Filtration assemblies, cassettes, systems, and methods for filtration and cell growth

Abstract

To provide new filtration and culturing devices for microbial enumeration.SOLUTION: A filtration assembly comprising: a) a funnel, b) a membrane frame comprising a porous membrane, and c) a base comprising a membrane support and an outlet, wherein the membrane frame is releasably attached to the funnel, the base is releasably attached to the funnel, during filtration liquid flows from the funnel through the membrane and membrane support to the outlet, and the membrane support keeps the membrane flat during filtration.SELECTED DRAWING: Figure 2

Description

[Background technology]

[0001] Many industries require the determination of the number of microorganisms in a sample. One method for determining the number of microorganisms in a sample involves capturing the microorganisms on a membrane, culturing the microorganisms on the membrane, and counting the number of colonies formed. Manipulation of the membrane can introduce non-sample microbial contaminants or microbial debris, or damage or create defects in the membrane, all of which can interfere with enumeration.

[0002] Therefore, there is a need for new filtration and culture devices for microbial enumeration. Summary of the Invention

[0003] The present invention provides devices, systems, and kits for filtering cells from a sample solution and then culturing the captured cells, for example, for imaging and counting colony forming units (CFUs). The filtration assemblies, cassettes, systems, and methods of the present invention are particularly suitable for incorporation into automated counting systems and processes.

[0004] In a first aspect, the present invention provides a filtration assembly including a funnel, a membrane frame including a porous membrane, and a base having a membrane support and an outlet. The membrane frame is releasably attached to the funnel, and the base is releasably attached to the funnel. During filtration, liquid flows from the funnel through the membrane and membrane support to the outlet. In some embodiments, the membrane support keeps the membrane flat during filtration.

[0005] In some embodiments, the funnel is attached to the base by a locking mechanism that releases when a portion of the outer wall of the base is pressed. In certain embodiments, the porous membrane is ultrasonically bonded or heat staked to the membrane frame. In other embodiments, the membrane frame is releasably attached to the funnel by a mating arrangement of a raised lip on the funnel and a recess in the membrane frame. In certain embodiments, the membrane frame includes a protruding ring that mates with a tab on the cassette base. In some embodiments, the membrane is black and / or non-fluorescent. In certain embodiments, the membrane is a black mixed cellulose ester membrane.

[0006] In some embodiments, the funnel is transparent and / or includes volumetric markings. In certain embodiments, the filtration assembly also includes a funnel lid that covers the opening to the funnel. In some embodiments, the funnel lid is hingedly attached to the funnel. In some embodiments, the membrane support is attached to the filter base. In certain embodiments, the filter base includes a plurality of supports configured to facilitate fluid flow to the outlet and / or to support the membrane support. In some embodiments, the membrane frame may also include fiducial markings.

[0007] In some embodiments, the filtration assembly further includes a washer. In certain embodiments, the washer is attached to the membrane support and / or is a thin film. In certain embodiments, the washer and the membrane support are a single molded part. In some embodiments, the washer includes a spring. In some embodiments, the washer and the membrane support are a single molded part.

[0008] In certain embodiments, the filtration assembly includes a cavity and / or a shim between the membrane and the membrane support. In certain embodiments, the membrane support and / or base are shaped to create the cavity.

[0009] A second aspect of the present invention provides a cassette base having a base layer including an outer wall, a first inner wall including a plurality of radially arranged gaps, and a second inner wall. The second inner wall has an outer ledge and defines a well, with the first inner wall disposed between the outer wall and the second inner wall. The cassette also includes a solid or semi-solid nutrient medium in the well. The medium has a flat growth area that is taller than the second inner wall. The cassette also includes a cassette lid that is releasably sealable to the base.

[0010] In some embodiments, the cassette lid is optically clear and non-fluorescent. In some embodiments, the cassette base is non-fluorescent. In certain embodiments, the cassette further comprises a third inner wall disposed between the first inner wall and the outer wall. In certain embodiments, the cassette also comprises a fourth inner wall between the third inner wall and the outer wall.

[0011] In some embodiments, the second interior wall includes a plurality of supports that project radially toward the first interior wall.

[0012] In some embodiments, the cassette includes a washer. In certain embodiments, the washer is attached to the cassette base and / or is a membrane. In certain embodiments, the washer and the cassette base are a single molded part. In some embodiments, the washer includes a spring.

[0013] In some embodiments, the cassette base is positioned to receive a membrane lowered onto the base at an angle of 1° to 75° relative to the flat growth area. In certain embodiments, the cassette base includes a ring supported by a plurality of springs arranged in a ring shape, with the tallest spring of the plurality of springs antipodal to the shortest spring of the plurality of springs, and the springs positioned therebetween tapered in height. In some embodiments, the cassette base includes features that engage with the membrane frame to create an initial angle of 1° to 75° between the membrane and the flat growth area.

[0014] Another aspect of the present invention provides a system for filtering and culturing cells. The system can include the filtration assembly embodiment of the first aspect and the cassette embodiment of the second aspect. The membrane frame is configured to detach from the filter base along with the funnel, attach to the cassette base, and detach from the funnel when attached to the cassette base.

[0015] In some embodiments of the system, the membrane frame includes a protruding ring and the cassette base includes tabs that mate with the protruding ring, hi some embodiments, when the membrane frame is attached to the cassette base, the membrane is placed in conformal contact with the flat growth area.

[0016] A further aspect of the present invention provides a method for determining the presence of microorganisms. The method includes attaching a filtration assembly according to any embodiment of the first aspect to a vacuum source. The method further includes flowing a sample fluid through the filtration assembly so that any cells in the fluid are retained on the membrane; removing a funnel attached to a membrane frame from the base; attaching the membrane frame to a cassette base according to the second aspect; and removing the funnel from the membrane frame. The membrane may then be incubated.

[0017] In some embodiments, the method further comprises imaging the membrane to detect any colonies formed from the retained cells.

[0018] In some embodiments, the filtration assembly includes a cavity, and the method includes applying pressure, e.g., from a vacuum, to cause the membrane to conform to the shape of the membrane support and / or cavity. In certain embodiments, the membrane maintains the shape of the membrane support and / or cavity after the funnel is removed from the base. In certain embodiments, the shaped membrane interacts with the cassette to prevent air bubble trapping during attachment of the membrane to the cassette base.

[0019] In some embodiments, the membrane initially contacts the flat growth area at a first point on its circumference and finally contacts the flat growth area at a second point on the circumference that is antipodal to the first point. In some embodiments, the membrane frame is initially lowered onto the base at an angle of 1° to 75° relative to the flat growth area. In certain embodiments, the cassette includes a feature (configuration) that engages with the membrane frame to position the membrane to contact the flat growth area at an angle of 1° to 75° relative to the flat growth area when the membrane frame is lowered onto the cassette base. In certain embodiments, the feature includes a tapering ring supported by a plurality of springs arranged in an annular shape, the tallest spring of the plurality of springs being antipodal to the shortest spring of the plurality of springs, and the plurality of springs arranged therebetween having tapering heights.

[0020] It is understood that the filtration assemblies, cassettes, systems, and methods described herein may include additional features beyond those specified herein, including any that are not inconsistent with the structure of the underlying filtration assembly, cassette, system, or method.

[0021] The term "about" as used herein refers to ±10% of the recited value. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows the filtration of the sample followed by transfer of the membrane to the base of the cassette for cell growth. [Figure 2] Figure 2A is a schematic diagram showing a funnel of the present invention with a lid, and Figure 2B is a schematic diagram showing an exploded view of a filtration assembly of the present invention, featuring a funnel with a funnel lid, a membrane frame with a membrane, and a base. [Figure 3] FIG. 3 is a schematic diagram showing (from left to right) an example of a membrane frame without a membrane, a filtration assembly base and membrane support (e.g., a flat sintered thermoplastic polymer disc, such as Porex®), and a cassette base. [Figure 4]FIG. 4 is a schematic diagram showing a cross section of the funnel, membrane frame, and base of the present invention when installed. [Figure 5] FIG. 5 is a schematic diagram showing a close-up of the funnel, membrane frame, and base. [Figure 6] FIG. 6 is a schematic diagram of an embodiment of the present invention featuring a transparent funnel with volumetric markings and a base featuring a push button on the outer wall of the base to release the funnel from the base. [Figure 7] Figure 7A is a schematic diagram of a stacked filtration assembly of the present invention without a lid, and Figure 7B is a schematic diagram of a stacked filtration assembly of the present invention with a lid. [Figure 8] Figure 8A is a schematic diagram of a filtration assembly of the present invention featuring an injection molded lid, and Figure 8B is a schematic diagram of a filter of the present invention featuring a thermoformed lid. [Figure 9] FIG. 9 shows a filtration assembly of the present invention having three different lids. [Figure 10] FIG. 10 is a schematic diagram showing a filtration assembly of the present invention having a hinged lid. [Figure 11] 11A-11B are schematic diagrams illustrating a staggered arrangement of filtration assemblies of the present invention. [Figure 12] 12A-12B are schematic diagrams illustrating a staggered arrangement of the filtration assembly of the present invention compared to a rectangular arrangement. [Figure 13] 13A-13B are schematic diagrams illustrating the staggered arrangement of filtration assemblies of the present invention on a tray of the present invention. [Figure 14] FIG. 14 is a schematic diagram showing a cross section of a filtration assembly with a funnel, membrane frame, and base as installed. [Figure 15] Figure 15A is a schematic diagram showing the top and bottom views of a funnel of the present invention, Figure 15B is a schematic diagram showing the top and bottom views of a base of the present invention, and Figure 15C is a schematic diagram showing the top and bottom views of a membrane frame (without membrane) of the present invention. [Figure 16]Figure 16A is a schematic diagram showing top and bottom views of a cassette base of the present invention, and Figure 16B is a schematic diagram showing a cross section of a funnel, membrane frame, and cassette base of the present invention as installed. [Figure 17] FIG. 17 is a schematic diagram showing a cross section of a filtration assembly with a funnel, membrane frame, and base as installed. [Figure 18] Figure 18A is a schematic diagram showing the top and bottom views of a funnel of the present invention, Figure 18B is a schematic diagram showing the top and bottom views of a base of the present invention, and Figure 18C is a schematic diagram showing the top and bottom views of a membrane frame (without membrane) of the present invention. [Figure 19] Figure 19A is a schematic diagram showing top and bottom views of a cassette base of the present invention, and Figure 19B is a schematic diagram showing a cross section of the funnel, membrane frame, and cassette base as installed. [Figure 20] FIG. 20 is a schematic diagram showing a close-up cross section of the funnel, membrane frame, and cassette base as they are installed. [Figure 21] FIG. 21 is a schematic diagram showing a close-up cross section of a portion of the filtration assembly, including a view of the funnel, membrane frame, and base as installed, and showing the seals and various tabs. [Figure 22] FIG. 22 is a schematic diagram showing a close-up cross section of the funnel, membrane frame, and cassette base of the present invention, including features on the cassette base for releasing tabs on the membrane frame. [Figure 23] FIG. 23 is a diagram of a cassette base of the present invention with a solid or semi-solid nutrient medium and a membrane in a membrane frame showing (top to bottom) that the membrane in the membrane frame is positioned at an angle on the solid or semi-solid nutrient medium. [Figure 24] FIG. 24 is a schematic diagram showing a cross section of a filtration assembly of the present invention. [Figure 25] FIG. 25 shows a cassette base of the present invention with a solid or semi-solid nutrient medium and a washer. [Figure 26]Figure 26A shows a partial cross section of the present invention during the filtration step of the method of the present invention, and Figure 26B shows the attachment of the membrane in the membrane frame to the cassette base. [Figure 27] FIG. 27 illustrates various advantageous features of the present invention. [Figure 28] Figures 28A-28D show various washers of the present invention. Figure 28A shows the washer as a part attached (e.g., by mechanical interengagement) to a membrane ring. Figure 28B shows the washer as a thin film attached (e.g., by mechanical interengagement) to a membrane frame. Figure 28C shows the washer and membrane frame as a single molded part. Figure 28D shows the washer as a thin film bonded (e.g., thermally) to the membrane frame. [Figure 29] Figures 29A-29C are diagrams of a washer of the present invention attached to a cassette base. Figure 29A shows the washer as an attached part that contacts the solid or semi-solid nutrient medium. Figure 29B shows the washer as a membrane attached to the cassette and in contact with the solid or semi-solid nutrient medium. Figure 29C shows the washer as a spring attached to the cassette. [Figure 30] 30A and 30B are photographs of a membrane of the present invention on a filtration assembly base of the present invention with an angled shim. [Figure 31] FIG. 31 is a photograph of a cassette base of the present invention that includes angled spring loaded elements to control the placement of the membrane during its transfer to the cassette base. [Figure 32] FIG. 32 is a schematic diagram showing a partial cutaway view of a filtration assembly of the present invention including a washer of the present invention. [Figure 33]33 is a schematic diagram showing a cross section of a cassette base of the present invention with a membrane in a membrane frame of the present invention with a thin film (e.g., 0.1 mm) of the present invention and a thicker (e.g., 0.45 mm) washer before and after laying the membrane on the solid or semi-solid nutrient medium in the cassette base. The thin film (e.g., 0.1 mm thick) conforms to the pressure of the membrane. The thicker (e.g., 0.45 mm) washer conforms to the pressure of the membrane, takes the shape of a Belleville washer, and bites into the solid or semi-solid nutrient medium. The thicker (e.g., 0.80 mm washer) is stiff and less conforms to the membrane under pressure, causing excessive stretching and biting into the solid or semi-solid nutrient medium. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention The present invention provides filtration assemblies, cassettes, systems, and methods for microbiological growth enumeration, e.g., of bacteria, fungi, and archaea. The devices, systems, and methods of the present invention are particularly suited to automated enumeration, allowing for the transfer of cells between components of the membrane that retains the cells in a manner that reduces the likelihood of damage to or fouling of the membrane.

[0024] Filtration Assembly One device of the present invention is a filtration assembly for filtering a sample and retaining any microorganisms that may be found therein (see, e.g., Figures 2B, 14, or 17). The filtration assembly of the present invention features a funnel (e.g., Figures 2A, 15A, or 18A), a membrane frame (e.g., the ring shown in Figures 3, 15C, or 18C) containing a porous membrane (e.g., a mixed cellulose ester membrane), and a base (e.g., Figures 3, 15B, or 18B) having a membrane support (e.g., a flat disk or a shaped disk with a flat portion of a sintered thermoplastic polymer) and an outlet (e.g., for connection to a vacuum source). The membrane frame is releasably attached to the funnel (e.g., by interlocking or overlapping features or a friction or jam fit, see, e.g., Figures 4 or 21), and the base is releasably attached to the funnel (e.g., with a locking mechanism). During filtration, liquid flows from the funnel through the membrane and membrane support to the outlet. The construction of the assembly allows the membrane frame to be removed from the base while still attached to the funnel for transfer to the cassette base in a manner that avoids direct handling of the membrane or membrane frame.

[0025] In some embodiments, the membrane support keeps the membrane flat during filtration, for example, by having a height that keeps the membrane support and membrane in conformal contact. Suitable materials that can be sintered to manufacture the membrane support include, for example, high-density or ultra-high-molecular-weight polyethylene, polyethersulfone, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, etc. Other thermoplastic polymers known in the art may also be used. In some embodiments, the membrane support is attached to the filter base (e.g., by adhesive or interlocking features), which prevents the membrane support from being removed along with the funnel and membrane frame. The membrane support may also be directly incorporated into the base, for example, during base manufacture. Alternatively, the membrane support allows the membrane to deform during filtration, for example, to fit into cavities in the membrane support.

[0026] In some embodiments, the funnel is attached to the base by a locking mechanism that releases when a portion of the exterior wall of the base is pressed (e.g., an unlatching button, e.g., FIG. 6). In one example, the exterior portion of the base wall may include a flexible hinge with an inward protrusion (e.g., a tab) that latches onto an outwardly protruding feature (e.g., a ring or ledge) on a portion of the exterior wall of the funnel, and pressing the hinge retracts the latch, releasing the funnel from the base. The hinge may also feature a post. Other suitable locking mechanisms include a deformable tab or catch that can be separated by pulling the funnel and base apart.

[0027] In certain embodiments, the porous membrane is ultrasonically bonded or heat-staked to the membrane frame. Other suitable bonding methods include adhesive bonding, mechanical retention, and the like. The membrane frame may be releasably attached to the funnel, for example, by a mating arrangement of a raised lip on the funnel and a recess in the outer wall of the membrane frame (e.g., FIG. 4). In certain embodiments, the membrane frame may include a protruding ring that mates with a tab on the cassette base. Alternatively, the tab may be on the membrane frame (e.g., FIG. 15C or FIG. 18C) and the ring may be on the cassette base (e.g., FIG. 20). Locking features (e.g., tabs or rings) on the membrane frame may be configured to hold the membrane frame more strongly to the funnel than to the base of the filtration assembly. The locking features (locking configurations) on the membrane frame may be the same features as the cassette base for attachment of the filtration assembly to the base, but the complementary feature(s) on the filtration assembly base may provide a weaker interaction than the corresponding feature(s) on the cassette base. The locking features (e.g., tabs) on the membrane frame may also be configured to interact with features on the cassette base that act to disengage the locking features from the funnel. The locking features on the membrane frame may be antipodal, for example, configured to first engage the cassette base with one locking feature and finally engage with a locking feature that is antipodal to the first locking feature.

[0028] The membrane frame includes a porous membrane on which cells are retained during filtration (see, e.g., Figure 2B). The membrane may be black and / or non-fluorescent so as not to interfere with imaging and counting. An exemplary membrane is a black mixed cellulose ester membrane. Other suitable membrane materials include, for example, cellulose, cellulose acetate, ethylene vinyl acetate, polystyrene, nitrocellulose, polyether ether ketone, nylon, polyolefins (e.g., polyethylene or polypropylene), polyacrylonitrile, polyethylene terephthalate (PET), polyethersulfone, track-etched polyester or polycarbonate, polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, and silicone copolymers. The membrane may also feature a surface coating, for example, to enable or promote cell attachment or colony growth. The choice of material and / or coating may depend on the type of cells expected to be retained. The membranes of the present invention have pore sizes sized to prevent the passage of microorganisms such as bacteria or yeast, e.g., about 0.45 μm, e.g., about 0.1-1 μm (e.g., 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, or 1 μm) (depending on the application). The membrane frame on the outside of the membrane may be made of any suitable material, e.g., plastic or metal, that allows for attachment to a filter and base.

[0029] The funnel may be transparent and / or include volumetric markings to help the user add the correct amount of sample fluid and enable volumetric data generation (i.e., CFU count per unit volume of sample fluid). The funnel may also include a seal on the inside (e.g., FIG. 2A) that presses against the membrane and defines the region of interest (e.g., FIG. 5), defining the area where cells may be found. The membrane may separate from the seal when the funnel and membrane frame are removed from the base, for example, to prevent the seal from interacting with the nutrient medium when the membrane frame is attached to the cassette base. The filtration assembly may also include a funnel lid that covers the opening to the funnel, which may be hinged onto the funnel itself. The funnel lid may serve to protect the membrane surface from contamination during storage or use. A hinged lid may be formed from a separate lid and funnel, for example, with each lid and funnel having features that clip together to form a hinge (see, e.g., FIG. 10). Such a configuration has advantages for storage and transport of multiple filtration assemblies, since they can be stacked more efficiently without the lid in the cover position (see, e.g., Figures 7A-7C). The lid of the present invention may be thermoformed (e.g., Figure 8B or Figure 9) or injection molded (e.g., Figure 8A or Figure 9) and may be of the same material as the funnel or another suitable material. The funnel and lid may be formed of any suitable material, such as plastic or metal.

[0030] The base connects the filtration assembly to a vacuum source, for example, via a tulip. Bases of the present invention may be configured to connect, for example, to a tulip valve, which allows fluid communication of the outlet to the vacuum source. In certain embodiments, the filter base includes a plurality of supports configured to facilitate fluid flow to the outlet and / or to support the membrane support. The filtration base may include features for connecting to a vacuum source, for example, a groove to receive the edge of a tulip valve or another sealing member.

[0031] Because the end use of the membrane frame may be for imaging colony growth, the membrane frame or membrane may also include fiducial markings to help maintain alignment of multiple images, for example over an incubation time series. The fiducial markings may also aid in alignment with a robotic handling system.

[0032] The membrane frame may be tilted at an angle of 1° to 75° (e.g., about 1° to 5°, about 1° to 10°, about 1° to 15°, about 10° to 15°, about 15° to 25°, about 20° to 30°, about 25° to 35°, about 30° to 40°, about 35° to 45°, about 40° to 50°, about 45° to 55°, about 50° to 60°, about 55° to 65°, about 60° to 70°, or about 65° to 75°), for example, about 1° to 37.5°, about 10° to 4 It may be configured (e.g., by having a positioning feature) to be initially lowered onto the base at an angle of 0°, about 15° to 45°, about 22.5° to 67.5°, about 40° to 70°, about 25° to 75°, or about 35° to 75°, for example, at least 5°, 10°, 15°, 20°, 25°, or 30°, for example, about 22.5°, about 30°, about 45°, about 60°, or about 67.5°.

[0033] The filtration assembly may include a washer disposed under the membrane, for example, around the inner edge of the membrane frame (see, e.g., Figures 28A-28D and 32). The washer may be a single element, e.g., an annular ring or partial ring (e.g., C-shaped), or multiple elements arranged annularly, e.g., as discontinuous rings. The washer may be, for example, plastic, e.g., Teflon, polypropylene, polyethylene, polyethylene terephthalate, polyester, polycarbonate, etc. The washer in the filtration assembly may be part of the membrane frame, e.g., attached to the membrane frame (e.g., by mechanical engagement or sealing, e.g., by adhesive or thermal bonding), or formed with the membrane frame (e.g., molded, e.g., as a single piece). The washer may reduce air bubble trapping under the membrane (e.g., during transfer to a cassette). The washer, which is part of the membrane support, may improve membrane flatness (e.g., when stretched on a solid or semi-solid nutrient medium). Washers of the present invention may include fluorescent material to show through as fiducial markings, for example, through holes in the membrane or membrane frame. The washers may be thin, e.g., less than about 0.2 mm thick, e.g., 0.1-0.2 mm, 0.5-0.15 mm, 0.01-0.05 mm, or about 0.05 mm, 0.07 mm, 0.1 mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, or about 0.15 mm thick. The washer may be a thicker, harder ring, e.g., thicker than 0.2 mm, e.g., about 0.2-0.3 mm, 0.25-0.35 mm, 0.3-0.4 mm, 0.35-0.45 mm, 0.4-0.5 mm, 0.45-0.55 mm, 0.5-0.6 mm, 0.55-0.65 mm, 0.6-0.7 mm, 0.65-0.75 mm, 0.7-0.8 mm, 0.75-0.85 mm, 0.8-0.9 mm, 0.85-0.95 mm, or 0.9-1 mm thick. The washer may be flat. The washer may be smooth. The washer may allow for reduced friction when the membrane is installed on the cassette base.

[0034] The filtration assemblies of the present invention may be configured to stretch the membrane so that it conforms to a flat surface (e.g., the surface of a membrane support) (see, e.g., Figures 24 and 26A). The filtration assembly may include a cavity between the membrane support and the membrane, into which the membrane is drawn when filtration occurs. The membrane support may be shaped to create the cavity, for example, with an edge that tapers upward around a central flat disk region. The inside of the base may similarly taper to accommodate or create the cavity. A shim in the base may shape the cavity. The shape of the membrane after filtration may be determined in whole or in part by the shim (see, e.g., Figures 30A and 30B). The shim may be an addition to the filtration base (e.g., resting on or attached to the filtration base, e.g., by mechanical engagement or sealing, e.g., by adhesive or thermal bonding), or may be part of the filtration base (e.g., molded together with the filtration base).

[0035] cassette The present invention provides cassettes for cell growth, e.g., on membranes described herein. The cassettes of the present invention include a cassette base having a base layer including an outer wall, a first inner wall including multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) radially arranged gaps, and a second inner wall (see, e.g., Figures 3, 16A, or 19A). The first inner wall is positioned between the second inner wall and the outer wall. The second inner wall defines a well for holding a solid or semi-solid nutrient medium. The second inner wall also includes an outer ledge, which may allow the well to be completely filled with nutrient medium. The solid or semi-solid medium, having a flat growth area that is higher than the second inner wall, ensures that the membrane remains flat for imaging by conformally pressing the membrane against the medium when the frame is attached to the base. The cassette base layer can also have one or more holes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) between the first inner wall and the outer wall (e.g., Figure 3 or Figure 19A). These holes can help prevent air bubbles from being trapped under the membrane when the membrane is attached to the cassette base. Air bubbles trapped between the membrane and medium can not only prevent areas of the membrane from receiving nutrients, but can also distort the membrane and disrupt imaging. The holes may also receive one or more tabs on the membrane frame as part of a locking mechanism between the membrane frame and the cassette base (e.g., see Figure 19B). The cassette base may include a feature that acts to disengage the interlocking features of the membrane frame and funnel when the funnel and membrane frame are pressed onto the cassette base (e.g., see Figures 21 and 22). The cassette also includes a cassette lid that can be releasably sealed to the base.

[0036] In some embodiments, the second interior wall can include multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) supports projecting radially toward the first interior wall. Such radial supports can help retain medium filled to the ledge and increase the diameter of the flat growth area. Other interior walls can also feature multiple supports projecting radially toward the outer wall, for example, to support a membrane frame.

[0037] The cassette may also have a third interior wall disposed between the first interior wall and the outer wall, and may further have a fourth interior wall between the third interior wall and the outer wall (see, e.g., FIG. 3). The cassette may include additional interior walls (e.g., 5, 6, 7, 9, or 10 or more). In some embodiments, one or more of the interior walls are configured to interact with a locking mechanism (e.g., tabs) between the membrane frame and the funnel to disengage the locking mechanism and release the funnel.

[0038] The solid or semi-solid nutrient medium may be any suitable medium. Examples include Sabouraud dextrose agar (SDA), R2A agar, tryptic soy agar (TSA), letheen, and plate count agar (PCA). Other media are known in the art. The flat growth area is typically at least 5 mm in diameter, e.g., 5 to 200 mm, e.g., 10 to 80 mm.

[0039] The cassette lid may be thermoformed or injection molded. The cassette lid is preferably optically transparent (e.g., to detect cells by light) and / or non-fluorescent (e.g., to allow detection of cells by autofluorescence), e.g., a cyclic olefin polymer lid. Other suitable transparent lid materials include PET, polymethyl methacrylate, ethylene tetrafluoroethylene, polystyrene, etc. The cassette may also include a cover lid, e.g., a flexible polymer (e.g., rubber) lid, to protect the cassette during transport.

[0040] The cassette base is preferably made of a non-fluorescent material, such as a black styrene-butadiene-copolymer, to prevent interference with imaging.

[0041] The cassette may include a washer (see, e.g., Figures 29A-29C and 33) on or connected to the second inner wall (e.g., positioned between the membrane and the second inner wall and / or the solid or semi-solid nutrient medium). The washer in the cassette may be a single element, e.g., an annular ring or partial ring (e.g., C-shaped), or multiple elements arranged annularly, e.g., as discontinuous rings. The washer may be, e.g., plastic, e.g., Teflon, polypropylene, polyethylene, polyethylene terephthalate, polyester, polycarbonate, etc. The washer in the cassette may be part of the cassette base, e.g., attached to the cassette base (e.g., by mechanical engagement or sealing, e.g., by adhesive or thermal bonding), or formed with the cassette base (e.g., molded, e.g., as a single piece). The washer may reduce air bubble trapping between the membrane and the solid or semi-solid nutrient medium. Washers can improve membrane flatness (e.g., when stretched over solid or semi-solid nutrient media). Washers that are part of the cassette base can improve membrane flatness (e.g., when stretched over solid or semi-solid nutrient media). Washers of the present invention can include fluorescent material to show through as fiducial markings, for example, through holes in the membrane or membrane frame. Washers can be thin (e.g., see FIG. 29B), e.g., less than about 0.2 mm thick, e.g., 0.1-0.2 mm, 0.5-0.15 mm, 0.01-0.05 mm, or about 0.05 mm, 0.07 mm, 0.1 mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, or about 0.15 mm thick. The washer may be a thicker (see, e.g., FIG. 29A), stiffer ring, e.g., thicker than 0.2 mm, e.g., about 0.2-0.3 mm, 0.25-0.35 mm, 0.3-0.4 mm, 0.35-0.45 mm, 0.4-0.5 mm, 0.45-0.55 mm, 0.5-0.6 mm, 0.55-0.65 mm, 0.6-0.7 mm, 0.65-0.75 mm, 0.7-0.8 mm, 0.75-0.85 mm, 0.8-0.9 mm, 0.85-0.95 mm, or 0.9-1 mm thick.The washer may be flat. The washer may be smooth. The washer may be a spring-loaded mounted part (see, e.g., FIG. 29C) (e.g., arranged to bend downward toward the nutrient medium under the pressure of the depressed membrane). If the washer is thin (e.g., about 0.1 mm thick), it may conform to the shape of the membrane and / or nutrient medium. If it is thicker, e.g., about 0.45 mm thick, the washer may conform to the pressure of the washer, take the shape of a Belleville washer, and bite into the nutrient medium. If the washer is thicker, e.g., about 0.8 mm, it may be less able to conform to the pressure of the membrane and bite into the nutrient medium. The washer may allow for reduced friction when the membrane is installed on the cassette base.

[0042] The cassette base may be configured to engage with the membrane frame such that the membrane frame and membrane are angled (e.g., 1°-75°) when the membrane contacts the solid or semi-solid nutrient medium. For example, the cassette base may include angled features such as angled slots, tabs, catches, cams, latches, or hooks that engage with the membrane frame at an angle relative to the surface of the medium.

[0043] The cassette base is tilted at an angle of 1° to 75° (e.g., about 1° to 5°, about 1° to 10°, about 1° to 15°, about 10° to 15°, about 15° to 25°, about 20° to 30°, about 25° to 35°, about 30° to 40°, about 35° to 45°, about 40° to 50°, about 45° to 55°, about 50° to 60°, about 55° to 65°, about 60° to 70°, or about 65° to 75°), for example, about 1° to 37.5°, about 10° to 40°, The membrane frame may be configured (e.g., by having positioning features) so that it can be initially lowered onto the base at an angle of about 15° to 45°, about 22.5° to 67.5°, about 40° to 70°, about 25° to 75°, or about 35° to 75°, e.g., at least 5°, 10°, 15°, 20°, 25°, or 30°, e.g., about 22.5°, about 30°, about 45°, about 60°, or about 67.5°.

[0044] In some embodiments, the cassette base may include rings (see, e.g., FIG. 31) with springs of various heights (e.g., positioned so that one side of the ring contacts the membrane or membrane frame before its antipodal point, thereby forcing the opposite side of the membrane to contact the nutrient medium first).

[0045] The cassette base, together with the membrane frame and solid or semi-solid medium, can act to stretch the membrane when the membrane frame is attached to the cassette base. The washers of the present invention can also help stretch the membrane.

[0046] Systems and Kits The present invention provides a system for filtering and culturing cells, e.g., for microbiological enumeration. The system includes a filtration assembly and a cassette of the present invention. In the system, the membrane frame of the filtration assembly is configured to detach from the base of the filtration assembly, attach to the cassette base, and detach from the funnel when attached to the cassette base. By leaving the membrane frame attached to the funnel until it is securely attached to the cassette base, the membrane is protected from contamination and damage during the transfer process.

[0047] The system of the present invention requires that the membrane frame be attachable to the cassette base in a manner that creates a stronger attachment than the membrane frame's attachment to the funnel. To accomplish this, the membrane frame may include a protruding ring, and the cassette base may include tabs that mate with the protruding ring to attach the membrane frame to the cassette base. Alternatively, the features may be reversed, and the cassette may feature a protruding ring, for example, from one of its interior walls, and the membrane ring may include a tab. Alternatively, or in addition, the cassette may include a feature (e.g., an inner ring shown in FIG. 22) that presses against a tab on the membrane frame or funnel (see, e.g., FIG. 21 ) to disengage the interlocking feature.

[0048] In some embodiments, when the membrane frame is attached to the cassette base, the membrane is placed in conformal contact with the growth region, i.e., the membrane and medium are kept pressed together. Maintaining the flatness of the membrane growth region, e.g., by pressing against the medium, is advantageous during imaging.

[0049] Kits may include one or more of the devices described herein. For example, if the funnel and / or cassette do not include a lid, the kits of the present invention may include one or more separate lids for covering the funnel and / or cassette. The lids may be included in the kit with the filtration assembly and cassette, or may be packaged separately. The kits may include both a filtration assembly and a cassette, or multiple filtration assemblies, or multiple cassettes, so that, for example, a microorganism-independent filtration assembly may be combined by the user with a microorganism-specific cassette. Alternatively, the filtration assembly may be microorganism-specific. The kits may include a flexible polymer cover to protect the cassette base during transport, and a separate lid that is clear and / or non-fluorescent for incubation and imaging. The kits may also include a washer as a separate component, as described herein.

[0050] The systems or kits of the invention may also include a tray for holding multiple filtration assemblies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more). In some embodiments, the system also includes a base arranged to hold six filtration assemblies, as shown, for example, in FIGS. 13A and 13B. The trays may be stackable with or without filtration assemblies. The trays may hold the filtration assemblies in a staggered layout (see, e.g., FIGS. 13A and 13B) or a rectangular layout (e.g., FIG. 12B). A staggered arrangement may allow stacked trays of filtration assemblies to occupy less volume than a rectangular arrangement, e.g., during shipping (see, e.g., FIG. 12A). The trays may be thermoformed or injection molded.

[0051] The filtration assemblies and cassettes of the present invention may be combined as part of kits and systems with various external components, such as vacuum pumps, aspirators, liquid handling robots, robotic arms, light sources (e.g., lasers), detectors, heaters (e.g., for incubation), coolers (e.g., for storage), reagents (e.g., for cell staining), and the like.

[0052] method The present invention provides a method for determining the presence of microorganisms (e.g., bacteria or yeast) in a sample, e.g., a water sample. An exemplary method includes first attaching a filtration assembly to a vacuum source, e.g., by connecting the base to a tulip valve connected to a vacuum pump or aspirator. The method then includes flowing a sample fluid through the filtration assembly so that any cells in the fluid are retained on the membrane. The funnel attached to the membrane frame is then removed from the base (e.g., by pressing a button to release the latch) before attaching the membrane frame to a cassette base of the present invention (e.g., as shown in Figure 16B or Figure 19B). The funnel is then removed from the membrane frame, and the cassette is incubated to allow any retained cells to grow. The method advantageously allows for the transfer of the membrane without handling and risk of damage or contamination. The method is particularly advantageous for integration into automated testing systems. An example of the method of the present invention is shown in Figure 1.

[0053] The methods of the invention may include using pressure, e.g., from a vacuum, to force the membrane to assume the shape of the cavity between the membrane and the membrane support (see, e.g., Figures 24, 26A, and 27). The membrane may maintain this shape after the membrane frame is removed from the base and until the membrane frame is attached to the cassette base (see, e.g., Figure 27). The shaped membrane may interact with the cassette (e.g., solid or semi-solid medium) to prevent air bubble trapping during attachment of the membrane to the cassette base (see, e.g., Figure 27).

[0054] In the methods of the present invention, the membrane may initially contact the flat growth area at a first point on its circumference and finally contact the flat growth area at a second point on the circumference that is antipodal to the first point (see, e.g., Figure 26B). Propagation of contact between the initial and final contact points at a continuously decreasing contact angle can act, for example, to minimize or eliminate bubble traps and wrinkles. The membrane frame is angled at an angle of 1° to 75° (e.g., about 1° to 5°, about 1° to 10°, about 1° to 15°, about 10° to 15°, about 15° to 25°, about 20° to 30°, about 25° to 35°, about 30° to 40°, about 35° to 45°, about 40° to 50°, about 45° to 55°, about 50° to 60°, about 55° to 65°, about 60° to 70°, or about 65° to 75°) relative to the flat growth area. , e.g., about 1°-37.5°, about 10°-40°, about 15°-45°, about 22.5°-67.5°, about 40°-70°, about 25°-75°, or about 35°-75°, e.g., at least 5°, 10°, 15°, 20°, 25°, or 30°, e.g., about 22.5°, about 30°, about 45°, about 60°, or about 67.5°. Asymmetrically positioned locking features (e.g., positioned at different heights or with different resistances) may be used to direct the angle of approach of the membrane frame (and membrane) during attachment of the membrane frame to the cassette base. The cassette and / or membrane frame may include features (e.g., angled slots, tabs, catches, cams, latches, hooks, etc., or an attached or inserted asymmetrically spring-loaded ring) that position the membrane in contact with the flat growth area such that contact propagates from an initial point to a final antipodal point (e.g., at an initial angle of about 1°-75°, e.g., at a decreasing angle) when the membrane frame is lowered onto the cassette base. The spring-loaded ring may be a ring supported by multiple springs arranged in an annular fashion, with the tallest spring of the multiple springs antipodal to the shortest spring of the multiple springs, and multiple springs positioned therebetween having tapering heights.

[0055] The method may further include imaging the membrane to detect any colonies formed from the retained cells, for example, by detecting autofluorescence of specific microorganisms, including small colonies of only a few hundred cells. Imaging may also include monitoring colony growth by taking a time series of images, for which embodiments of the invention featuring fiducial markings are particularly suitable. The cassette may be advantageously imaged with a Growth Direct® automated microbial detection system (Rapid Micro Biosystems).

[0056] Other embodiments are within the claims.

Claims

1. below, a) Funnel; b) a membrane frame containing a porous membrane; c) a base containing a membrane support and an outlet 1. A filtration assembly comprising: A filtration assembly wherein the membrane frame is releasably attached to the funnel, the base is releasably attached to the funnel, and during filtration, liquid flows from the funnel through the membrane and membrane support to an outlet, and the membrane support keeps the membrane flat during filtration.

2. 10. The filtration assembly of claim 1, wherein the funnel is attached to the base by a locking mechanism that is released when a portion of the outer wall of the base is pressed.

3. 3. The filtration assembly of any one of claims 1 to 2, wherein the porous membrane is ultrasonically bonded or heat staked to the membrane frame.

4. 4. A filtration assembly according to any one of claims 1 to 3, wherein the membrane frame is releasably attached to the funnel by a mating arrangement of a raised lip on the funnel and a recess in the membrane frame.

5. The filtration assembly of any one of claims 1 to 4, wherein the membrane frame includes a protruding ring that mates with a tab on the cassette base.

6. 6. The filtration assembly of any one of claims 1 to 5, wherein the membrane is black and / or non-fluorescent.

7. The filtration assembly of any one of claims 1 to 6, wherein the membrane comprises a black mixed cellulose ester membrane.

8. The filtration assembly of any one of claims 1 to 7, wherein the funnel is transparent and / or includes volumetric markings.

9. The filtration assembly of any one of claims 1 to 8, further comprising a funnel cover covering an opening to the funnel.

10. 10. The filtration assembly of claim 9, wherein the funnel lid is hinged onto the funnel.

11. The filtration assembly of any one of claims 1 to 10, wherein the membrane support is attached to a filter base.

12. 12. The filtration assembly of any one of claims 1 to 11, wherein the filter base comprises a plurality of supports configured to facilitate fluid flow to the outlet and / or to support the membrane support.

13. The filtration assembly of any one of claims 1 to 12, wherein the membrane frame further comprises fiducial markings.

14. The filtration assembly of any one of claims 1 to 13, wherein the filtration assembly further comprises a washer.

15. 15. The filtration assembly of claim 14, wherein the washer is attached to a membrane support and / or is a thin film.

16. 16. The filtration assembly of claim 14 or 15, wherein the washer and membrane support are a single molded part.

17. The filtration assembly of any one of claims 14 to 16, wherein the washer comprises a spring.

18. 16. The filtration assembly of claim 14 or 15, wherein the washer and membrane support are a single molded part.

19. 17. The filtration assembly of any one of claims 1 to 16, further comprising a cavity and / or shim between the membrane and the membrane support.

20. 18. The filtration assembly of claim 17, wherein the membrane support and / or the base are shaped to create a cavity.

21. below, a) a cassette base, i. a base layer including an outer wall, a first inner wall including a plurality of radially arranged gaps, and a second inner wall including an outer ledge and defining a well, the first inner wall being disposed between the outer wall and the second inner wall; ii. A solid or semi-solid nutrient medium disposed in the well and having a flat growth area, the growth area being higher than the second inner wall. a cassette base including: b) a cassette lid releasably sealable to the base Includes cassette.

22. 22. The cassette of claim 21, wherein the cassette lid is optically clear and non-fluorescent.

23. 23. The cassette of any one of claims 21 to 22, wherein the cassette base is non-fluorescent.

24. 24. The cassette of any one of claims 21 to 23, further comprising a third inner wall disposed between the first inner wall and the outer wall.

25. 25. The cassette of claim 24, further comprising a fourth inner wall between the third inner wall and the outer wall.

26. 26. The cassette of any one of claims 21 to 25, wherein the second inner wall includes a plurality of supports that project radially toward the first inner wall.

27. The cassette of any one of claims 21 to 26, further comprising a washer.

28. 28. The cassette of claim 27, wherein the washer is attached to the cassette base and / or is a membrane.

29. 27. A cassette according to claim 25 or 16, wherein the washer and the cassette base are a single moulded part.

30. A cassette according to any one of claims 27 to 29, wherein the washer comprises a spring.

31. 31. The cassette of any one of claims 27 to 30, wherein the cassette base is positioned to receive a membrane lowered onto the base at an angle of between 1° and 75° relative to the flat growth area.

32. 32. The cassette of claim 31, wherein the cassette base includes a ring supported by a plurality of springs arranged in an annular fashion, the tallest spring of the plurality of springs being symmetrical with the shortest spring of the plurality of springs, and the springs arranged therebetween having gradually decreasing heights.

33. 33. The cassette of any one of claims 21 to 32, further comprising features that engage with the membrane frame to provide an initial angle of between 1° and 75° between the membrane and the planar growth area.

34. below, a) a filtration assembly according to any one of claims 1 to 20, and b) a cassette according to any one of claims 21 to 33 1. A system for filtering and culturing cells, comprising: A system in which the membrane frame is configured to detach from the filter base along with the funnel, attach to the cassette base, and detach from the funnel when attached to the cassette base.

35. 35. The system of claim 34, wherein the membrane frame includes a raised ring and the cassette base includes tabs that mate with the raised ring.

36. 36. The system of any one of claims 34-35, wherein the membrane frame is attached to the cassette base such that the membrane is placed in conformal contact with the planar growth area.

37. below, a) attaching a filtration assembly according to any one of claims 1 to 20 to a vacuum source; b) flowing the sample fluid through a filtration assembly, whereby any cells in the fluid are retained on the membrane; c) removing the funnel attached to the membrane frame from the base; d) attaching a membrane frame to a cassette base according to any one of claims 21 to 33; e) removing the funnel from the membrane frame; and f) Incubating the cassette 1. A method for determining the presence of a microorganism, comprising:

38. 38. The method of claim 37, further comprising imaging the membrane to detect any colonies formed from the retained cells.

39. 40. The method of claim 38, wherein the filtration assembly includes a cavity and step (a) includes applying pressure to cause the membrane to conform to the shape of the cavity.

40. 40. The method of claim 39, wherein the membrane maintains the shape of the cavity after the funnel is removed from the base.

41. 41. The method of claim 40, wherein the shaped membrane interacts with the cassette to prevent air bubble trapping during step (d).

42. 42. The method of any one of claims 37 to 41, wherein during step (d), the membrane initially contacts the flat growth area at a first point on its circumference and finally contacts the flat growth area at a second point on its circumference that is antipodal to the first point.

43. 43. The method of claim 42, wherein during step (d), the membrane frame is initially lowered onto the base at an angle of between 1° and 75° relative to the flat growth area.

44. 44. The method of claim 43, wherein the cassette includes features that engage with the membrane frame to position the membrane in contact with the flat growth area at an angle of 1° to 75° relative to the flat growth area when the membrane frame is lowered onto the cassette base.

45. 45. The method of claim 44, wherein the feature comprises a ring supported by a plurality of springs arranged in an annular fashion, the tallest spring of the plurality of springs being antipodal to the shortest spring of the plurality of springs, and the springs arranged therebetween having tapering heights.

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