Cell culturing device

Abstract

The invention features devices and kits for capturing and culturing microorganisms (e.g., bacteria, fungi, or protists) and methods of using the devices and kits to detect microorganisms in environmental and other samples. The device includes a nutrient media having a flat growth area on which microorganisms can grow. Samples are collected by contacting the device with any environmental sample, e.g., rolling device on a work surface or exposing device to air, or by filtering a sample through a membrane. Microorganisms deposited on the membrane derive nutrients from the underlying media and grow into colonies that can then be detected using methods known in the art. The detected colonies can be imaged digitally or with film.

Claims

1. A cassette comprising: a circular base with a bottom surface and side wall; a circular lid; a circumferential rim integral with the circular lid or the circular base; a plurality of detents, wherein, if the rim is on the circular lid, the plurality of detents are on a surface on the circular base, and vice versa, and whereby the circular lid and circular base are secured by inter-engagement between the circumferential rim and the plurality of detents resulting from axial compression; and at least one centering detent, positioned to apply pressure on the circumferential rim, wherein the plurality of detents are attached to the surface by a first ridge and the circumferential rim rests in a gap between the plurality of detents and the surface, and wherein the at least one centering detent is attached to a second ridge extending from the surface and distinct from the first ridge.

2. The cassette of claim 1, wherein the plurality of detents are deflected as the circumferential rim passes the plurality of detents.

3. The cassette of claim 1, wherein the plurality of detents are snap detents.

4. The cassette of claim 3, wherein the plurality of snap detents have a pointed protrusion to increase force per area applied to the circumferential rim.

5. The cassette of claim 1, wherein the at least one centering detent is in the circular base.

6. The cassette of claim 1, further comprising an optically clear window in the circular lid.

7. The cassette of claim 1, wherein the circular base contains a nutrient media for microorganisms.

8. The cassette of claim 1, wherein the circumferential rim is continuous around the circular base or the circular lid.

9. A cassette comprising: a circular base with a bottom surface and side wall; a circular lid; a circumferential rim, wherein the circumferential rim comprises at least one kerf; and at least one ball detent and at least one secondary detent, wherein, if the rim is on the circular lid, the at least one ball detent and at least one secondary detent are on the circular base, and vice versa, whereby the circular lid and circular base are secured by inter-engagement between the circumferential rim and the at least one ball detent resulting from axial compression and whereby interaction between the at least one secondary detent and the kerf inhibits rotation of the lid relative to the base.

10. The cassette of claim 9, further comprising at least one centering detent positioned to apply pressure on the circumferential rim.

11. The cassette of claim 9, further comprising an optically clear window in the circular lid.

12. The cassette of claim 9, wherein the circular base contains a nutrient media for microorganisms.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A is an exploded view of a device of the invention. FIG. 1B is a photograph of a device with the cover removed.

(2) FIGS. 2A-2B are cross-sectional views of a device of the invention. FIG. 2A is a cross-sectional view of a device with a protective lid installed before use of the device. FIG. 2B is a cross-sectional view of a device with an optical lid installed.

(3) FIGS. 3A-3B are cross-sectional views showing details of attachment of a membrane to a base. FIG. 3A is a cross-sectional view of a part of a device showing an expansion trough. Heat-seal the membrane to the outer ramp of plastic, as shown. A trough feature is bridged to allow expansion of the membrane without wrinkling. Also traps liquid moisture formed in attachment. In this approach, organisms are corralled to the desired USP 25 S-CM area by means of film or hydro edge coating of the membrane area outboard of the 57 mm. FIG. 3B is a schematic depiction of a part of a device showing the location of a film or edge coating and expansion trough.

(4) FIG. 4 is a depiction of a base of a device of the invention including a region for application of a bar code, mechanically supporting elements (ribs), a double circumferential side wall, indentations (V features) for alignment and gripping, and a feature for optical alignment.

(5) FIG. 5A is an exploded view of a device of the invention including a protective lid, a circumferential film with fiducial marks (dashes), a base with nutrient media and a growth area, and a bar code label. FIG. 5B is a photograph of a device of the invention including a circumferential film with fiducial marks (dashes), a base with nutrient media and a growth area, and a membrane with circumferential fiducial through holes overlaying the growth area and partially overlaying the circumferential film.

(6) FIG. 6 is a schematic depiction of a device of the invention including an optical lid with a circumferential rim and a base with three first detents (Snap detent, Detail A of FIG. 6) and three second detents (Centering detents, Detail B of FIG. 6).

(7) FIG. 7 is a schematic depiction of a device of the invention including a lid with a kerfed circumferential rim (Detail D in FIG. 7) and a base with three first detents (Ball detents, Detail B in FIG. 7), and three third detents (Centering detents, Detail C in FIG. 7), and nine second detents (Detail A in FIG. 7) that are configured to fill within the kerfs.

(8) FIG. 8 is a schematic depiction of a device of the invention including a lid having a discontinuous circumferential rim (see Detail A in FIG. 8) and a base with two types of detents that are complementary to the discontinuous rim of the lid. The detents of the base are separated by about 40 degrees relative to the circumference. The first detents (see Detail C of FIG. 8) and second detents (see Detail B of FIG. 8) engage the discontinuous rim. The lid also has a visual indicator (rim feature). The figure shows eight discontinuous circumferential rims at 45° intervals. All eight circumferential rims secure with either the first or second detents (Detail A).

(9) FIG. 9 is a schematic depiction of a device of the invention including a lid that has two discontinuous, sloping circumferential rims and a base with three first detents and two stops (Cam Stop, Detail A in FIG. 9).

(10) FIG. 10 is a photograph of a device of the invention including a base, a membrane overlaying a nutrient media, and a circumferential area which slopes to the base. In addition, fiducial marks (dots) can be seen in the circumferential area.

(11) FIG. 11 is a photograph of microbial colonies grown on a device and visible to the human eye.

(12) FIGS. 12A-12C are a set of images showing the use of a device in monitoring different types of environmental samples: air (FIG. 12A), surfaces such as work areas (FIG. 12B), and garments such as gloves or sleeves (FIG. 12C).

(13) FIG. 13A is an exploded view of a filtration device including a funnel, a membrane with fiducial marks, a mesh support pad, and a filtration base. FIG. 13B is a photograph of an assembled filtration device.

(14) FIGS. 14A-14D are a set of images showing the monitoring of a liquid sample using a filtration device and a device of the invention. FIG. 14A is a photograph of the assembled filtration device, a protective lid, and a device of the invention. FIG. 14B is a photograph of filtering a sample through the membrane within the filtration device. FIG. 14C is a photograph of the disassembled filtration device, showing the funnel, membrane on the filtration base, protective lid, and a device of the invention. FIG. 14D is a photograph showing the transfer of the membrane to a device of the invention, in which the membrane is placed with the fiducial marks on the circumferential rim and fiducial marks (circumferential through holes) in the membrane allow for alignment of multiple images take of the membrane.

DETAILED DESCRIPTION OF THE INVENTION

(15) The invention features devices and kits for capturing and culturing microorganisms (e.g., bacteria, fungi, or protists) and methods of using the devices and kits to detect microorganisms in environmental samples. The device is useful for rapid environmental monitoring and can be used to collect microorganisms, for example, by rolling the device on a surface. The device is then incubated to allow any microorganisms collected to grow into colonies, which are indicative of microbial contamination.

Device

(16) The cell culturing devices of the invention facilitate the sample collection, sample growth, and detection of microorganisms within a sample. Devices of this invention allow for efficient, cost effective, and robust microorganism monitoring for a wide variety of applications.

(17) The device, e.g., as shown in (FIGS. 1A-1B), includes a base that contains nutrient media overlaid by a porous membrane. The membrane is in conformal contact with the nutrient media. The membrane and nutrient media form a flat growth area surrounded by a sloping circumferential area. In preferred embodiments, the membrane allows nutrients to pass through and sustain microbial growth in the growth area but not in the circumferential area. In one aspect of the invention, the device is useful for automated detection of microorganisms, e.g., using the Growth Direct™ system for rapid colony counting, e.g., as described in U.S. Publication No. 2003/0082516, which is hereby incorporated by reference. The device may be of any appropriate size, e.g., that of a RODAC™ plate.

(18) The base may be substantially non-radiative and non-reflective and may be made of any suitable material, e.g., polystyrene or other plastic. The base may also have a circumferential groove (also referred to as an expansion trough) that can be used for attachment of a membrane (e.g., as shown in FIGS. 2 and 3). The base may be produced by methods known in the art, e.g., injection molding.

(19) The device, e.g., the base and/or the lid, may include indentations (e.g., as shown in FIG. 4) or protrusions for gripping or alignment on the device. In certain embodiments, the device is not compressible in the lateral direction. For example, the device may include one or more mechanically supporting elements (FIG. 4) to maintain the flatness of the growth area during handling.

(20) The device includes a porous membrane, e.g., one having fluorescence properties commensurate with detection of autofluorescent microbial microcolonies. For example, the membrane is substantially non-radiative and non-reflective for detection of autofluorescent microbial microcolonies. Membranes may be manufactured from materials including cellulose, cellulose acetate, polystyrene, polyethylene, polycarbonate, polyethylene terephthalate (PET), polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, nylon, and silicone copolymer. The choice of membrane depends, in part, on the type of cell to be cultured (e.g., microorganisms that grow attached to a surface (anchorage-dependent), microorganisms that grow in suspension (anchorage-independent), or microorganisms that grow as attached to a surface or in suspension), degree of permeability, and rate of transfer of fluids and gases. An exemplary membrane is a black mixed cellulose ester membrane (Sartorius AG).

(21) The membrane is placed over the nutrient media so that the membrane and media are in conformal contact. The membrane and nutrient media form a flat growth area raised above the base with a circumferential sloping area around the edges. Such a design makes the device suitable for contact testing, e.g., by rolling the device on a surface. The membrane and nutrient media form a growth area that is flat across an area, e.g., of 10, 15, 20, 25, 30, 35, or 50 cm.sup.2, preferably at least at least 25 cm.sup.2. The membrane on the nutrient media has a flatness of about 100 to 600 μm, e.g., 200 to 350 μm, e.g., about 300 μm, as fabricated or 300 to 500 μm, e.g., about 450 μm, after collection of sample. The membrane is preferably factory installed and stays wet for the life of the product. The membrane has pores so that microorganisms deposited on the membrane may obtain nutrients from the underlying nutrient media. Examples of membrane pore sizes are 0.45 μm and 0.22 μm.

(22) Solid or semi-solid nutrient growth media can be employed in the present device. Examples include Sabouraud dextrose agar (SDA), R2A agar, tryptic soy agar (TSA) letheen, and plate count agar (PCA). The media may be poured onto the base in a molten liquid state and then allowed to solidify into a flat growth area that is raised above the base and a circumferential area that slopes from the edge of the flat growth area to the base. The flatness of the growth area may be controlled by surface tension and by filling normal to gravity. The flatness of the growth area may also be achieved using several alternate methods. For example, one method to achieve a flat growth area includes pouring molten nutrient media onto the underside of a pre-attached, wet membrane. In this alternate method, the membrane is pre-attached to a base that has an opening on the bottom. The opening is used to fill molten nutrient media, e.g., agar. This opening is then sealed post filling by a cover or film. The membrane is circumferentially sealed to prevent leakage. The membrane expands or inflates during the filling process and may be shaped by trapping within a nest or cavity of appropriate shape. Another method to achieve flatness is to pre-bow the base mid section downward by approximately 150 to 200 μm, e.g., using vacuum. The nutrient media is poured, and, once the media solidifies with a concave surface, the bowing force is released and the growth area springs back to the flat state. Alternatively, the nutrient media may be a liquid media held in a porous matrix, which is shaped to have a flat growth area and sloping circumferential area.

(23) The membrane is preferably secured to prevent peeling during use. The membrane may be installed by heat sealing to the base, e.g., by bridging over a circumferential groove. The groove (FIG. 3A) allows expansion of the membrane without wrinkling. The groove also traps any moisture formed during the attachment. Alternatively, the membrane may be attached to the device using a circumferential film, e.g., of PET, co-PET, acetate, or polyimide, that may be used to define the edges of the growth area. The film may be made black. The film may be substantially non-radiative and/or non-reflective. The film may contain several layers (FIG. 3B). Such layers allow for heat fusion to the membrane without damaging the membrane, low fluorescence, and/or low reflectance. A film may also cover the circumferential area to prevent growth of microorganisms outside the flat growth area. This arrangement is beneficial in confining growth to an area of the device, e.g., for imaging or visual inspection.

(24) In alternative design, a device does not include an integral membrane. This device also includes a nutrient media raised above the base, a flat growth area, a film overlaying the circumferential area of the nutrient media (FIG. 5A and 5B), and the circumferential area that slopes from the edge of the flat growth area to the base. The device includes a film that overlays the circumferential area (in whole or in part), and the film has an opening to expose a portion of the flat growth area. The opening in the film and the nutrient media form a growth area that is flat across an area, e.g., of 10, 15, 20, 25, 30, 35, or 50 cm.sup.2, preferably at least at least 25 cm.sup.2. Suitable films include any non-porous or hydrophobic plastic or material, e.g., PET, co-PET, acetate, or polyimide. The film may be made black. The film may be substantially non-radiative and/or non-reflective. The growth area provides a surface where a porous membrane, e.g., exposed, to a sample may be overlaid. The nutrient media provides sustained growth of organisms on the top surface of the overlaid membrane, thereby providing a device for cell culturing and detection of contamination.

(25) The device also includes a lid. The lid is for example a protective lid (FIG. 2A) that does not contact the membrane and prevents contamination of the device prior to use. The lid may also be an optical lid (FIG. 2B) with an optically clear window for imaging the growth area, e.g., with visible or UV light. An exemplary material for the lid is Zeonor® 1060R (polycycloolefin resin; Zeon Chemicals LP). Glass may also be employed. The optical lid may also be used as a protective lid prior to use. The lid mates to the base to seal the membrane and nutrient media from outside contamination. The lid may be produced by methods known in the art, e.g., injection molding. All or part of the lid may be non-radiative and/or non-reflective.

(26) A lid secures to the base using securing members present on both the lid and the base. The lid and base may secure or engage reversibly, in which the lid and base may be separated and reattached multiple times. Securing the lid to the base affixes the lid relative to the base in the axial direction (z-axis), thereby sealing the device. Securing members may provide alignment of the lid relative to the base securing in the lateral directions (x-axis and y-axis). Preferably, the lid protects the base and also prevents rotation of the lid relative to the base, e.g., to less than 50 μm.

(27) A lid may be secured to the base with axial compression. For example, a circumferential rim (FIG. 6) on the lid and detents (snap detents, FIG. 6, Detail A) on the base may be used to secure the lid to the base once an axial force is applied to the lid. The detent is deflected as the circumferential rim passes the detent. The circumferential rim rests in a gap between the snap detent and the bottom surface of the base. The snap detent (Detail A of FIG. 6) may have a pointed protrusion to reduce the contacting surface area of the snap detent, thus increasing the force per area applied to the circumferential rim. In addition to the snap detents, centering detents can be used that apply pressure to the lateral edge of a circumferential rim (Detail B of FIG. 6), thus fixing the position of the lid. The reproducibility of the optical lid position relative to the base is important for improving the consistency of automated sample imaging. Securing and preventing movement of the optical lid improves the reproducibility of the automated image analysis.

(28) In an alternative embodiment, the base may include multiple ball detents (Detail B of FIG. 7), multiple second detents (Detail A of FIG. 7), and multiple centering detents, (Detail C of FIG. 7), around perimeter of the base. The lid is secured to the base first detent by a circumferential rim (FIG. 7). The second detents help align the lid relative to the base. The kerfed circumferential rim (Detail D of FIG. 7) prevents rotation of the lid relative to the base because the second detents (Detail A of FIG. 7) fill the kerfs of the rim.

(29) A lid may be secured to the base with a rotational motion of less than or equal to 90 degrees. For example, the lid may be have a series of discontinuous circumferential rims (FIG. 8, Detail A). The discontinuous circumferential rims may or may not be evenly distributed around the perimeter, each about 40 degrees apart (FIG. 8). A series of detents may be located on the base (FIG. 8). Two variations of detents are shown (Detail C and Detail B of FIG. 8), but a single type may be employed. The first detent engages to the top edge (distal edge adjacent to top surface of the lid) of the circumferential rim in the portion opposite the vertical portion of the discontinuous circumferential rim. The second detent engages to the top edge (distal edge adjacent to top surface of the lid) of the circumferential rim.

(30) A lid may also be secured to the base with a rotational motion of less than or equal to 90 degrees. For example, the base includes multiple detents (Detail A of FIG. 9) sized and shaped to reversibly secure to a discontinuous, sloped circumferential rim (FIG. 9). The first detent has a horizontal length with a top surface that prevents lid from being installed in wrong location. The first detent facilitates a jam fit by providing an upper boundary that increasingly interacts with the variable height (sloped) discontinuous circumferential as the lid is twisted relative to the base. A stop (cam stop, Detail A of FIG. 9) provides a rotational boundary for the discontinuous circumferential rim. The increasing height of the circumferential rim engages the first detent as the rim is turned about 90 degrees, thereby inducing a jam fit. The cam stop prevents over-rotation, providing a stop for the leading edge of the circumferential rim.

(31) Additional non-limiting exemplary securing members and mechanisms for securing a lid to the base include: threads, clamps, gaskets, magnets, crown caps, and friction fits. For example, a lid of the device of the invention may be configured with a series of threads. A base of the invention may be configured with a complimentary series of threads, for securing the lid to the base.

(32) The protective lid and the optical lid may attach to the same base using the same or different mechanisms. The securing members may be on the lid or the base. For example, the circumferential rim may be on the lid, and the detents on the base. Alternatively, the detents may be on the lid, and the circumferential rim on the base. Circumferential rim may also be on the outer or inner perimeter of the side wall of the lid or the base.

(33) The device may include features that indicate successful securing of the lid to the base. For example, a rim feature on a lid (FIG. 8) may be above a base and be visible before the lid is secured to the base. Once the lid is secured to the base, the rim feature is not visible above the base, thereby indicating a successful union of the lid and the base. The device may include securing members compatible with automation. For example, a securing member may be sized and shaped such that robotic transfer arms may engage a securing member, thereby facilitating the automated use of the device.

(34) The device may also include a fiducial mark, e.g., printed fluorescent material, embossed fluorescent material, radiative plastic, or a through hole exposing fluorescent media, material, or plastics. Other fiducial marks are known in the art. The fiducial mark may be outside the growth area (FIG. 5B and FIG. 10). The fiducial mark may help align a membrane with the device (FIG. 5B). A fiducial mark may be used to align multiple images acquired from the same device, e.g., taken at different times, or the fiducial mark may be used to align the device within an imager. The fiducial marks may be at any position on the device that is suitable for imaging, e.g., the base, the circumferential area, the growth area, or the lid.

(35) The device may also have a unique ID label imprinted or affixed on the device to aid in automated handling or sample tracking, e.g., by the Growth Direct™ system. The ID label may be a bar code or 2D barcode. The ID label may be used to track the device identity, device compatibility with instruments and protocols, device expiration date, sterilization history, and other information of interest.

Methods of Use

(36) The devices can be used to monitor the presence of microorganisms, e.g., in the environment. Environmental samples may include, without limitation, air, surfaces, and garments. The devices and kits of the invention may be used in any situation where microbial contamination needs to be rapidly detected, e.g., laboratories, hospitals, manufacturing areas, and “clean rooms” for nanotechnology manufacturing and applications. Exemplary surface samples include surfaces of stainless steel, glass or granite work surfaces, walls, floors and equipment surfaces. Surface may also include anatomical structures such as fingers and foreheads. Exemplary garment samples include jacket sleeves, gloves, chest plate and any other portion of wearable garment. The method may include: contacting the growth area of the device with a volume of air or a surface; incubating the device to allow growth of microorganisms (incubation may occur at, above, or below room temperature); and determining the extent of growth of microorganisms, e.g., by manual counting or by automated counting of colonies (as shown in FIG. 11). Any culturable microorganism, including bacteria, cyanobacteria, protozoa, and fungi may be employed in conjunction with the device described herein. The device can be used for aerobic and anaerobic testing.

(37) The sample may be collected by rolling the device so that the circumferential area and the growth area contact the surface. The surface can be, e.g., work surfaces such as a laboratory surface or industrial surface (FIG. 12B), garments (FIG. 12C), or gloves (FIG. 12C). In another aspect of the invention, air samples can be tested by passing a volume of air over the growth area, e.g., in an air monitor (FIG. 12A) or by leaving the device uncovered for a period of time.

(38) After sample collection, the device is typically covered using the optical cover and is incubated for microorganisms to grow, e.g., in an incubator at temperatures above or below room temperature. In one embodiment, after sample collection, the device is placed within the Growth Direct™ system for incubation and imaging. The device may be imaged at predefined intervals of time, and microorganisms may be detected by suitable methods known in the art, e.g., fluorescence (via autofluorescence or stains), reflectance, or absorbance. Alternatively, vital stains may be introduced into the nutrient media and absorbed into the microorganisms during growth. Detection may be repeated to discern growing colonies from non-growing microorganisms or debris. Images of microorganisms may be recorded, either digitally or with film. The optical lid of the device may be removed manually or using automation and replaced with a protective lid during storage. The protective lid may reversibly secure the base using one or more securing members. An optical lid may reversibly secure the base using one or more securing members.

(39) Alternatively the sample may be collected by filtering a sample through a membrane and then applying the membrane to the device of the invention. For example, a membrane and filtration device (FIG. 13A and 13B, and FIG. 14A) may be used to filter a liquid sample, thus collecting any contaminating microorganisms on the membrane. As shown in FIGS. 13A-13B, an exemplary filtration device includes a funnel that traps a membrane to a filtration base. The membrane is supported by a mesh support pad to maintain flatness during filtration. Other filtration devices are known in the art, e.g., as described in WO 2007/038478. A sample of interest is passed through the membrane to collect microorganisms (FIG. 14B). The membrane is removed from the filtration device and overlaid onto the nutrient media of a device configured to accept a membrane (FIGS. 14C-14D). The membrane may be aligned relative to the fiducial marks on the film (dashes). The membrane may include fiducial marks, e.g., through holes in the region outside of the filtered area. Such through holes can be used to expose fluorescent media, plastic, or printed material that is otherwise covered by the membrane. The membrane may be larger diameter than the exposed growth area and overlap the circumferential film. The membrane may adhere to the growth area by wetting from the nutrient. The membrane and device are then covered, incubated, and imaged as described above.

(40) Other methods and instruments for manual or automated colony counting that can be used with the device are known in the art.

Kit for Environmental Monitoring and Filtering

(41) The invention also features a kit which includes the device, a protective lid, and an optical lid. The kit may be shipped with a protective cover installed on the device. The device with the protective cover and the optical cover may be packaged separately or together in sterile packaging. In use, the protective cover is removed, the sampling is done, and the optical cover is then installed.

(42) Alternatively, the invention also features a kit which includes the device, a protective lid, a membrane, and an optical lid. The kit may be shipped with a protective cover installed on the device. The device with the protective cover, membrane and the optical cover may be packaged separately or together in sterile packaging. In use, the protective cover is removed, the sampling is done, and the optical cover is then installed. Such kits may also include a filtration device as discussed herein.

Other Embodiments

(43) All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

(44) Other embodiments are in the claims.