Tubing set with dual pressure regulating valve

Abstract

A microbial enumeration kit or system is provided having a plurality of cassettes with upper and lower chambers separated by a membrane, the kit further comprising a media reservoir fluidly connected to the cassettes by a tubing set, and a dual pressure valve mechanically coupled to the tubing set and configured to vary the pressure within the cassette to cause fluid from the fluid reservoir to pass through or interact with the membrane.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 387,172, filed December 13, 2022, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Many industries require measuring the number or presence of microorganisms in a sample. This is often referred to as microbial enumeration or detection. One method for measuring the number of microorganisms in a sample (or detecting their presence) is to expose a filtration membrane to the sample, trap the microorganisms in the sample on the membrane, culture the trapped microorganisms, and, optionally, count the number of colonies that grow during the culture.

[0003] Membrane filtration is therefore a commonly used method for concentrating and enumerating microorganisms present in a liquid sample, whereby a known volume of the sample is filtered through a membrane filter of defined pore size, retaining the microorganisms on the filter surface.

[0004] First, the liquid sample to be analyzed is typically diluted to reduce the microbial load and ensure the presence of single colonies on the filter. The membrane filter is selected with an appropriate pore size depending on the expected size of the microorganisms to be counted. Commonly used pore sizes are 0.45 μm or 0.2 μm.

[0005] The membrane filter is placed in a filter holder or manifold designed for microbial analysis. The filter holder is connected to a vacuum source, creating a pressure differential across the membrane. A known volume of diluted sample is aseptically poured or pipetted onto the membrane filter and placed in the filter holder. When a vacuum is applied, the liquid portion of the sample passes through the filter, leaving the microorganisms on the filter surface.

[0006] For efficient recovery of microorganisms, the filter is rinsed or washed with an appropriate solution (e.g., sterile buffered saline) to remove any residual sample or contaminants.

[0007] After filtration, the membrane filter containing the retained microorganisms is aseptically transferred to a suitable culture medium, such as an agar plate or broth. The selection of the medium depends on the specific requirements of the microorganisms to be counted. The culture medium containing the membrane filter is then incubated under appropriate conditions (temperature, time, and atmospheric conditions) to promote the growth of viable microorganisms. The incubation period is determined based on the expected growth characteristics of the target microorganisms.

[0008] After the incubation period, the membrane filters are examined and the number of visible microbial colonies growing on the filter surface is counted. The colonies are distinguished based on characteristics such as size, shape, color, and morphology. From the number of colonies obtained from the membrane filter, the number of viable microorganisms in the original sample is calculated, taking into account the dilution factor and the volume of the filtered sample.

[0009] Membrane filtration is particularly useful when sample volumes are large and microbial loads are low. It can concentrate microorganisms on the filter, making colony detection and enumeration easier than with direct plating. This method is widely adopted in various industries, including pharmaceuticals, food and beverages, water quality testing, and environmental monitoring.

[0010] An example of an automated processing system for enumeration and / or detection of microorganisms is the GROWTH DIRECT™ system offered by RAPID MICROBIOSYSTEMS™, located in Lowell, Massachusetts.

[0011] To ensure consistency in microbial counts, the physical properties of the membrane must be controlled when it is exposed to the sample and when the microorganisms are cultured on the membrane. Summary of the Invention

[0012] During microbial counting, a membrane (e.g., inside a cassette, etc.) is exposed to a sample, and microorganisms in the sample are captured on the membrane. Generally, the present disclosure provides improvements to systems or kits used to capture microorganisms on a membrane for the purpose of microbial counting. One challenge in microbial counting is controlling the distribution of the sample across the membrane. Some conventional systems use a vacuum to "pull" the sample through the membrane and into a chamber. One such vacuum system uses an open funnel that contains the sample, which can introduce contaminants other than the sample and invalidate the counting process. Furthermore, such systems require tension on the membrane and allow space for the membrane to expand.

[0013] The present disclosure provides a closed microbial counting kit (or system) that addresses the above-mentioned challenges to provide a more consistent microbial counting process. Specifically, the present disclosure provides a system including multiple cassettes, each having a membrane. The cassettes are fluidly connected to a closed reservoir by a tubing set. The tubing set includes a dual-state pressure valve positioned to maintain the membrane in a relatively "flat" state during exposure to a sample. Maintaining the membrane in a relatively flat state ensures that the entire membrane is exposed to (or wetted by) the sample. Furthermore, the dual-state pressure valve is positioned to ensure that rinsing fluid is properly filtered through the membrane while the membrane is exposed to the sample, and that the sample remains within a designated chamber of the cassette. [Brief explanation of the drawings]

[0014] To easily identify the description of a particular element or act, the most significant digit(s) of a reference number refers to the number of the figure in which that element is first introduced. [Figure 1A] FIG. 1A illustrates a subject embodiment according to one embodiment. [Figure 1B] FIG. 1B illustrates a subject embodiment according to one embodiment. [Figure 1C]FIG. 1C illustrates a subject embodiment according to one embodiment. [Figure 2] FIG. 2 illustrates a subject matter embodiment according to one embodiment. [Figure 3] FIG. 3 illustrates a subject matter embodiment according to one embodiment. [Figure 4] FIG. 4 illustrates a subject matter embodiment according to one embodiment. [Figure 5] FIG. 5 illustrates a subject matter embodiment according to one embodiment. [Figure 6] FIG. 6 illustrates a subject matter embodiment according to one embodiment. [Figure 7] FIG. 7 illustrates a subject matter embodiment according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0015] 1A illustrates a microorganism counting system 100 that can be provided in accordance with embodiments of the present disclosure. The microorganism counting system 100 includes a cassette 102, a dual pressure valve 104, and a reservoir 106 fluidly connected by a tubing set 108. Generally, the cassette 102 is any of a variety of containers or devices configured to allow for the growth of microorganisms, and the reservoir 106 is a fluid container configured to hold a rinse fluid, a sample containing microorganisms, etc. During use, fluid (not shown) flows from the reservoir 106 through the tubing set 108 into the cassette 102, and the dual pressure valve 104 maintains the pressure within the cassette 102 at one of two pressure states to ensure that the cassette 102 is filled with fluid from the reservoir 106 as intended, as described in more detail below.

[0016] 1B shows cassette 102 in more detail. As shown in this figure, cassette 102 has an upper chamber 110 and a lower chamber 112 with a membrane 114 disposed between the chambers. One of upper chamber 110 or lower chamber 112 can be filled with a growth medium for the microorganisms (e.g., nutrient broth, lysogeny broth medium, or the like). Additionally, cassette 102 has at least one port 116 to which tubing set 108 can connect, allowing fluid from reservoir 106 to enter cassette 102.

[0017] In some embodiments, membrane 114 can be a filter configured to capture microorganisms from a sample of fluid in reservoir 106. Operation of membrane 114 may require a predetermined pressure to ensure that the membrane is secure (e.g., does not vibrate or tear) and is sufficiently covered with fluid. An example process for capturing microorganisms on membrane 114 is described below.

[0018] FIG. 1C shows the dual pressure valve 104 in more detail. As shown in this figure, the dual pressure valve 104 includes a manually operated clamp that, in one actuation state, compresses the tubing set 108, increasing the pressure on one side of the tubing set 108, and in another actuation state, expands the tubing set 108, decreasing the pressure on one side of the tubing set 108. In some embodiments, the tubing set 108 includes a vent port 118 located on one side of the dual pressure valve 104 to stabilize the pressure in the tubing set 108 on one side of the dual pressure valve 104 at atmospheric pressure, while the pressure in the tubing set 108 on the other side of the dual pressure valve 104 is maintained at a higher pressure. Additionally, the tubing set 108 can include a flow restrictor 120 configured to restrict fluid flow below a certain pressure and a coupler 122 located to couple to the port 116. In some embodiments, the coupler 122 can be a low-pressure check valve or can be open to the atmosphere.

[0019] FIG. 2 illustrates a method 200 for processing a sample for microbial counting. Method 200 can be used with microbial counting system 100 or other systems similar to microbial counting system 100. Generally, method 200 can be performed such that a membrane or filter (e.g., membrane 114 of microbial counting system 100) is sufficiently covered by the sample to capture microorganisms from the sample for growth and counting. Method 200 can begin at block 200. In block 202, "Load sample into upper chamber of multi-chamber cassette having membrane between upper and lower chambers," the upper chamber 110 is filled with fluid from reservoir 106. For example, reservoir 106 can be movable such that gravity allows fluid to flow from reservoir 106 through tubing set 108 to upper chamber 110. In some examples, reservoir 106 can include a one-way valve that allows air to enter reservoir 106 while allowing fluid to exit. In particular examples, reservoir 106 may include a filter and / or a valve to prevent the fluid within reservoir 106 from becoming contaminated by airborne microorganisms.

[0020] Continuing with block 204, "Increase pressure in upper chamber relative to lower chamber," the pressure in the upper chamber 110 increases relative to the lower chamber 112, causing the fluid to flow through the membrane 114 sufficiently to cover the membrane 114 and trap microorganisms in the fluid on the membrane 114.

[0021] 3 illustrates a microbial counting system 300 according to an embodiment of the present disclosure. The microbial counting system 100 includes a cassette 302, dual pressure valves 304a, 304b, and 304c, and reservoirs 306a, 306b, 306c, and 306d fluidly connected by a tubing set 308. Multiple distinct reservoirs are provided, including a pre-rinse reservoir 306a, a sample reservoir 306b, a post-rinse reservoir 306c, and a media reservoir 306d. Sample reservoir 306b can contain a "sample" liquid, solid, gel, or aerosol medium, or, alternatively, a liquid medium to be analyzed by the microbial counting method contemplated herein. Media reservoir 306d can contain a liquid microbial growth medium. Microbial counting system 300 further includes an atmospheric vent 316 coupled to upper chamber 310 (although in some embodiments, atmospheric vent 316 can be omitted to allow for pressurized sample preparation). In some embodiments, microbial counting system 300 can include a fluid pump configured to pump fluid within tubing set 308. However, for clarity, the fluid pump is not shown in this figure.

[0022] During operation, fluid from reservoirs 306a, 306b, 306c, and 306d can flow (e.g., by pumping, gravity, etc.) from the reservoirs to cassette 302. Additionally, dual pressure valves 304a, 304b, and 304c can be actuated to vary the pressure in upper chamber 310 relative to the pressure in lower chamber 312, or vice versa.

[0023] In another embodiment, media from media reservoir 306d can pass through dual pressure valve 304a. In these embodiments, lower chamber 312 can be used as a vent path.

[0024] 4 illustrates a method 400 that can be implemented to capture microorganisms from a sample for microbial enumeration. Method 400 can be used with microbial counting system 300 or other systems similar to microbial counting system 300. In general, method 400 can be implemented to ensure that a membrane or filter (e.g., membrane 314 of cassette 302) is sufficiently covered by a sample (e.g., fluid from sample reservoir 306b) to capture microorganisms from the sample for the purposes of growing and enumerating the microorganisms.

[0025] Method 400 may be performed by a controller having a memory storing logic for carrying out the method and a processor configured to execute the logic. The controller may be incorporated into microbial counting system 300 or may be a separate device. While Figure 4 depicts a sequence of specific steps in a particular order, one skilled in the art will recognize that an exemplary method may perform more steps, omit some steps, and / or perform steps in a different order.

[0026] Method 400 may begin at block 402. In block 402, "Fill Cassette with Pre-Rinse Fluid," cassette 302 may be filled with pre-rinse fluid from pre-rinse reservoir 306a. In particular, upper chamber 310 of cassette 302 may be filled with fluid from pre-rinse reservoir 306a during a period when the pressure in upper chamber 310 is low (e.g., 1.5 pounds per square inch (PSI) above atmospheric pressure or less, 0.5 PSI to 1.5 PSI above atmospheric pressure, or the like).

[0027] Continuing with block 404, "Temporarily increase pressure in upper chamber to force liquid through membrane," temporarily increases pressure in upper chamber 310, forcing fluid in cassette 302 (e.g., fluid added in block 402) through membrane 314 and into lower chamber 312. For example, dual pressure valve 304b can be closed to pinch (or restrict) tubing set 308 between upper chamber 310 and atmosphere vent 316, increasing pressure in upper chamber 310. In some examples, closing dual pressure valve 304b increases pressure to between 0.5 PSI and 4 PSI. In some embodiments, pressure in the upper chamber can be increased for a set period of time (e.g., 30 seconds, 60 seconds, 90 seconds, etc.), while in other examples, pressure in the upper chamber can be increased until a condition is met (e.g., liquid flows through membrane 314 to lower chamber 312).

[0028] Continuing with block 406, "Return cassette to low pressure state," causes the upper chamber 310 of the cassette 302 to return to a low pressure state (e.g., atmospheric pressure plus 1.5 PSI or less). Continuing with block 408, "Fill cassette with sample fluid," causes the cassette 302 to be filled with sample fluid from the sample reservoir 306b. In particular, the upper chamber 310 of the cassette 302 can be filled with fluid from the sample reservoir 306b while the pressure in the upper chamber 310 is in the low pressure state.

[0029] Continuing with block 410, "Temporarily increase pressure in upper chamber to force liquid through membrane," temporarily increases pressure in upper chamber 310, forcing fluid in cassette 302 (e.g., fluid added in block 408) through membrane 314 and into lower chamber 312. For example, dual pressure valve 304b can be closed to pinch (or restrict) tubing set 308 between upper chamber 310 and atmosphere vent 316, increasing pressure in upper chamber 310. In some examples, closing dual pressure valve 304b increases pressure to between 0.5 PSI and 4 PSI. In some embodiments, pressure in the upper chamber can be increased for a set period of time (e.g., 30 seconds, 60 seconds, 90 seconds, etc.), while in other examples, pressure in the upper chamber can be increased until a condition is met (e.g., liquid flows through membrane 314 to lower chamber 312).

[0030] Continuing with block 412, "Return cassette to low pressure," causes the upper chamber 310 of the cassette 302 to return to a low pressure state (e.g., atmospheric pressure plus 1.5 PSI or less). Continuing with block 414, "Fill cassette with post-rinse fluid," causes the cassette 302 to be filled with post-rinse fluid from the post-rinse reservoir 306c. In particular, the upper chamber 310 of the cassette 302 can be filled with fluid from the post-rinse reservoir 306c during the period when the pressure in the upper chamber 310 is in the low pressure state.

[0031] Continuing with block 416, "Increase pressure in upper chamber to force liquid through membrane," increases the pressure in the upper chamber 310, forcing fluid in the cassette 302 (e.g., fluid added in block 414) through the membrane 314 and into the lower chamber 312. For example, the dual pressure valve 304b can be closed to pinch (or restrict) the tubing set 308 between the upper chamber 310 and the atmospheric vent 316, increasing the pressure in the upper chamber 310. In some examples, closing the dual pressure valve 304b increases the pressure to between 0.5 PSI and 4 PSI. In some embodiments, the pressure in the upper chamber can be increased for a set period of time (e.g., 30 seconds, 60 seconds, 90 seconds, etc.), while in other examples, the pressure in the upper chamber can be increased until a condition is met (e.g., liquid flows through the membrane 314 to the lower chamber 312).

[0032] Continuing with block 418, "Fill cassette with media fluid," the cassette 302 can be filled with media fluid from the media reservoir 306d. In particular, while the pressure in the upper chamber 310 is in a high pressure state, the lower chamber 312 of the cassette 302 can be filled with fluid from the media reservoir 306d.

[0033] Continuing with block 420, "Increase pressure in lower chamber to force media fluid to interact with membrane," increases the pressure in lower chamber 312, forcing media fluid in cassette 302 (e.g., media fluid added in block 418) to interact with membrane 314. For example, dual pressure valve 304c can be closed to pinch (or restrict) tubing set 308 between lower chamber 312 and media reservoir 306d, increasing the pressure in lower chamber 312. Additionally, dual pressure valve 304a can be closed to pinch (or restrict) tubing set 308 between the other reservoir and upper chamber 310, increasing the pressure in lower chamber 312. In some examples, the pressure in lower chamber 312 can be increased from 0.5 PSI to 4 PSI. In some embodiments, the pressure in the upper chamber can be increased for a set period of time (e.g., 30 seconds, 60 seconds, 90 seconds, etc.). While in other instances, the pressure in the lower chamber can be increased until a condition is met (eg, movement of liquid through tubing set 308 stops, etc.).

[0034] In some embodiments, method 400 can include the further step of separating cassette 302 from tubing set 308 and treating the cassette with a microbial culture process (e.g., exposure to light, darkness, heat, moisture, etc.).

[0035] FIG. 5 illustrates a microbial counting system 500 that includes multiple cassettes. In particular, microbial counting system 500 includes cassettes 502a, 502b, and 502c, each of which is coupled to a reservoir 506 through a tubing set 508. Cassettes 502a, 502b, and 502c can be filled with fluid from reservoir 506 using similar processes described elsewhere herein (e.g., with respect to FIGS. 2 and 4). Tube set 508 is coupled (e.g., mechanically or in a similar manner) to a single dual pressure valve 504, which can be utilized to effect pressure changes within cassettes 502a, 502b, and 502c, as described elsewhere herein. Additionally, microbial counting system 500 can include multiple reservoirs 506 (e.g., such as microbial counting system 300 of FIG. 3).

[0036] In other examples, multiple dual pressure valves 504 may be provided. For example, FIG. 6 shows a microbial counting system 600 including cassettes 602a, 602b, and 602c, each of which is coupled to a reservoir 606 through a tubing set 608. Cassettes 602a, 602b, and 602c may be filled with fluid from reservoir 606 using similar processes described elsewhere herein (e.g., with respect to FIGS. 2 and 4). Tube set 608 is coupled (e.g., mechanically or in a similar manner) to multiple dual pressure valves. In particular, tubing set 608 is coupled to dual pressure valves 604a, 604b, and 604c. In this manner, the pressures within cassettes 602a, 602b, and 602c may be individually controlled.

[0037] As mentioned above, in some embodiments, exemplary microbial counting systems (e.g., microbial counting system 100, microbial counting system 300, microbial counting system 500, microbial counting system 600, etc.) may include a pump. FIG. 7 illustrates a microbial counting system 700 according to an embodiment of the present disclosure. The microbial counting system 700 includes a cassette 702, a dual pressure valve 704, and a reservoir 706 fluidly connected by a tubing set 708. A pump 710 is further provided to assist in moving fluid (e.g., via capillary action, etc.) from the reservoir 706 to the cassette 702 through the tubing set 708. An atmospheric vent 712 is further provided. During operation, fluid can flow from the reservoir 706 to the cassette 702. Additionally, dual pressure valve 704 can be actuated to increase or decrease the pressure in the upper and / or lower chambers of cassette 702 as described herein to assist in wetting the membrane within cassette 702 with fluid from reservoir 706.

[0038] It should be noted that the exemplary devices shown in the block diagrams above represent one functionally illustrative example of many potential embodiments. Thus, the division, omission, or inclusion of block functions shown in the accompanying figures does not imply that the hardware components, circuits, software, and / or elements for implementing those functions are necessarily divided, omitted, or included in the embodiments.

[0039] Some embodiments may be described using the phrase "in one embodiment" or "embodiment" and their derivatives. These terms mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Multiple appearances of the phrase "in one embodiment" in various places in the specification do not necessarily refer to the same embodiment. Furthermore, unless otherwise specified, it is recognized that the features described above can be used in any combination. Thus, features described separately can be used in combination with each other unless they are expressly stated to be incompatible with each other.

[0040] Some embodiments may be described using the terms "coupled" and "connected," along with their derivatives. These terms are not necessarily intended as synonyms for each other. For example, some embodiments may be described using the terms "connected" and / or "coupled" to indicate that two or more elements are in direct physical or electrical contact with each other. However, the term "coupled" may also mean that two or more elements are not in direct contact with each other, but yet still cooperate or interact with each other.

[0041] It is emphasized that the Abstract of the Disclosure is provided to allow the reader to quickly grasp the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. Moreover, in the foregoing Detailed Description, various features are grouped together in a single embodiment to streamline the disclosure. This method of disclosure should not be interpreted as reflecting an intention that the claimed embodiment requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in some, but not all, features of a single disclosed embodiment. Accordingly, the following claims are incorporated into the Detailed Description, with each claim standing on its own as a standalone embodiment. In the appended claims, the terms "including" and "in which" are used as plain English versions of "comprising" and "wherein," respectively. Furthermore, the terms "first," "second," "third," etc. are used merely as labels and are not intended to impose numerical requirements on their objects.

[0042] The above description includes examples of the disclosed architecture. Of course, it is not possible to describe every conceivable combination of components and / or techniques, but one of ordinary skill in the art will recognize that many further combinations and permutations are possible. Accordingly, the novel architecture is intended to encompass all such changes, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

1. Filling at least one cassette with a sample fluid; increasing the pressure within the at least one cassette; reducing the pressure in the at least one cassette; filling the cassette with a media fluid; increasing the pressure in the at least one cassette.

2. the cassette comprising an upper chamber, a lower chamber, and a membrane disposed between the upper chamber and the lower chamber; The method comprises: filling the upper chamber of the at least one cassette with the sample fluid; and increasing the pressure in the upper chamber of the at least one cassette to force the sample fluid through the membrane into the lower chamber.

3. filling the upper chamber of the at least one cassette with a pre-rinse fluid; increasing the pressure in the upper chamber of the at least one cassette.

4. filling the upper chamber of the at least one cassette with a post-rinse fluid; increasing the pressure in the upper chamber of the at least one cassette.

5. filling the lower chamber of the at least one cassette with the media fluid, which may be a solid, liquid, gel, gas, or aerosol; and increasing the pressure in the lower chamber of the at least one cassette to force the media fluid to interact with the membrane.

6. 3. The method of claim 2, wherein the pressure in the upper chamber increases by 1 pound per square inch (PSI) or more, or by 4 PSI or more.

7. at least one cassette; a fluid reservoir; a tubing set fluidly connecting the fluid reservoir to the at least one cassette; a dual pressure valve mechanically coupled to the tubing set and configured to vary the pressure within the at least one cassette.

8. Filling the at least one cassette with a sample fluid; increasing the pressure within the at least one cassette; reducing the pressure within the at least one cassette; Filling the at least one cassette with a media fluid; The system of claim 7 , further comprising a controller configured to increase the pressure in the at least one cassette.

9. The system of claim 7 , wherein the cassette comprises an upper chamber, a lower chamber, and a membrane disposed between the upper and lower chambers.

10. The controller filling the upper chamber of the at least one cassette with the sample fluid; 10. The system of claim 9, further configured to increase pressure in the upper chamber of the at least one cassette to force the sample fluid through the membrane to the lower chamber.

11. The controller filling the upper chamber of the at least one cassette with a pre-rinse fluid; The system of claim 7 , further configured to increase the pressure in the upper chamber of the at least one cassette.

12. The controller filling the upper chamber of the at least one cassette with a post-rinse fluid; The system of claim 7 , further configured to increase the pressure in the upper chamber of the at least one cassette.

13. The controller filling the lower chamber of the at least one cassette with the media fluid, which may be a solid, liquid, gel, gas, or aerosol; The system of claim 7 , further configured to increase the pressure in the lower chamber of the at least one cassette to force the media fluid to interact with the membrane.

14. 8. The system of claim 7, wherein the pressure in the upper chamber increases by more than 1 pound per square inch (PSI), or more than 4 PSI.

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