HK1204649B - Cassette for sterility testing - Google Patents

Description

Cartridge for sterility testing

Cross reference to related applications

This application claims U.S. provisional application No. 61/556,390 filed on 7/9/2011 and U.S. provisional application No.61/624,499 filed on 16/4/2012, each of which is incorporated herein by reference.

Technical Field

The present invention relates to the field of cell growth and detection.

Background

In many industries, particularly in the food, beverage, healthcare, electronics and pharmaceutical industries, it is important to rapidly analyze the extent to which a sample is contaminated with microorganisms (such as bacteria, yeast or molds).

One microbial cultivation technique, known as microbial enumeration or colony counting, quantifies the number of microbial cells in a sample. Microbial enumeration methods based on in situ microbial replication generally produce a visually detectable "colony" for each culturable microbial cell or cell population in a sample, referred to as a colony forming unit or CFU. Thus, counting visible colonies allows a microbiologist to accurately determine the number of microbial CFUs in a sample. To perform microbial enumeration, bacterial cells may be spread on the surface of nutrient agar in Petri dishes ("agar plates") and cultured under conditions that allow for in situ bacterial replication. Microbial counts are simple, very sensitive, inexpensive and quantitative, but are typically slow as well. The long time required results in increased costs for healthcare and manufacturing.

Additional incubation devices and methods are needed to perform microbial enumeration.

Disclosure of Invention

The present invention provides a device, called a cassette, for growing cells. In one aspect, the invention features a cell culturing device that includes a housing containing: a cover having a light transmissive window (the cover may or may not be removable); a fluid distribution channel, for example, which is a single channel or connected to multiple channels; a sample injection port fluidly connected to the fluid distribution channel; a base containing a porous media pad; and a media injection port fluidly connected to the media pad. The lid mates to the base to form a sterile seal; the fluid distribution channel is disposed on a media pad viewable through the optical window; and the sample fluid introduced into the fluid distribution channel is uniformly distributed to the media pad, e.g., via a plurality of channels. In certain embodiments, the device further comprises a membrane disposed on the media pad, wherein cells in the sample fluid remain on the membrane and are observable through the optical window. Alternatively, the media pad may have sufficient porosity to function as a separator. The space between the cover and the diaphragm may be pressurized. In other embodiments, the cartridge further comprises a drainage port. Oxygen scavengers sufficient to render the interior of the device oxygen free may also be included. In certain embodiments, the cartridge further comprises an actuator for the oxygen scavenger, e.g., activated by flipping the lid over or by a pull tab or push rod accessed through a membrane located on top of the cartridge. The cassette of the present invention may include a pressure relief valve and/or the base may further include a channel that relieves pressure in the media pad upon introduction of fluid. The cartridge may further comprise a media distribution channel connected to the media injection port and optionally having a plurality of outlets around the circumference of the media pad, wherein the media introduced via the media injection port is evenly distributed to the media pad via the media distribution channel. The media distribution channel may be formed, in whole or in part, by an insert in the base, such as the fluid ring depicted in the figures. The cartridge may also include an oxygen indicator. In certain embodiments, the fluid distribution channel comprises, for example, a helical channel connected to a plurality of channels. When the fluid distribution channel is a single channel, it may include an angled circumferential region around the media pad. The cartridge may further comprise a cover disposed on top of the fluid dispensing channel. The cover may shape the fluid flow to achieve uniform distribution to the media pad through the single channel. The cartridge may further include a vent port for venting the interior of the cartridge when liquid is introduced. The vent port may be self-sealing, for example, until connected to a tubing set of the present invention.

In a related aspect, the invention features a kit for detecting cells that includes the cassette of the invention, and a tubing set that includes tubing having a connector with a needle and that mates to a sample injection port, a media injection port, a vent port, or a drain port. The connector may further include a septum through which the needle passes, and the connector may mate to a sample injection port, a media injection port, a vent port, or a drain port and seal the port with the septum when the needle and tubing are removed. The kit may further include a second tubing set and optionally a third tubing set having a connector with a needle, wherein the connector of the tubing set mates to one of the sample injection port, the media injection port, the vent port, and the drain port. The tubes of the first tube set, the second tube set and the optional third tube set may share a common inlet or outlet. The connector may include a clip that snaps into the cassette. The tubing set may further comprise a plurality of individual tubes for performing at least two of: sample introduction, media introduction, drainage and aeration. The connector may comprise a needle for each conduit. When one conduit is used for aeration, the conduit may include a filter (e.g., to prevent release of bacteria or fluid) and/or a pressure relief valve.

The cartridges and kits of the present invention may be used in any method for growth, assay or maintenance (maintaine) of cells, including counting, detecting, diagnosing or treating a response.

Other features and advantages will be apparent from the following description, and from the claims.

Drawings

Fig. 1A and 1C are exploded views of the cartridge. Fig. 1B is a cross-section of the cartridge. FIG. 1D is a cross-section of the base of the cartridge showing the media injection port, the drainage port, the media pad, and the media distribution channel. FIG. 1E is a view of the cassette with the lid removed showing the tray for the oxygen scavenger. As shown in the inset, the base includes two stops. The first stop allows the lid to seal the cartridge without activating the oxygen scavenger. By flipping the lid over to the second stop, a tab on the lid pierces a seal on the oxygen scavenger. FIG. 1F is a diagram of an alternative lid embodiment.

Fig. 2A is an exploded view of one embodiment of a cassette. Fig. 2B is a cross-section of the cartridge. Figure 2C is a diagram of a cartridge for aerobic and anaerobic use.

Fig. 3A is an exploded view of one embodiment of a cassette. Fig. 3B is a cross-section of an aerobic box. Figure 3C is a cross section of an oxygen-free cell. Figure 3D is a diagram of a cartridge for aerobic and anaerobic use.

Fig. 4A is an exploded view of one embodiment of a cassette. Fig. 4B is a cross section of an oxygen-free cell.

Fig. 5A-5B are top views of a cartridge including a fluid distribution channel having a helical channel with two helices.

Fig. 5C is a top view of a cassette including an outer guide channel and an inner guide channel having a plurality of channels.

Fig. 5D is a top view of a cassette including an outer channel and an inner channel having a single channel.

Fig. 6A is a diagram of a fluid distribution channel having multiple channels with a cover in place. Fig. 6B is a view of the fluid distribution channel with the cover removed. Fig. 6C is a view of the cover.

Fig. 7A is a view of a fluid distribution channel with a single channel with the cover in place. Fig. 7B is a view of the fluid distribution channel with the cover removed. Fig. 7C is a view of the cover.

FIG. 8 is a view of the base of the cartridge.

Fig. 9A is a schematic depiction of a tubing set of the present invention. The tubing set has three connectors that mate to the cassette, and a safety sheathed needle for use in sample or media transport or waste removal. Fig. 9B is a schematic depiction of a tubing set, the septum of which remains in the cassette after use. In this example, the connector includes a groove that snaps onto a corresponding feature of a port on the enclosure.

Fig. 10A is a schematic depiction of a tubing set of the present invention. The tubing set has three connectors (e.g., branches) that terminate in a keyed needle clamp that mates to the cassette, and a safety sheathed needle (not shown) for use in sample or media transport or waste removal. There are two fluid lines per cassette. Fig. 10B is a schematic depiction of a tubing set installed in a cassette. The keyed needle clamp provides that the tubing set can only be inserted in a predefined orientation.

Fig. 11A is a schematic depiction of a tubing set of the present invention. The tubing set has three connectors (e.g., branches) that terminate in a keyed needle clamp that mates to the cassette, and a safety sheathed needle (not shown) for use in sample or media transport or waste removal. There are three fluid lines per cassette. The vent lines are shown coupled to a common valve and filter, but the tube sets may employ separate vent lines for each cassette. Fig. 11B is a schematic depiction of a tubing set installed in a cassette. The keyed needle clamp provides that the tubing set can only be inserted in a predefined orientation.

Figure 12 shows one possible variation of a packaged test kit in which the breathable access panel is removed.

FIG. 13 is a series of photomicrographs showing the growth of bacteria in the cassette of the present invention.

The figures are not necessarily to scale.

Detailed Description

The invention features devices for capturing and growing cells (e.g., microorganisms or cells containing microorganisms) and methods of using these devices. One example is a cartridge containing a nutrient medium, which can be used in an automated Rapid counting system, such as a Growth Direct ­ system (Rapid Micro Biosystems, bedford, massachusetts), for example, as described in U.S. publication No.2003/0082516, which is incorporated herein by reference. In one embodiment, the present invention provides a fully contained closed loop sterility test that allows the end user to filter samples through a membrane (e.g., 0.45 μm), add nutrient media to support Growth and image the cartridge, e.g., on a Growth Direct system, without exposing the samples or other internal components to possible external contamination. The cartridge can be used in aerobic or anaerobic conditions. Multiple cartridges may be packaged together in a kit, for example, at least one cartridge would be configured to perform an aerobic test and one configured to perform an anaerobic test. The present invention also provides a tubing set to allow for the introduction of a sample, nutrient medium, and/or the removal of excess fluid. The tubing set may also allow for uniform distribution of samples among multiple cassettes.

Box

In general, the cassette of the present invention will comprise: a cover having a light transmissive window; a fluid distribution channel; a sample injection port fluidically connected to the fluid distribution channel; a base containing a porous media pad; and a media injection port fluidly connected to the media pad. The sample injection port is typically located on the side of the cap, but may also be located on the top. In certain embodiments, the cover is made of a light transmissive material. Alternatively, the light transmissive window is housed within the optical frame.

In certain embodiments, the fluid distribution channel comprises a plurality of channels. Various views of such a cassette are shown in fig. 1A-1F. The figure shows a cover with an optically transparent window, a base containing a media pad, a sample injection port, a media injection port, and a fluid distribution channel comprising a stabilizing channel, and a plurality of channels for distributing a sample to the media pad. Also shown are drainage ports and a septum, which may or may not be removable. Typically, the lid mates to the base to form a sterile seal, which may or may not be hermetic. The septum is positioned on the media pad so as to be viewable through the optical window. Fig. 1F shows a cover made of a light transmissive material or having a light transmissive window housed within an optical frame. The sample injection port is located on the top of the cap, but may also be located on the side.

Various views of the alternative cassette shown in fig. 2A-2C. The figure shows a cover with an optically transparent window, a base containing a media pad, a sample injection port, a media injection port, and a fluid distribution channel comprising a stabilizing channel, and a plurality of channels for distributing a sample to the media pad. Also shown are drainage ports and a septum, which may or may not be removable. Typically, the lid mates to the base to form a sterile seal, which may or may not be air tight. The septum is positioned on the media pad so as to be viewable through the optical window. Fig. 2C shows a cover having a light transmissive window received within an optical frame. Alternatively, the cover is made of a light-transmissive material. The sample injection port is located on the side of the cap, but may also be located on the top. This cartridge features a pressure relief valve in the lid.

Various views of another cassette are shown in fig. 3A-3D. The figure shows a cover with an optically transparent window, a base containing a media pad, a sample injection port, a media injection port, and a fluid distribution channel comprising a stabilization channel and a plurality of channels for distributing a sample to the media pad. Also shown are drainage ports and a septum, which may or may not be removable. Typically, the lid mates to the base to form a sterile seal, which may or may not be air tight. The septum is positioned on the media pad so as to be viewable through the optical window. Fig. 3B and 3C show the aerobic and anaerobic versions of this cassette. Fig. 3D shows a cover having a light transmissive window received within an optical frame. Alternatively, the cover is made of a light-transmissive material. The sample injection port is located on the side of the cap, but may also be located on the top. The lid also includes a vent port (fig. 3D).

The cartridge of the present invention may include a fluid distribution channel that delivers fluid to the media pad through a single channel. Such a cassette is shown in fig. 4A-4B. The figure shows a cover with an optically transparent window, a base containing a media pad, a sample injection port, a media injection port, and a fluid distribution channel comprising a stabilizing channel for distributing a sample to the media pad. Also shown are drainage ports and a septum, which may or may not be removable. Typically, the lid mates to the base to form a sterile seal, which may or may not be air tight. The septum is positioned on the media pad so as to be viewable through the optical window. The sample injection port is located on the side of the cap, but may also be located on the top. The lid also includes a vent port.

The fluid distribution channel may or may not include a helical guideway. The spiral guide channel is designed to smooth out excess turbulence and distribute the sample evenly to the media pad or a membrane positioned on top of the media pad, e.g. via a plurality of channels. As shown in fig. 5A-5C, the guide channel typically comprises two loops around the device, but three or more loops may be employed. When present, the channels can provide fluid to the media pad via a single channel or multiple channels (fig. 5A-5D). Fig. 5D shows an alternative cartridge that employs a single fluid distribution channel. As shown in fig. 5D, the cassette includes a sloped surface that circumferentially surrounds the media pad. The fluid flows around the surface and onto the media pad. In a cassette having multiple channels to the media pads, the multiple channels may also be formed on or in the sloped circumferential surface. The cartridge may or may not include a member covering the fluid distribution channel with or without the helical guide or channels, e.g., fig. 1A-4B. An exemplary covering for a plurality of channels is shown in fig. 6A-6C. Fig. 6A shows a fluid distribution channel having multiple channels with the cover in place. Fig. 6B shows a fluid distribution channel having multiple channels. Fig. 6C shows a cover for multiple channels. An exemplary cover for a single fluid distribution channel is shown in fig. 7A-7C. Fig. 7A shows a fluid distribution channel with a single channel with the cover in place. Fig. 7B shows a single fluid distribution channel. Figure 7C shows a cover for a single channel. This cover includes a fluid manipulator that reduces the column height of the fluid distribution channel as it enters the sloped circumferential region. The fluid manipulator may also spread the fluid along the surface leading to the media pad. The cartridge may further comprise a splash shield positioned over the fluid distribution channel, wherein the sample is delivered to the media pad. The splash shield may form a port of the cover or be a separate component. The edges of the media pad and diaphragm (if present) are typically covered by a fluid distribution channel or cover or splash shield to prevent the edges from being imaged.

The media pad is designed to accommodate media for the growth or maintenance of cells. In certain embodiments, the media pad is sized to contain enough media for the cells to grow for one week, two weeks, or more. Media is delivered to the pad via a media injection port. The media injection port is typically located on the side or bottom of the base. The cartridge may also include a media distribution channel connected to the media injection port. The media distribution channel may have a plurality of outlets around the perimeter of the media pad to distribute the media evenly to the media pad. An exemplary chassis with a media pad is shown in fig. 8.

When the medium is introduced into the cartridge, the medium is a liquid, and the medium may remain as a liquid in the pad or gel or otherwise set within the pad. Examples include LB liquid medium or sapouraud (Sabouraud) glucose agar (SDA), R2A agar, Tryptic Soy Agar (TSA), Plate Count Agar (PCA), Schaedler blood agar, or similar medium without an agar coagulating agent. The membrane may be disposed on the media pad, for example, between the fluid distribution channel and the pad. The membrane has pores sufficient to retain the cells of interest while allowing fluid to pass through. Examples of pore sizes are 0.45 μm and 0.22 μm. The septum may be separate from the media pad or integral to the media pad. Alternatively, the surface of the media pad may be fabricated or treated to create the separator.

The cassette may or may not include an oxygen scavenger that renders the cassette oxygen free (e.g., fig. 1E, 2A, and 3A). The oxygen scavenger is typically stored in a sealed tray or compartment within the cartridge, the exact location of which within the cartridge is not critical. Once the sample and media have been delivered to the cartridge, the seal may then be broken. Various methods for breaking a seal are known in the art. In one embodiment, the sealed compartment is located near a protrusion on the lid (or base). The cap can be flipped over so that the protrusion pierces the seal on the scavenger (fig. 1E). Actuation may also be by a pull tab that is accessed by a membrane or septum located on the outside of the cartridge (fig. 2A and 3A). Exemplary oxygen scavengers include iron oxide, glucose oxidase or similar agents. The cartridge may also include an indicator of internal oxygen content, which is located in the interior of the cartridge. Suitable indicators are known in the art.

The inlet and outlet ports of the cartridge are preferably self-sealing, e.g., rubber septa or other self-closing valves. As discussed below, the cartridge may be provided without a self-sealing portion that is installed prior to use. In addition to the sample injection port and the media injection port, the cartridge may include a drainage port, for example, on the bottom or side of the base. The cassette may or may not include a pressure relief valve to control the maximum pressure inside the cassette. The space between the cover and the diaphragm may also be pressurized, for example, to prevent excess media from pooling on top of the pad or leaking through the diaphragm. The base may also include channels or other areas to allow for pressure relief during introduction of media to the pad.

Preferably, the cartridges are capable of being stacked in a carrier, e.g., a carrier designed to transfer and introduce a set of cartridges to an automated imaging instrument. Such automated handling of the cassettes may include transportation, docking between the cassettes and the carrier, positioning for automated handling, and robotic transfer capabilities. The cassette may also be designed to allow repeatable mechanical positioning, i.e., repeatedly being able to return the same cassette to the same position for automated imaging.

The cassette may also include design features to facilitate alignment of the multiple images. The imaging fiducial markers include a through-going aperture in a fluorescent plastic or medium. The imaging fiducial markers also include printed or embossed fluorescent material on the cassette. Other fiducial markers are known in the art.

Materials for making the various components of the cartridge are known in the art. Such materials include plastics, polymers, metals, glass and ceramics. In various embodiments, the cartridge facilitates automated imaging of autofluorescent microbial microcolonies containing less than 500 cells, for example, by employing materials with fluorescence properties comparable to such detection. An exemplary material is black K-Resin @ (styrene-butadiene-copolymer; Chevron Phillips Corp.). The cassette may also employ a transparent cover having fluorescence properties comparable to detection of autofluorescent microbial microcolonies. An exemplary material for the cover is Zeonor 1060R (polycycloolefin resin; Zeon Chemicals LP, Inc.). Glass may also be used. A porous membrane having fluorescence properties comparable to the detection of autofluorescent microbial microcolonies can also be employed. The septum may be made of a material comprising: cellulose, cellulose acetate, polystyrene, polyethylene, polycarbonate, polyethylene terephthalate, polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, nylon, and silicone copolymer. The choice of membrane depends in part on the type of cells to be cultured (e.g., microorganisms growing attached to the surface (depending on anchoring), microorganisms growing in suspension (independent of anchoring), or microorganisms attached to or growing in suspension), the degree of permeability, and the transfer rates of fluids and gases. An exemplary membrane is a black mixed cellulose ester membrane (Sartorius AG). The non-imaging portion of the cassette may be made of any suitable material, for example, acrylonitrile butadiene styrene or acrylonitrile styrene. An exemplary media pad is formed from sintered polyethylene (Porex corporation) that can provide a predefined pore size and pore space.

Pipe set

The present invention also provides a tubing set that allows for sterile connection to the cassette. The tubing set includes at least one connector that mates with an inlet or outlet port of the cassette of the present invention. The other end of the conduit may be open, for example, for draining or sliding to a nozzle or other fluid source or sink. Alternatively, the other end may contain a connector, such as a Luer lock, needle, or similar fitting. The tubing set may be designed to deliver fluid from one source to or remove fluid from multiple cassettes or inlets. Each tubing set may be actuated by a separate pump, for example, a peristaltic pump, or multiple tubing sets may be actuated by a single pump.

In one embodiment, the connector that mates with the cartridge includes a needle that is surrounded by a shield such that a tip of the needle is spaced rearwardly relative to an edge of the shield. The shield mates to a port on the cartridge and the needle provides a fluid connection to deliver or remove fluid. In the particular embodiment shown in fig. 9A-9B, the connector includes a septum that surrounds the needle. The connector mates to a port on the cassette and locks into place. Once fluid delivery or removal is complete, the needle and tubing can be removed, leaving the septum in place, thereby sealing the cassette (fig. 9B).

In another embodiment shown in fig. 10A-10B, the connector includes two separate fluid lines, i.e., tubes, each having its own needle and configured to prevent improper installation into the cassette. In this configuration, the connector snaps into place, providing positive feedback to the user that proper insertion has been achieved (fig. 10B). The number of fluid lines may be increased as required for a particular application. 11A-11B show an example of a tubing set that includes three fluid lines, one of which provides a pressure relief. The pressure relief line may include a pressure relief valve and a filter, as shown. Once fluid delivery or removal is complete, the needle and tubing can be removed by gently squeezing the connector while pulling it away from the cassette.

The cartridge may be additionally accessed by making additional connections with the needle. The connector may mate with the cartridge by any suitable mechanism, such as threads, luer lock, friction fit, and snap fit. One or more tubing sets (e.g., each for sample and media transport and waste removal) may also be packaged together with one or more cassettes in a kit.

The tubes of the tube bank may be made of any suitable material, such as polyethylene, polytetrafluoroethylene and Tygon @flexibletubes. The connector and needle may be made of metal (e.g., stainless steel), plastic, ceramic, or a combination thereof.

Application method

The cassettes and tubing sets of the invention can be used for cell growth or maintenance, including detection, enumeration, diagnosis, and therapeutic response. Exemplary fields of use include testing microbial loads of liquids, air or surface samples; testing the microbial load of an industrial sample, a sterile pharmaceutical product sample, a non-sterile pharmaceutical product sample; and the anaerobic microbial load of the test sample. Any culturable cell type can be sampled in conjunction with the cassettes described herein, including bacteria, cyanobacteria, protozoa, fungi, mammalian cells, plant cells, or other eukaryotic cells. The cassette can be used for aerobic and anaerobic tests. The cassettes may be packaged in sterile kits or sterilized by the end user (fig. 12). The cassette will typically be used in a laboratory environment using a laminar flow hood or isolation chamber.

In a typical experiment, the cassette is sterilized, or the cassette is set to be pre-sterilized. The pre-rinse fluid, the sample medium fluid, and the post-rinse fluid are introduced through the sample injection port. After entering the cartridge, the fluid will travel through a fluid distribution channel, which may comprise a spiral stabilizing channel to smooth out excess turbulence before the fluid passes through the face of the membrane. The introduction of these fluids into the sealed chamber through the face of the septum can cause residual air trapped in the cartridge to compress as the column of fluid rises, resulting in a protective barrier to the underside of the optical window. After the sampling and rinsing steps are completed, additional air may be pumped into the cartridge to ensure that all fluid is forced through the septum and/or media pad. This can result in the chamber above the diaphragm being pressurized.

The nutrient medium is then pumped into the media pad via the media injection port. The media is absorbed by the media pad and provides a source of food for a prescribed period of time, for example, at least 7 or 14 days. A septum, for example, having a pore size of 0.45 μm, and pressurization of the chamber between the cap and the septum may be used to prevent excess nutrient medium from passing through the septum.

When a drain port is present, excess sample or media fluid may be removed from the cartridge via the drain port. Alternatively, excess sample fluid may be removed via the media injection port. A preset amount of media may also be delivered via a media injection port, with gas displaced inside the cartridge vented through a sample injection port, a vent port, or a pressure relief valve. Other configurations are possible.

The cartridge is preferably capable of handling large volumes of fluid, for example, 2 liters of sample and 2 liters of rinse solution. The exact amount of fluid will depend on the sample.

The cassette may be sterilized by any suitable method. Gas sterilization, for example by ethylene oxide, may be performed by: pressurizing the cartridge with gas, retaining the gas for a predetermined amount of time, and evacuating the gas under high vacuum.

In one embodiment, after the filtration process and nutrient transfer are complete, the cassette is placed in an incubator at a predefined temperature (e.g., within a Growth Direct chamber system) and stored while awaiting imaging. At predefined time intervals, the cartridge is automatically removed and sent through an imaging station where it is subjected to a high intensity excitation light of a specific wavelength. In response, any microbial growth present on the membrane will naturally fluoresce. The fluorescence image is captured by means of an optical filter and a CCD camera and the fluorescing object is recorded. Over time, subsequent images are captured, and these fluorescing objects are measured and monitored to measure growth. Those that meet the growth criteria are considered colonies. Other fluorescing objects are characterized as debris.

The invention will now be further described with respect to certain preferred embodiments.

The cassette of the present invention houses a 0.45 micron black mixed cellulose ester filter membrane supported by a media pad made of sintered polyethylene beads. Samples containing mixed microorganisms were pumped via a peristaltic pump through Tygon S-50-HL tubing into the sample injection ports of the cartridges. During sample addition, the tubing on the media injection port is sealed and the tubing on the drainage port is opened. Fluid D (peptone-tween 80 flush Fluid) was pumped during the following sample addition, followed by addition of air to force all Fluid through the septum and pressurizing the upper chamber to 10 psi. The sample injection port is then sealed with a clamp and the media injection port is opened. Liquid Schaedler blood media was added via the media injection port and pumped into the media pad under the septum to replace the Fluid D rinse with growth media.

The tubing was then removed from all ports and the ports were sealed with a sealing film (Parafilm). The cassette was incubated at 32.5 ℃. The cassette was manually placed in the imager at different time intervals (incubation between images). About 600 watts/cm²The excitation light at 460-500nm is provided by a blue LED, which is tuned by an optical bandpass filter. The 505-550nm bandpass filter allows the emitted light to be captured by a CCD camera.

To perform these experiments, the lid was removed before the cartridge was placed in the imager and replaced before continuing the incubation. The imager captures nine tiles at various points in time, and the images are stitched together to display the complete box. Alignment of the cartridge is performed by eye and manually.

The time series in fig. 13 shows fluorescence images of colonies of microorganisms grown in the cassette. Although debris particles are initially present, the fluorescence of the growing microorganisms increases over time. The growing fluorescent spots can be detected as growing colonies using software algorithms and can be identified when they are still small, in part due to the resolution of the non-magnifying imaging system. The final panel in fig. 13 shows the image of the cassette obtained with a digital video camera under normal illumination at the end of the incubation period. As can be seen by comparing the last two panels, a one-to-one correspondence between fluorescent colonies and regular images.

OTHER EMBODIMENTS

All publications, patents and patent applications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention will be described in conjunction with specific embodiments, it will 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 which are obvious to those skilled in the art are intended to be within the scope of the invention.

Other embodiments are within the claims.

Claims (31)

1. A cell culturing device comprising a housing, the housing comprising:

i) a cover having a light transmissive window;

ii) a fluid distribution channel;

iii) a sample injection port fluidically connected to the fluid distribution channel;

iv) a base comprising a porous media pad;

v) a media injection port fluidly connected to the media pad; and

vi) an optional membrane disposed on the media pad,

wherein the lid mates to the base to form a sterile seal; the fluid distribution channel is disposed on the media pad; the dielectric pad or the optional membrane is viewable through the optically transmissive window; and the sample fluid introduced into the fluid distribution channel is uniformly distributed to the media pad.

2. The device of claim 1, further comprising a membrane disposed on the media pad, wherein cells in the sample fluid remain on the membrane and are viewable through the optically transparent window.

3. The device of claim 2, wherein the space between the cap and the septum is pressurizable.

4. The device of any one of claims 1-3, further comprising a drain port.

5. The device of any one of claims 1-3, further comprising an oxygen scavenger sufficient to oxygen-free the interior of the device.

6. The device of claim 5, further comprising an actuator for the oxygen scavenger.

7. The device of claim 6, wherein the actuator is activated by flipping the lid or by a pull tab.

8. The device of any of claims 1-3, further comprising a pressure relief valve.

9. The device of any one of claims 1-3, wherein the base further comprises a channel that releases pressure in the media pad when fluid is introduced.

10. The device of any one of claims 1-3, wherein the cover is removable.

11. The device of any one of claims 1-3, further comprising a media distribution channel connected to the media injection port and having a plurality of outlets around a circumference of the media pad, wherein media introduced via the media injection port is evenly distributed to the media pad via the media distribution channel.

12. The device of any one of claims 1-3, further comprising an oxygen indicator.

13. The device of any one of claims 1-3, wherein the fluid distribution channel comprises a helical channel.

14. The device of any one of claims 1-3, wherein the fluid distribution channel comprises a plurality of channels, and wherein sample fluid introduced into the fluid distribution channel is uniformly distributed to the media pad via the plurality of channels.

15. The device of any one of claims 1-3, wherein the fluid distribution channel is a single channel.

16. The device of claim 15, wherein the fluid distribution channel comprises an angled circumferential region around the media pad.

17. The device of any one of claims 1-3, further comprising a cover disposed on top of the fluid distribution channel.

18. The apparatus of claim 17, wherein the cover shapes the fluid flow to achieve uniform distribution to the media pad through a single channel.

19. The device of any one of claims 1-3, further comprising a vent port for venting the interior of the device upon introduction of liquid.

20. The device of claim 19, wherein the vent port is self-sealing.

21. A kit for detecting cells comprising the device of any one of the preceding claims, and a first tubing set comprising tubing having a connector with a needle and mated to the sample injection port, the media injection port, vent port, or drain port.

22. The kit of claim 21, wherein the connector further comprises a septum through which the needle passes.

23. The kit of claim 22, wherein the connector mates to the sample injection port, media injection port, vent port, or drainage port and seals the port with the septum when the needle and tubing are removed.

24. The kit of claim 21, further comprising a second tubing set and an optional third tubing set, each comprising tubing having a connector with a needle, wherein the connectors of the second tubing set and the optional third tubing set mate to one of the sample injection port, media injection port, vent port, and drain port.

25. The kit of claim 24, wherein the tubes of the first, second, and optional third tube sets share a common inlet or outlet.

26. The kit of claim 21, wherein the connector comprises a clip that snaps into the device.

27. The kit of claim 21, wherein the first tubing set further comprises a plurality of individual tubes for at least two of: sample introduction, media introduction, drainage and aeration.

28. The kit of claim 27, wherein the connector comprises a needle for each conduit.

29. The kit of claim 27, wherein one of the conduits is for venting.

30. The kit of claim 27, wherein the conduit for venting comprises a filter.

31. The kit of claim 27, wherein the conduit for venting comprises a pressure relief valve.