is inserted into recesses provided in the suction filtration attachment, the first centrifugal filtration attachment, and the second centrifugal filtration attachment. 
     An inner diameter of the filtration nozzle  31  can be set to be equal to or greater than at least 0.5 mm and can be set to be increased as much as possible from the viewpoint of allowing a substance having passed through the filter  21  to pass therethrough without capturing the substance and from the viewpoint of efficiently discharging the liquid. By setting the inner diameter of the filtration nozzle  31  to, for example, 0.5 mm to 10 mm, provided is the filtration nozzle  31  which is excellent in compatibility with an inner diameter of a general-purpose tube used for suction. 
     An opened portion of the filtration nozzle  31  is provided to the operator in a state of being sealed with the sealing material  35  until immediately before use by the operator. Thus, an inside of the first filter cup attachment  3  is prevented from coming into contact with outside air or coming into contact with the operator or a surface of an object. The sealing material  35  can be removed by the operator. The sealing material  35  can be, for example, a tape made of metal or resin. 
       FIG.  3 B  is a schematic cross-sectional view illustrating another configuration example of the first filter cup attachment  3 . In the example illustrated in  FIG.  3 B , the attachment joint hole  33  has a structure suitable for the second filter cup attachment  4  including the claw-like connector. 
     In the example illustrated in  FIG.  3 B , the filter cup insertion portion  34  has a slightly tapered structure in which an inner diameter decreases along a direction of the filtration nozzle  31 . As a result, the filter cup  2  can be more easily removed from the first filter cup attachment  3 . 
     In the example illustrated in  FIG.  3 B , the outer diameter of a formed portion of the filtration nozzle  31  is smaller than the outer diameter of the body portion  32 . The opened portion of the filtration nozzle  31  is sealed with the sealing material  35  in the form of a cap (for example, made of resin). 
       FIGS.  3 C and  3 D  are schematic cross-sectional views illustrating another configuration example of the first filter cup attachment  3 . In the example illustrated in  FIG.  3 C , a recessed portion  36  is provided on an outer peripheral surface of the body portion  32 , and in the example illustrated in  FIG.  3 D , a protrusion portion  37  is provided on the outer peripheral surface of the body portion  32 . As a result, improved is ease of gripping the body portion by the operator, for example, in attaching and detaching the first filter cup attachment  3  to and from the suction filtration attachment, the first centrifugal filtration attachment, or the second centrifugal filtration attachment. 
     Configuration Example of Second Filter Cup Attachment 
       FIG.  4 A  is a schematic front view of the second filter cup attachment  4 .  FIG.  4 B  is a schematic side view of the second filter cup attachment  4 . In  FIGS.  4 A and  4 B , a cross section of the second filter cup attachment  4  is partially illustrated. 
     As illustrated in  FIG.  4 A , the second filter cup attachment  4  includes the lid  41 , a body portion  42 , and a claw-like connector  50 . The body portion  42  is provided with a filter cup introduction hole  43 , a flange holding portion  44 , a first fixing member  45 , a lid fixing hole  46 , and a second fixing member  47 . 
     A step as the flange holding portion  44  is provided in the filter cup introduction hole  43 . An inner diameter of the filter cup introduction hole  43  at a lower stage is smaller than an inner diameter at an upper stage than the flange holding portion  44 . The flange  22  of the filter cup  2  inserted from the filter cup introduction hole  43  abuts on the flange holding portion  44  to serve as a stopper. Thus, the filter cup  2  is held at a predetermined position. When the inner diameter of the filter cup introduction hole  43  and the outer diameter of the container portion  23  of the filter cup  2  are substantially the same, these components are press bonded and fixed. The first fixing member  45  is inserted into a step portion of the filter cup introduction hole  43 , and an outer periphery of the container portion  23  under the flange  22  is brought into close contact with the filter cup introduction hole. As a result, the fixing performance of the filter cup  2  is improved. The first fixing member  45  is, for example, an O-ring, a sealing tape, or the like. 
     When the lid  41  is closed, a projection portion  49  of the lid  41  enters into the lid fixing hole  45 , and the lid is temporarily closed at a location where a taper angle of the lid fixing hole  46  changes. In this position, there is a minute gap into which the outside air flows, and thus, the lid is in a semi-closed state (first closed state). When the lid  41  is further pushed into the lid fixing hole  46 , a sealed state for blocking the outside air is formed. This state is set to a fully closed state (second closed state). A position of the lid  41  in the semi-closed state is referred to as a “semi-closed position.” The semi-closed position is a position required to create a necessary air flow during suction filtration. On the other hand, a position of the lid  41  in the fully closed state is referred to as a “fully closed position”. The fully closed position is a position necessary for preventing contamination of airborne microorganisms when being taken out from a sterile operation zone to a peripheral zone. The second fixing member  47  is, for example, an O-ring, a sealing tape, or the like, and prevents unintended opening of the lid  41 . As described above, the lid  41  can take the two-stage closed state. 
     A knob  48  is a recess provided in an upper surface of the lid  41 . The operator hooks his or her finger on the knob  48  during an opening and closing operation of the lid  41 . As illustrated in  FIG.  4 A , the knob  48  has a recessed shape, but may have a protruding shape. 
     The claw-like connector  50  is provided to project from a bottom surface of the body portion  42 . As illustrated in  FIG.  4 B , the claw-like connector  50  has a substantially U-shape curved outward. When the second filter cup attachment  4  is connected to the above-described first filter cup attachment  3 , the claw-like connector  50  is inserted into the attachment joint hole  33 . At this time, a curved portion of the claw-like connector  50  is distorted, and joined to the attachment joint hole  33  illustrated in  FIG.  3 B  by using a relationship between a key and a key hole. 
       FIG.  4 C  is a schematic front view illustrating another configuration example of the second filter cup attachment  4 . In the example illustrated in  FIG.  4 C , the lid  41  is in the form of a hinge mechanism  52  that opens and closes at an angle of, for example, 90° or more and less than 180° with respect to the opening surface of the filter cup  2 . Provided is the lid  41  which is excellent in the operability of opening and closing also by such a form. A knob  53  corresponding to a protrusion portion is provided in the lid  41  of the hinge mechanism  52 . In the example illustrated in  FIG.  4 C , a tubular connector  51  is provided on the bottom surface of the body portion  42 . The tubular connector  51  is a tubular male screw, and is screwed to the attachment joint hole  33  (female tap shape) illustrated in  FIG.  3 A . 
     Conclusion of First Embodiment 
     As described above, the filtration cartridge  1  according to the first embodiment includes the filter cup  2  having the filter  21  that captures bacteria at the bottom portion, the first filter cup attachment  3  that covers the lower portion and the bottom surface of the filter cup  2  and has at least one filtration nozzle  31  (through-hole) at the bottom portion, and the second filter cup attachment  4  that covers the upper portion of the filter cup  2  and has the lid  41  installed with a space at a position away from the uppermost surface of the filter cup  2 . With such a configuration, the lid  41  can be closed at the time of use or transportation by the operator. Since the lid  41  does not come into contact with the opening portion of the filter cup  2 , contamination of the filter cup  2  from the outside can be prevented. Since the operator may open the lid  41  when adding the test sample or the reagent to the filter cup  2 , the operation is also simple. 
     At the time of producing the filtration cartridge  1 , it is possible to assemble the filter cup  2 , the first filter cup attachment  3 , and the second filter cup attachment  4  under an environment of grade A of the sterile operation zone described above. Since the lid  41  is sealed and the filtration nozzle  31  is sealed with the sealing material  35  at the time of being provided to the operator, it is possible to maintain a level of grade A for the filter cup  2  disposed inside even when the filtration cartridge  1  is transported from a zone of grade B or the like to a zone of grade A. As a result, it is not necessary to bring a device such as a centrifuge or an incubator into grade A. In addition, the sterile operation is facilitated without increasing a risk of environmental break due to deterioration of a complete sterile environment in grade A due to bringing the device. 
     As described above, according to the filtration cartridge  1  of the first embodiment, it is possible to suppress false positive due to environmental contamination, and it is possible to easily perform a highly sensitive and accurate microbial test (particularly, MF method). 
     Second Embodiment 
     In a second embodiment, the suction filtration attachment connected to the filtration cartridge will be described. 
     Configuration Example of Suction Filtration Attachment 
       FIG.  5    is a schematic diagram illustrating a state where a suction filtration attachment  100  and the filtration cartridge  1  are connected. In  FIG.  5   , a cross section is partially illustrated. As illustrated in  FIG.  5   , the suction filtration attachment  100  includes a support block  101 , an opening and closing valve  102 , and a pipe connector  103  (connection structure portion). The suction filtration attachment  100  can be made of, for example, a resin such as polypropylene, polystyrene, or polyethylene. 
     The first filter cup attachment  3  is fitted to the support block  101 , and thus the first filter cup attachment is supported. A flow path  104  is provided inside the support block  101 . One end portion of the flow path  104  is connected to the filtration nozzle  31  of the first filter cup attachment  3 . The other end portion of the flow path  104  is connected to a suction device (not illustrated) via the opening and closing valve  102  and the pipe connector  103 . In other words, the flow path  104  connects the filtration nozzle  31  and the pipe connector  103 . As a result, the liquid sample added to the filter cup  2  passes through the filter  21  of the filtration cartridge  1  and is filtered. The liquid (filtrate) other than microorganisms captured by the filter  21  passes through the flow path  104  of the suction filtration attachment  100  and then is discharged from the pipe connector  103 . 
     Third Embodiment 
     In a third embodiment, the centrifugal filtration attachment connected to the filtration cartridge will be described. 
     Configuration Example of Centrifugal Filtration Attachment 
       FIG.  6 A  is a schematic diagram illustrating a state where a first centrifugal filtration attachment  200  and the filtration cartridge  1  are connected. In  FIG.  6 A , a cross section is partially illustrated. As illustrated in  FIG.  6 A , the first centrifugal filtration attachment  200  includes a first fitting portion  201  and a waste liquid tube  202  (container). The first centrifugal filtration attachment  200  can be made of, for example, a resin such as polypropylene, polystyrene, or polyethylene. The body portion  32  of the first filter cup attachment  3  is fitted to the first fitting portion  201 . The waste liquid tube  202  is inserted into the first fitting portion  201  on a side opposite to a side where the body portion  32  is fitted. 
     By setting in a centrifuge the first centrifugal filtration attachment  200  and the filtration cartridge  1  in a state of being connected to each other and then performing centrifugal separation, the liquid sample added to the filter cup  2  passes through the filter  21  and is filtered. A filtrate is collected in the waste liquid tube  202 . 
       FIG.  6 B  is a schematic diagram illustrating a state where a second centrifugal filtration attachment  300  and the filtration cartridge  1  are connected. In  FIG.  6 B , a cross section is partially illustrated. As illustrated in  FIG.  6 B , the second centrifugal filtration attachment  300  includes a second fitting portion  301  and a measurement tube  302  (container). The body portion  32  of the filtration cartridge  1  is fitted to the second fitting portion  301 . The measurement tube  302  is inserted into the second fitting portion  301  on a side opposite to a side where the body portion  32  is fitted. 
     By setting in a centrifuge the second centrifugal filtration attachment  300  and the filtration cartridge  1  in a state of being connected to each other and then performing centrifugal separation, the liquid sample added to the filter cup  2  passes through the filter  21  and is filtered. A filtrate is collected in the measurement tube  302 . 
       FIG.  6 C  is a schematic diagram illustrating a modification example in which the first centrifugal filtration attachment  200  also serves as the body portion  32 . A illustrated in  FIG.  6 C , the body portion  42  of the second filter cup attachment  4  is connected to the first fitting portion  201  of the first centrifugal filtration attachment  200 . In this case, the first fitting portion  201  is provided with an attachment joint hole for connecting the claw-like connector or the tubular connector of the body portion  42 . The state illustrated in  FIG.  6 C  is realized, for example, by the operator removing the first filter cup attachment  3  of the filtration cartridge  1  and then connecting the first centrifugal filtration attachment  200 . 
       FIG.  6 D  is a schematic diagram illustrating a modification example in which the second centrifugal filtration attachment  300  also serves as the body portion  32 . As illustrated in  FIG.  6 D , the body portion  42  of the second filter cup attachment  4  is connected to the second fitting portion  301  of the second centrifugal filtration attachment  300 . The other points are similar to those in  FIG.  6 C . 
     Fourth Embodiment 
     In a fourth embodiment, a microbial test method using the above-described filtration cartridge will be described. Here, it is supposed that the filtration cartridge of the present disclosure is applied to pharmaceutical water management. A microbial management standard of pharmaceutical water is managed as treatment standard values with an allowable amount of microorganisms contained in water, based on the pharmacepoeia of each country. In the Japanese Pharmacopoeia, maintenance of a pharmaceutical water production line is required when normal water exceeds 100 CFU/mL, purified water exceeds 10 CFU/mL, and injection water exceeds 10 CFU/100 mL. As the treatment standard values in the United States Pharmacopeia, 500 CFU/mL is defined for drinking water, 100 CFU/mL is defined for purified water, and 10 CFU/100 mL is defined for injection water. As the treatment standard values in the European Pharmacopeia, 100 CFU/mL is defined for purified water, 10 CFU/100 mL is defined for highly purified water, and 10 CPU/100 mL is defined for injection water. 
     Thus, in the following description, three kinds of normal water, purified water, and injection water are used as test samples, in order to perform a microbial test of pharmaceutical water conformity to the test of the Japanese Pharmacopoeia. 1 mL of normal water and purified water is used as a test volume, and only 100 mL of injection water is used. 
     In the microbial test of pharmaceutical water, it is possible to apply various microbial rapid test methods such as a fluorescence staining method. However, it is supposed, in the present embodiment, to use rapid test method by an ATP luminescence method using a high-sensitivity ATP test kit (Lumione (registered trademark) rapid microbial test reagent kit (manufactured by Hitachi High-Tech Solutions Corporation)) and a high-sensitive ATP measurement device (Lumione BL-2000 (manufactured by Hitachi High-Tech Solutions Corporation)). The high-sensitivity ATP test kit includes an active erasure liquid, a cleaning liquid, a luminescent liquid, an extraction liquid, a positive control (ATP 100 amol/50 μL extraction liquid), a measurement tube, and a dedicated rack thereof (microbial rapid test apparatus that ensures quality of pharmaceutical products Lumione BL-2000, 2018, vol. 100, No. 6). 
     Microbial Test Environment 
       FIG.  7    is a schematic diagram illustrating a biological clean room  70 . It is possible to perform the rapid test method by the ATP luminescence method in the biological clean room  70 . As illustrated in  FIG.  7   , the biological clean room  70  is provided with a safety cabinet  71  of grade A as an important zone and a sterile operation zone  79  of grade B. The sterile operation zone  79  is installed with a suction pump  72  (suction device) and peripheral tools (waste liquid trap  73  and suction pump tube  74 ), an incubator  75 , a centrifuge  76 , and a measurement device  77 . The operator in the sterile operation zone  79  can arbitrarily access the safety cabinet  71 , the suction pump  72 , and the peripheral tools (waste liquid trap  73  and suction pump tube  74 ), the incubator  75 , the centrifuge  76 , and the measurement device  77 . 
     Here, grade A means a local sterile operation zone where an operation that needs to prevent a risk of contamination of products at a high level is performed. On the other hand, grade B means a multipurpose zone where an operation that needs to prevent the risk of contamination of products at a relatively high level is performed. That is, this zone of grade B is a zone where there are a person, an instrument, a device, and the like that carry in a container, a raw material, and an intermediate product after sterilization stored to be able to maintain sterility and that directly get involved in the sterile operation zone. In a typical sterile room, the zone of grade B is a surrounding environment of grade A. 
     The arrangement of  FIG.  7    is merely an example. An actual site is installed with infrastructure facilities other than the facilities illustrated in  FIG.  7    to maintain cleanliness. Cleaning and environmental monitoring in the biological clean room  70  are appropriately performed. For example, airborne bacteria are managed at less than 1 CFU/m 3  in the safety cabinet  71  corresponding to grade A. The zone of grade B is a test zone in which airborne bacteria are managed at 10 CFU/m 3  or less. 
     The operator can bring the test sample, the filtration cartridge  1 , the suction filtration attachment  100 , and at least one of the first centrifugal filtration attachment  200  and the second centrifugal filtration attachment  300 , the test reagents, and a sample and reagent dispensing tool (for example, micropipette or Pipetman) to the sterile operation zone  79  via a pass box  78 , and can introduce these devices in a state of being sterile-packed in the safety cabinet  71 . The filtration cartridge  1  can be at least doubly packaged to remove one packaging before introduction into the safety cabinet  71  and the remaining packaging after introduction into the safety cabinet  71 . The filtration cartridge  1  is packaged through sterilization steps such as gamma ray sterilization, EOG sterilization, and autoclaving. 
     The test reagents are the activity erasure liquid (hereinafter, referred to as a reagent A), the cleaning liquid (hereinafter, referred to as a reagent B), and the extraction liquid (hereinafter, referred to as a reagent C) included in the high-sensitivity ATP test kit. The luminescent liquid is set at a predetermined position of the measurement device  77 . 
     Rapid Microbial Test Using Filtration Cartridge and Suction Filtration Attachment 
       FIG.  8    is a flowchart illustrating the rapid microbial test method using the filtration cartridge  1  and the suction filtration attachment  100  ( FIG.  5   ). 
     Step S 11 : Bacteria Capturing 
     In step S 11 , the operator captures bacteria on the filter  21  of the filtration cartridge  1 . Details of this step S 11  are as follows. 
     First, the operator introduces the test sample, the filtration cartridge  1 , the suction filtration attachment  100 , the test reagents, and the sample and reagent dispensing tool into the safety cabinet  71 , opens the filtration cartridge  1  and the suction filtration attachment  100 , removes the sealing material  35  of the filtration nozzle  31 , and connects the filtration cartridge  1  and the suction filtration attachment  100 . One filtration cartridge  1  and one suction filtration attachment  100  are used for each of the test samples (1 mL of normal water, 1 mL of purified water, and 100 mL of injection water). 
     Next, as preparation for a filtration step of the test sample, the operator pulls out only the pipe connector  103  attached to the suction filtration attachment  100  from a front shutter of the safety cabinet  71  to the zone of grade B, connects the pipe connector to the suction pump tube  74  in the zone of grade B, and drives the suction pump  72 . 
     Next, the operator opens the lid  41  of the second filter cup attachment  4  in the safety cabinet  71  to inject the test sample into the filter cup  2  from a container containing the test sample by a pipetting operation. After injecting a required amount of test sample into the filter cup  2 , the operator closes the lid  41  of the second filter cup attachment  4  to the semi-closed position, and opens the opening and closing valve  102  of the suction filtration attachment  100  to start the filtration of the test sample. After starting the filtration, the operator visually observes the transparent or translucent suction pump tube  74 . The operator opens the lid  41  when the flow of the liquid cannot be visually observed, and closes the lid  41  to the fully closed position after confirming that the liquid does not remain on the filter  21 . Since there is no residual liquid, the operator determines that the filtration is completed, closes the opening and closing valve  102  of the suction filtration attachment  100 , disconnects the suction filtration attachment  100  and the suction pump  72 , to stop the filtration. 
     With the operations so far, liquid components of the test sample (1 mL of normal water, 1 mL of purified water, and 100 mL of injection water) pass through the filter  2 . When there are bacteria in the filter  21 , the liquid components are concentrated and the bacteria is captured by the filter  21 . 
     Step S 12 : ATP Activity Erasure Reaction 
     In step S 12 , the operator performs an ATP activity erasure reaction by using the reagent A of the Lumione test kit. Details of this step S 12  are as follows. 
     The operator opens the lid  41  of the second filter cup attachment  4  to add a predetermined addition amount of reagent A by a pipetting operation, and then closes the lid  41  of the second filter cup attachment  4  to the fully closed position. In order to take out from the safety cabinet  71  the filtration cartridge  1  to which the suction filtration attachment  100  is connected, the operator removes the opening and closing valve  102  and the pipe connector  103  from the suction filtration attachment  100 , and holds the opening and closing valve  102  and the pipe connector  103  in the safety cabinet  71 . The operator introduces into the incubator  75  (zone of grade B) the filtration cartridge  1  to which the suction filtration attachment  100  is connected, to incubate the filtration cartridge  1  at 37° C. for 40 minutes. After 40 minutes, the operator takes out from the incubator  75  the filtration cartridge  1  to which the suction filtration attachment  100  is connected, to perform alcohol cleaning. After the alcohol cleaning, the operator re-introduces the filtration cartridge into the safety cabinet  71 , and then connects the opening and closing valve  102  and the pipe connector  103 . After opening the lid  41  of the second filter cup attachment  4  to the semi-closed position, the operator opens the opening and closing valve  102  of the suction filtration attachment  100  to filter the reagent A. The operator visually observes the suction pump tube  74 , opens the lid  41  when the flow of the liquid cannot be visually observed, and confirms that the liquid does not remain on the filter  21 . Thereafter, the operator closes the opening and closing valve  102  of the suction filtration attachment  100  to stop filtration. 
     Step S 13 : Cleaning 
     In step S 13 , the operator cleans the filter  21  by using the reagent B of the Lumione test kit. Details of this step S 13  are as follows. 
     The operator opens the lid  41  of the second filter cup attachment  4  to add a predetermined addition amount of reagent B by a pipetting operation. The operator brings the lid  41  of the second filter cup attachment  4  to the semi-closed position and opens the opening and closing valve  102  of the suction filtration attachment  100  to filter the reagent B. The operator visually observes the suction pump tube  74  and opens the lid  41  when the flow of the liquid cannot be visually observed. The operator confirms that the liquid does not remain on the filter  21  and closes the opening and closing valve  102  to stop the filtration. 
     Step S 14 : ATP Extraction 
     In step S 14 , the operator extracts ATP by using the reagent C of the Lumione test kit to complete the preparation of an ATP measurement sample. Details of this step S 14  are as follows. 
     The operator opens the lid  41  of the second filter cup attachment  4  to add the reagent C in an addition amount of 50 μL defined in the manual of the kit by a pipetting operation. After adding the reagent C, the operator brings the lid  41  of the second filter cup attachment  4  to the fully closed position, and then holds the reagent C at a room temperature for about several to 10 minutes in a state of being immersed in the filter  21 . Thereafter, the operator opens the lid  41  to collect the entire amount (50 μL) of reaction liquid (hereinafter, referred to as a “reagent C reaction liquid”) after the reaction between the reagent C and the bacteria from the filter  21  by a pipetting operation. The operator dispenses the collected reaction liquid into the measurement tube. The operator sets the measurement tube in a dedicated tube rack and covers the tube rack. 
     Finally, the operator introduces the tube rack into the measurement device  77  installed in grade B to perform ATP measurement. 
     ATP Luminescence Measurement 
     The high-sensitive ATP measurement device (Lumione BL-2000 (manufactured by manufactured by Hitachi High-Tech Solutions Corporation)) is used as the measurement device  77 . When the operator sets the luminescent liquid of the high-sensitivity ATP test kit (Lumione rapid microbial test reagent kit (manufactured by Hitachi High-Tech Solutions Corporation)) in the measurement device  77  and sets a dedicated tube rack, to start measurement, the measurement device  77  sequentially measures the reagent C reaction liquid contained in up to 24 measurement tubes. 
       FIG.  9    is a graph of a typical photon counting time course obtained by automatically dispensing the luminescent liquid into the measurement tube into which the reagent C reaction liquid is introduced. An ATP amount is calculated by integrating light emission amounts of the photon counting time course for a certain time and normalizing an average value of the light emission amounts as a relative light unit (RLU). 
     The operator prepares at least three blank samples obtained by directly dispensing 50 μL of reagent C into the measurement tube in advance. The operator also prepares at least one standard positive sample obtained by dispensing a positive control (ATP 100 amol/50 μL extraction liquid) included in the test kit into the measurement tube. The operator quantifies an ATP amount of an actual test sample by measuring these samples before measurement of the reagent C reaction solution as the actual test sample and by creating a two-point calibration curve of 0 amol and 100 amol. This flow of the ATP measurement is standard performed in accordance with a manual performed by the measurement device of the Lumione BL-2000. 
     When an ATP amount corresponding to one viable bacteria is set to 1 amol or more and measurement is performed by using this amount as a threshold value, it can be determined that there are microorganisms in the sample when the ATP amount exceeding 1 amol is detected. 
     Rapid Microbial Test Using Filtration Cartridge and Centrifugal Filtration Attachment 
       FIG.  10    is a flowchart illustrating the rapid microbial test method using the filtration cartridge  1  and the first centrifugal filtration attachment  200  ( FIG.  6 A ) and the second centrifugal filtration attachment  300  ( FIG.  6 B ). 
     Step S 21 : Bacteria Capturing 
     In step S 21 , the operator captures bacteria on the filter  21  of the filtration cartridge  1 . Details of this step S 21  are as follows. 
     First, the operator introduces the test sample, the filtration cartridge  1 , the first centrifugal filtration attachment  200 , the second centrifugal filtration attachment  300 , the test reagents, and the sample and reagent dispensing tool into the safety cabinet  71 , opens the microbial test filtration cartridge  1  and the first centrifugal filtration attachment  200 . Then, the operator removes the sealing material  35  of the filtration nozzle  31 , and connects the filtration cartridge  1  and the first centrifugal filtration attachment  200 . 
     Next, the operator opens the lid  41  of the second filter cup attachment  4  in the safety cabinet  71  to inject the test sample into the filter cup  2  from a container containing the test sample by a pipetting operation. 
     After injecting a required amount of test sample into the filter cup  2 , the operator closes the lid  41  of the second filter cup attachment  4  to the fully closed position. Then, the operator takes out from the safety cabinet  71  the filtration cartridge  1  to which the first centrifugal filtration attachment  200  is connected. Further, the operator sets the filtration cartridge in the centrifuge  76  (zone of grade B) to perform centrifugal filtration. A centrifugal force (×g) can be equal to or less than 2000×g not to damage bacteria. Since a time required for centrifugation depends on the volume and viscosity of the test sample, it is necessary to grasp a time during which the whole amount is filtered, in advance for each test sample. The condition in the present embodiment is 1000×g for 1 minute. 
     After the centrifugation is stopped, the operator takes out from the centrifuge  76  the filtration cartridge  1  to which the first centrifugal filtration attachment  200  is connected. Then, after spraying the filtration cartridge  1  to which the first centrifugal filtration attachment  200  is connected with alcohol to perform cleaning, the operator introduces the filtration cartridge into the safety cabinet  71 . Next, the operator opens the lid  41  of the second filter cup attachment  4  and confirms that there is no residual liquid. Here, when the liquid remains, the centrifugal filtration by the centrifuge  76  may be performed again. 
     With the operations so far, liquid components of the test sample (1 mL of normal water, 1 mL of purified water, and 100 mL of injection water) pass through the filter  21 . When there are bacteria in the filter  21 , the liquid components are concentrated and captured by the filter  21 . 
     Step S 22 : ATP Activity Erasure Reaction 
     In step S 22 , the operator performs the ATP activity erasure reaction by using the reagent A of the Lumione test kit. Details of this step S 22  are as follows. 
     The operator opens the lid  41  of the second filter cup attachment  4  to add a predetermined addition amount of reagent A by a pipetting operation. Then, the operator closes the lid  41  of the second filter cup attachment  4  to the fully closed position. Next, the operator introduces into the incubator  75  (zone of grade B) the filtration cartridge  1  to which the first centrifugal filtration attachment  200  is connected, to incubate the filtration cartridge  1  at 37° C. for 40 minutes. After 40 minutes, the operator takes out from the incubator  75  the filtration cartridge  1  to which the first centrifugal filtration attachment  200  is connected. Then, the operator sets the filtration cartridge in the centrifuge  76  (zone of grade B) to perform centrifugal filtration. A centrifugal force (×g) can be equal to or less than 2000×g not to damage bacteria. Since a time required for centrifugation depends on a degree of clogging of the filter  21  by components of the test sample, it is necessary to grasp a time for filtering the entire amount of reagent A, in advance for each test target. The condition in the present embodiment is 1000×g for 1 minute. 
     After the centrifugation is stopped, the operator takes out from the centrifuge  76  the filtration cartridge  1  to which the first centrifugal filtration attachment  200  is connected. After spraying the filtration cartridge with alcohol to clean the filtration cartridge, the operator introduces the filtration cartridge into the safety cabinet  71 . Next, the operator opens the lid  41  of the second filter cup attachment  4  and confirms that there is no residual liquid. Here, when the liquid remains, the centrifugal filtration by the centrifuge  76  may be performed again after the lid  41  is closed to the fully closed position. 
     Step S 23 : Cleaning 
     In step S 23 , the operator cleans the filter  21  by using the reagent B of the Lumione test kit. Details of this step S 23  are as follows. 
     The operator opens the lid  41  of the second filter cup attachment  4  to add a predetermined addition amount of reagent B by a pipetting operation. Then, the operator closes the lid  41  of the second filter cup attachment  4  to the fully closed position. The operator takes out from the safety cabinet  71  the filtration cartridge  1  to which the first centrifugal filtration attachment  200  is connected. The operator sets the filtration cartridge in the centrifuge  76  (zone of grade B) to perform centrifugal filtration. A centrifugal force (×g) can be equal to or less than 2000×g not to damage bacteria. Since a time required for centrifugation depends on a degree of clogging of the filter by components of the test sample, it is necessary to grasp a time for filtering the entire amount of reagent B, in advance for each test target. The condition in the present embodiment is 1000×g for 1 minute. 
     After the centrifugation is stopped, the operator takes out from the centrifuge  76  the filtration cartridge  1  to which the first centrifugal filtration attachment  200  is connected. After spraying the filtration cartridge with alcohol to clean the filtration cartridge, the operator introduces the filtration cartridge into the safety cabinet  71 . Next, the operator opens the lid  41  of the second filter cup attachment  4  to confirm that there is no residual liquid. Here, when the liquid remains, the centrifugal filtration by the centrifuge  76  may be performed again. 
     Step S 24 : ATP Extraction and ATP Luminescence Measurement 
     In step S 24 , the operator extracts ATP by using the reagent C of the Lumione test kit to complete the preparation of the ATP measurement sample. Details of this step S 24  are as follows. 
     After opening the lid  41  of the second filter cup attachment  4  and confirming that there is no residual liquid, the operator opens the second centrifugal filtration attachment  300 . Next, the operator removes the first centrifugal filtration attachment  200  and then attaches the second centrifugal filtration attachment  300  to the filtration cartridge  1 . The operator opens the lid  41  and adds the reagent C in an addition amount of 50 μL defined in the manual of the kit by a pipetting operation. Then, the operator closes the lid  41  to the fully closed position to hold the reagent C at a normal temperature for several to 10 minutes in a state of being immersed in the filter  21 . 
     After the reaction with the reagent C, the operator takes out from the safety cabinet  71  the filtration cartridge  1  to which the second centrifugal filtration attachment  300  is connected. Then, the operator set the filtration cartridge in the centrifuge  76  (zone of grade B), to perform centrifugal filtration. A centrifugal force (×g) can be equal to or less than 2000×g not to damage bacteria. Since a time required for centrifugation depends on a degree of clogging of the filter by components of the test sample, it is necessary to grasp a time required for filtering the entire amount (50 μL) of reagent C such that 40 μL or more of reagent C can be collected by centrifugation, in advance for each test target. The condition in the present embodiment is 1000×g for 1 minute. 
     After the centrifugation is stopped, the operator takes out from the centrifuge  76  the filtration cartridge  1  to which the second centrifugal filtration attachment  300  is connected. After spraying the filtration cartridge evenly with alcohol to clean the filtration cartridge, the operator introduces the filtration cartridge into the safety cabinet  71 . Next, the operator opens the lid  41  of the second filter cup attachment  4  and confirms that there is no residual liquid. Here, when the liquid remains, the centrifugal filtration be the centrifuge  76  may be performed again. After confirming that there is no residual liquid in the filter  21 , the operator closes the lid  41  to the fully closed position and takes out the measurement tube  302  containing the reagent C reaction liquid from the second centrifugal filtration attachment  300 . Then, the operator inputs the measurement tube in the dedicated tube rack to cover the tube rack. 
     Finally, the operator introduces the tube rack into the measurement device  77  installed in grade B to perform ATP measurement. 
     Rapid Microbial Test Using Filtration Cartridge, Suction Filtration Attachment, and Centrifugal Filtration Attachment 
       FIG.  11    is a flowchart illustrating the rapid microbial test method using the filtration cartridge  1 , the suction filtration attachment  100  ( FIG.  5   ), the first centrifugal filtration attachment  200  ( FIG.  6 A ) and the second centrifugal filtration attachment  300  ( FIG.  6 B ). 
     This method is also test method using a funnel, and is suitable for filtering a large volume of test sample. In the reagent reaction using the reagent A, the reagent B, and the reagent C, centrifugal filtration has better liquid breakage and less carryover than suction filtration. Thus, there is an advantage that higher accurate and higher sensitive test can be performed in many cases. On the other hand, since centrifugal filtration applies a load to the filter  21  rather than suction filtration, it is necessary to consider a case where viable bacteria are damaged or bacteria are dead. Thus, it is necessary to determine centrifugation conditions in consideration of a centrifugal force (×g), a centrifugation time, a type of the test sample, and damage to a body cell to be detected. 
     Step S 31 : Bacteria Capturing 
     In step S 31 , the operator captures bacteria on the filter  21  of the filtration cartridge  1 . Details of this step S 31  are as follows. 
     First, the operator introduces into the safety cabinet  71  the test sample, the filtration cartridge  1 , the suction filtration attachment  100 , the first centrifugal filtration attachment  200 , the second centrifugal filtration attachment  300 , the funnel, the test reagents, and the sample and reagent dispensing tool. Then, the operator opens the microbial test filtration cartridge  1 , the suction filtration attachment  100 , and the funnel. Further, the operator removes the sealing material  35  of the filtration nozzle  31  to connect the filtration cartridge  1  and the suction filtration attachment  100 . The operator opens the lid  41  of the second filter cup attachment  4  to insert the funnel into the filter cup  2 . 
     Next, as preparation for the filtration step of the test sample, the operator pulls out only the pipe connector  103  attached to the suction filtration attachment  100  from the front shutter of the safety cabinet  71  to the zone of grade B. Then, the operator connects the pipe connector to the suction pump tube  74  in the zone of grade B to drive the suction pump. Next, in the safety cabinet  71 , the operator opens a cap of the funnel and injects the test sample into the funnel by a pipetting operation from the container containing the test sample or directly pours the test sample into the funnel from the container containing the test sample. When the test sample is directly poured, it is convenient to pour a required amount of test sample based on a scale of the funnel. After injecting a required amount of test sample into the funnel, the operator closes the lid to the semi-closed position in order to prevent contamination while creating a differential pressure with the cap of the funnel. Then, the operator opens the opening and closing valve  102  of the suction filtration attachment  100  to start the filtration of the test sample. After starting the filtration, the operator visually observes that the sample in the transparent or translucent container of the funnel decreases and visually observe the flow of the liquid in the suction pump tube  74 . 
     When it is determined that the entire amount of test sample is filtered, the operator removes the funnel and opens the lid  41  of the second filter cup attachment  4 , to confirm that the liquid does not remain on the filter  21 . Then, the operator closes the lid  41  to the fully closed position. Since there is no remaining amount, the operator determines that the filtration is completed. Then, the operator closes the opening and closing valve  102  of the suction filtration attachment  100  and disconnects the suction filtration attachment  100  and the suction pump  72  to stop the filtration. 
     The operation using the suction filtration attachment  100  has been described so far. In the operation, the liquid component of the test sample (for example, 100 mL or more of injection water) passes through the filter  21 . When there are bacteria in the filter  21 , the bacteria are concentrated and captured by the filter  21 . When the reaction of the reagent A in next step S 32  is performed, in order to avoid dilution of a reagent concentration caused by carryover by a minute amount of test sample as much as possible, the reagent A may be added after the residual liquid amount of the test sample is reduced as much as possible, by replacing the suction filtration attachment  100  with the first centrifugal filtration attachment  200 , and by adding the filtration step using the centrifugal force. 
     Step S 32 : ATP Activity Erasure Reaction 
     In step S 32 , the operator performs the ATP activity erasure reaction by using the reagent A of the Lumione test kit. Details of this step S 32  are as follows. 
     The operator opens the first centrifugal filtration attachment  200 , removes the suction filtration attachment  100 , and attaches the first centrifugal filtration attachment  200  to the filtration cartridge  1 . 
     Subsequent operations are similar to the operations in step S 22  illustrated in  FIG.  10   . Thus, description thereof is omitted. 
     Step S 33 : Cleaning 
     Step S 33  is similar to step S 23  illustrated in  FIG.  10   . Thus, description thereof is omitted. 
     Step S 34 : ATP Extraction and ATP Luminescence Measurement 
     Step S 34  is similar to step S 24  illustrated in  FIG.  10   . Thus, description thereof is omitted. 
     Conclusion of Fourth Embodiment 
     As described above, the filtration cartridge  1  can easily open and close the lid  41 . The operation of dispensing the test sample and the reagents is performed in the zone of grade A. In performing the operation using the device such as the centrifuge  76  or the incubator  75  used for a pre-treatment of a specimen, it is possible to bring the device in a direct support zone of grade B in grade A in a sealed state where the lid  41  is closed, or another support zone (grade C or D) in some cases. Accordingly, it is not necessary to bring the device such as the centrifuge  76  or the incubator  75  into the grade A. It is also possible to easily perform the sterile operation without increasing a risk of environmental break due to degradation of a completely sterile environment in grade A due to bringing the device. 
     In the fourth embodiment, although the microbial test method by the ATP luminescence measurement has been described, the microbial test method using the filtration cartridge  1  can also be applied to other rapid microbial test methods (solid phase cytometry, flow cytometry, immunological methods, nucleic acid amplification methods, fluorescence (staining) methods, and the like). 
     Fifth Embodiment 
     In a fifth embodiment, an automated device that automatically performs the microbial test method in  FIG.  8    is proposed. 
       FIG.  12    is a schematic diagram illustrating a microbial test apparatus  500  according to a fifth embodiment. As illustrated in  FIG.  12   , the microbial test apparatus  500  includes a control device  501 , a stage  502 , a tube rack  503 , a lid opening and closing lever  504 , a dispenser  505 , and a movable valve waste liquid port  506 . 
     The filtration cartridge  1  to which the suction filtration attachment  100  is connected is placed on the stage  502 . 
     A reagent tube  507  in which the reagent is accommodated and the measurement tube  302  in which the test sample is accommodated are arranged in the tube rack  503 . 
     The lid opening and closing lever  504  is configured to be movable in three axial directions by actuators  508  and  509 , and is used to open and close the lid  41  of the filtration cartridge  1 . 
     The dispenser  505  is configured to be movable in three axial directions by actuators  510  and  511 . The dispenser  505  dispenses the test sample from the measurement be  302  to the filtration cartridge  1 , and dispenses the reagent from the reagent tube  507  to the filtration cartridge  1 . 
     The movable valve waste liquid port  506  is provide with the opening and closing valve  102  of the suction filtration attachment  100 . The movable valve waste liquid port  506  is configured to be movable in a horizontal direction by an actuator  512 . The actuator  512  also controls the opening and closing of the opening and closing valve  102 . 
     The control device  501  controls operations of the actuators  508  to  512  based on a predetermined instruction value. 
     The microbial test apparatus  500  can be installed, for example, in the above-described zone of grade A. The microbial test apparatus  500  can be defined as an apparatus that performs only operations to be performed in the zone of grade A (for example, the operation of dispensing the test sample and the reagent), but may be defined as an apparatus that can perform operations that can be performed in the zone of grade B. 
     Microbial Test Method 
     The microbial test method according to the present embodiment is substantially similar to the method illustrated in  FIG.  8   . The control device  501  operates the actuators  508  to  512  to perform the operations by the operator. 
     Conclusion of Fifth Embodiment 
     As described above, the microbial test apparats  500  according to the fifth embodiment automatically performs the microbial test using the filtration cartridge  1  and the suction filtration attachment  100 . As a result, since the number of manual operations by the operator is reduced, the microbial test is more convenient. 
     Sixth Embodiment 
     In a sixth embodiment, an automated device that automatically performs the microbial test method in  FIG.  10    is proposed. 
       FIG.  13    is a schematic diagram illustrating a microbial test apparatus  600  according to a sixth embodiment. As illustrated in  FIG.  13   , in the microbial test apparatus  600 , a control device  501 , a tube rack  503 , a lid opening and closing lever  504 , a dispenser  505 , and actuators  508  to  511  are similar to the components of the microbial test apparatus  500  ( FIG.  12   ) according to the fifth embodiment. 
     The microbial test apparatus  600  includes a centrifugal filtration attachment rack  601  and a swing rotor  602 . The centrifugal filtration attachment rack  601  holds the first centrifugal filtration attachment  200  not illustrated) and the second centrifugal filtration attachment  300 . The swing rotor  602  holds the filtration cartridge  1 . The centrifugal filtration attachment rack  601  and the swing rotor  602  are configured to be rotatable. The centrifugal filtration attachment rack  601  is configured to be vertically movable. The swing rotor  602  is configured to change an orientation of the filtration cartridge  1 . The driving of the centrifugal filtration attachment rack  601  and the swing rotor  602  is controlled by the control device  501 . 
     When the filtration cartridge  1  is attached to the first centrifugal filtration attachment  200  and the second centrifugal filtration attachment  300 , the centrifugal filtration attachment rack  601  is raised in a state where the second filter cup attachment  4  is aligned with the first fitting portion  201  and the second fitting portion  301 . 
     When a centrifugal operation of the filtration cartridge  1  is required, the lid of the filtration cartridge  1  to which the second centrifugal filtration attachment  300  is attached is set to the fully closed position. Then, an orientation of the filtration cartridge  1  is changed in the horizontal direction and the filtration cartridge  1  is rotated by the swing rotor  602 . As a result, since a centrifugal force is generated in a radial direction of the swing rotor  60 , the liquid added to the filtration cartridge  1  is filtered. As described above, when the centrifugal filtration is performed, since it is not necessary to move the filtration cartridge  1  to the centrifuge, there is no risk of contamination associated with the movement, and the operation is also simplified. 
     Microbial Test Method 
     The microbial test method according to the present embodiment is substantially similar to the method illustrated in  FIG.  10   . The operations by the operator are executed by the control device  501  operating the actuators  508  to  511 , the centrifugal filtration attachment rack  601 , and the swing rotor  602 . 
     Conclusion of Sixth Embodiment 
     As described above, the microbial test apparatus  600  according to the sixth embodiment automatically performs the microbial test using the filtration cartridge  1  and the second centrifugal filtration attachment  300 . As a result, since the number of manual operations by the operator is reduced, the microbial test becomes more simplified. 
     Modification Examples of Fifth and Sixth Embodiments 
     Although not illustrated, the microbial test method (method using both the suction filtration attachment  100  and the centrifugal filtration attachments  200  and  300 ) illustrated in  FIG.  11    can also be performed by using the microbial test apparatus including both the configuration of the fifth embodiment and the configuration of the sixth embodiment. 
     Modification Example 
     The present disclosure is not limited to the above-described embodiments, and includes various modification examples. The aforementioned embodiments are described in detail in order to facilitate easy understanding of the present disclosure, and are not limited to necessarily include all the described components. A part of a certain embodiment can be replaced with a configuration of another embodiment. The configuration of another embodiment can be added to the configuration of a certain embodiment. A part of the configuration of another embodiment can be added to, deleted from, or replaced with a part of the configuration of each embodiment. 
     REFERENCE SIGNS LIST 
       1  filtration cartridge 
       2  filter cup 
       21  filter 
       22  flange 
       23  container portion 
       24  support plate 
       3  first filter cup attachment 
       31  filtration nozzle 
       32  body portion 
       33  attachment joint hole 
       34  filter cup insertion portion 
       35  sealing material 
       36  recessed portion 
       37  protrusion portion 
       4  second filter cup attachment 
       41  lid 
       42  body portion 
       43  filter cup introduction hole 
       44  flange holding portion 
       45  first fixing member 
       46  lid fixing hole 
       47  second fixing member 
       48  knob 
       49  protrusion portion 
       50  clay-like connector 
       51  tubular connector 
       52  hinge mechanism 
       53  knob 
       70  biological clean room 
       71  safety cabinet 
       72  suction pump 
       73  waste liquid trap 
       74  suction pump tube 
       75  incubator 
       76  centrifuge 
       77  measurement device 
       78  pass box 
       79  sterile operation zone 
       100  suction filtration attachment 
       101  support block 
       102  opening and closing valve 
       103  pipe connector 
       104  flow path 
       200  first centrifugal filtration attachment 
       201  first fitting portion 
       202  waste liquid tube 
       300  second centrifugal filtration attachment 
       301  second fitting portion 
       302  measurement tube 
       500  microbial test apparatus 
       600  microbial test apparatus