HK1190109B - Fixation filter assembly - Google Patents

Description

Stationary filter assembly

Cross reference to related applications

This application claims the benefit of U.S. patent application No.12/907,330, filed on 19/10/2011, the contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to sample collection and disposal in a fluid environment, and more particularly to the collection and immobilization of microorganisms and specific samples in a water ecosystem. More broadly, the present disclosure relates to sampling in any aquatic environment, such as in a reservoir and/or any natural or artificial water storage configuration, and with respect to any industry that may be related to water quality.

Background

Sample collection and disposal in a fluid environment is undoubtedly the subject of various reports. Marine research typically requires continuous information about the marine food chain, which may generally include (1) aquatic bacteria and protists, (2) phytoplankton, (3) zooplankton, and (4) fish and higher forms, crustaceans, reptiles, marine mammals, and the like, where, in ascending order, each may become the food source for the next. Bacteria, protists, phytoplankton and zooplankton can be measured in the ocean and studied for their relevance and impact on fisheries and environmental health. Information about their abundance and vertical and horizontal distribution in continental-shelf waters, deep sea and inland waters may also be required. Obtaining data accurately, continuously, and at a wide level of spatial coverage remains an existing problem.

It may be noted that the united states coast guard is operable to define relevant biological species (from the perspective of invasive species in ship ballast water) as follows: zooplankton, organic matter with the minimum size more than or equal to 50 mu m; phytoplankton/protozoa, organic matter with a minimum size less than 50 μm and more than or equal to 10 μm; bacteria, organic matter of minimum size < 10 μm (this operating group will also include a variety of smaller photosynthetic phytoplankton and cyanobacteria).

Accordingly, there is a need for methods and apparatus to automate the collection of samples for key analyses including enumeration, phylogenetic nature, molecular and metabolic effects and viability of various microbial, phytoplankton and zooplankton components of the food chain. More specifically, the method may include automated field rate studies involving tracer incubation and sample preservation.

Disclosure of Invention

In a first exemplary embodiment, the present disclosure relates to a filter assembly for filtrate flow, the filter assembly comprising: for having a density D₁And comprises a flow having a density D₂A reservoir of a second fluid; an interior space for a filter media; and a reservoir plate separating the reservoir from the interior space, wherein the reservoir plate comprises at least two openings in communication with the reservoir and the interior space. Due to the density difference of the first fluid and the second fluid, the second fluid in the reservoir is able to exchange with the first fluid in the inner space by convection.

In a second exemplary embodiment, the present disclosure is directed to a method for measuring a particle from a particle having a first density D₁The method comprising the step of providing a filter assembly comprising an inlet and an outlet and a reservoir comprising a density D₂Of the second fluid. A filter media is positioned within the filter assembly between the inlet and the outlet of the filter assembly, wherein the filter media is positioned away from the reservoir and within an interior space separated from the reservoir by a reservoir plate. The reservoir plate comprises one opening for the flow of filtrate and at least two openings communicating with the reservoir and the inner space. The filtrate may then be passed through a filter assembly and the microorganisms and/or particulate material collected on the filter media and exposed to a second fluid under the following conditions: (1) when the filtrate has a density D₁Greater than D₂While filtrate flows out of the interior space into the reservoir, and a second fluid displaces filtrate in the interior space; or (2) the density D of the filtrate₁Is less than D₂When the second fluid flows from the reservoir into the interior space, and the second flowThe body replaces filtrate in the interior space. In the latter case, the filter assembly may be inverted to facilitate the flow of the second fluid into the interior space to replace the filtrate.

Drawings

The features, operation, and advantages of the present invention will be better understood from the following detailed description of the preferred embodiments when read in conjunction with the accompanying drawings, in which:

FIG. 1 is a front view of a filter assembly of the present disclosure;

FIG. 2 is a perspective view of the filter assembly of FIG. 1;

FIG. 3 is an exploded view of the filter assembly of FIG. 1;

FIG. 3A is an enlarged view of a portion of the reservoir plate of the filter assembly of FIG. 1;

FIG. 4 is a cross-sectional view of the filter assembly of FIG. 2 taken along line 4-4;

5A-5D are schematic diagrams illustrating four stages in the filtration process of the filter assembly of the present disclosure; while

Fig. 6 is a time-lapse illustration of a laminar convective flow process, seen down on the filter media, where the dye is used to replace the preservative (see below) and has a density less than the media being filtered.

Detailed Description

Still other objects and advantages of the present invention will become readily apparent to those skilled in this art from the following detailed description, wherein there is shown and described a preferred embodiment of this invention. As will be realized, the invention is capable of other and different embodiments and its several details are capable of modifications in various respects, all without departing from the invention. Accordingly, the description is illustrative in nature and not restrictive.

Leading edge microbial ecology is increasingly emphasizing the collection of samples for subsequent metagenomic (DNA, ribosomal RNA [ rRNA ]; quantitative genotyping of resident microbial biological regions), macrotransgenic (messenger RNA [ mRNA ]; assessment of the role of those metabolic genes) and macroprotein (identity and numerical value of the protein gene products produced) analyses that describe the identity and role of microorganisms in their environment. One potential benefit of the methods herein is that such information can now be collected without the need to culture the organism in a laboratory, which currently captures less than 1% of the viable microorganisms present in the environment. The collection of particulate material will therefore be critical (essential) and since samples are currently collected on site for a period of time, a method for chemical preservation of such material is also achieved which is now compatible with metagenomic, macrotransgenic and macroproteomic analysis.

The filter assembly of the present disclosure may thus have a wide potential use for the collection and preservation of filtered samples that are collected by robotic time sequential water samplers for subsequent molecular analysis. The methods and apparatus of the present disclosure are applicable to any water sampling condition, such as deep sea sampling, and may also be compatible with automated time sequential instrumentation. In addition, the sampling herein is suitable for collecting biological and/or organic and/or inorganic particulate components, such as suspended sediment, debris, snow material, fecal material, and the like. Since bacteria will be a component of such a collection material, it is important that such a collection is preserved in a manner that prevents bacterial decomposition. If bacterial decomposition is not prevented, the composition and physical properties of the inanimate material under study will be altered.

The filter assembly of the present disclosure is shown in a front view in fig. 1. The assembly 10 is a stand-alone in-line filter that, in addition to collecting particulate samples onto the surface of the filter media, is also capable of chemically preserving the collected particulate samples by means of a physical process that does not require moving parts. The expression "no moving parts" is to be understood to mean that no individual elements of the filter assembly need to be moved in order to provide filtration and preservation. Instead, the filter assembly utilizes laminar convective flow driven by the difference in density between the user-selected chemical preservative (fixing agent), which can be retained in the reservoir of the filter during sampling, and the aqueous medium from which the sample is to be filtered.

As used herein, "laminar convective flow" or "laminar convective flow" refers to a relatively smooth gravitational flow of a fluid that results from a relative difference in density of the respective fluids, and thus may be alternatively referred to as gravitational convective flow. Gravity on the two fluids of different densities in the common space may cause the higher density fluid to sink and the lower density fluid to move to an over-head (over-riding) position relative to the higher density fluid.

As used herein, "preservative" or "fixative" refers to a material used to preserve the structure of a freshly collected biological sample in a state that most closely represents the structure and composition of the original fresh state. These materials can chemically react with tissue macromolecules to form intermolecular and intramolecular crosslinks that increase the structural stability of the macromolecule. They may also inactivate autolytic enzymes and may make tissues more resistant to enzymatic degradation from autolysis (autolysis) and microbial activity. These preservatives typically comprise at least two components, one or more active ingredients (fixing agents) and a carrier, which is often a buffer system. The carrier may comprise materials such as metabolic poisons (azides, cyanides), divalent cations (Ca, Mg), penetrants (DMSO), surfactants (detergents-e.g. TWEEN), electrolytes or non-electrolytes, which are added to adjust permeability.

Depending on the subject matter studied, the preservatives can be, for example, alcohol-based (methanol, ethanol, propanol, butanol), aldehydesBasic (formaldehyde, glutaraldehyde), acid-based (acetic acid, dilute sulfuric acid), or mercury-based (mercuric chloride). Another preferred type-available from Ambion, Austin, TexasA solution, which is an aqueous non-toxic tissue storage agent that rapidly penetrates into tissue to stabilize and protect cellular DNA and RNA.The solution eliminates the need to immediately process the tissue sample or freeze the sample in liquid nitrogen for later processing. The tissue mass may be harvested and submergedIn solution for storage without compromising the quality or quantity of RNA/DNA obtained after subsequent RNA/DNA isolation.

The filter assembly 10 is a self-contained module with no moving parts, which may also include an inlet 34 and an outlet 36, the inlet 34 and outlet 36 may be fluidly connected to appropriate sample dispensing valves and flow sources (pumps, pipettes, not shown). Active transport of the preservative for the filtered sample occurs within the filter assembly 10, making application of the process very simple and adaptable for various filtering applications.

Fig. 2 is a perspective view of the filter assembly of fig. 1.

Fig. 3 is an exploded view of the filter assembly of fig. 1 and 2, showing, from bottom to top, an annular reservoir 20, which may contain a user-selected chemical preservative, a sealed reservoir plate 22, a filter support plate 26, and an overhead sample inlet plate 30, the filter support plate 26 supporting a filter media 28, the sample inlet plate 30 serving to close the assembly 10. The expression "reservoir plate" may be understood as any structure that serves to isolate the filter medium from the reservoir and also allows fluid to flow through the opening(s) therein to supply preservative to the filter medium after filtration has taken place, as will be explained more fully below.

The named elements may be joined together by means of a threaded cap 32, which threaded cap 32 may be screwed into an external thread on the outside of the reservoir 20. A second O-ring 42 may be mounted on the reservoir to seal it with the cap 32. The third O-ring 44 may be located on top of a central post 60, the central post 60 being located in the center of the reservoir, and may seal the reservoir 20 to the bottom of the reservoir plate 22. The reservoir plate 22 may also include an outlet 29 for filtrate to flow from the dead space (dead space) 50. A fourth O-ring 46 may be mounted on the underside of the sample inlet plate 30 to further seal the assembly 10. It should be noted that other means for mechanically engaging the elements (e.g., clamping means, bolting means, etc.) may be provided.

The named elements other than the filter media 28 may be molded from a relatively inert plastic (e.g., seawater, fresh water, or saline solution for the media being filtered), such as poly (vinyl chloride) (PVC). Other thermoplastics may include polyolefins such as polyethylene, polypropylene, and the like. The filter media may comprise any of a variety of filter materials including, but not limited to, for example, glass fibers, cellulose acetate fibers, or polycarbonate membranes that have been irradiated and etched to provide a plurality of relatively fine pores. One form of polycarbonate film is under the trade name Nuclecore^TMSold and available from Whatman, Ltd, the polycarbonate film includes defined pore sizes. The thickness of the fiber filter may be about 0.007-0.010 inches, while the thickness of the polycarbonate film may be about 0.002-0.003 inches. The nominal pore size of the glass fiber filter may be about 0.8 μm, for cellulose acetate filters 0.22-0.45 μm, and for polycarbonate membranes about 0.1-0.45 μm.

The filter assembly 10 may preferably have standard dimensions for sampling, such as 22mm diameter, 47 or 50mm diameter and 147mm diameter. Accordingly, the filter diameter may be in the range of 15mm to 200 mm.

FIG. 4 is a cross-sectional view of the filter assembly of FIG. 2 taken along line 4-4. Of particular note, the inner surfaces 31, 23 of the sample inlet plate 30 and the reservoir plate 22, respectively, each of which inner surfaces 31, 23 may preferably possess a conical taper to smoothly direct fluid flow through the filter assembly 10 and to trap the filter media 28 within the small back-to-back conical interior dead space 50. Such a taper may be in the range of 1-15 deg., preferably 5-10 deg., to minimize the volume of the dead space. The expression conical taper is understood to mean a surface feature that changes at a constant rate from a relatively large diameter to a relatively small diameter. It will also be appreciated that the inner surfaces 31 and 23 may be concave, meaning that they may also vary from a relatively larger diameter to a relatively smaller diameter, but not at a constant rate. The use of conical tapers and/or concave surface features may provide for relatively improved laminar flow of fluids, such as the preservative fluids described herein.

Likewise, fluid communication between the annular reservoir volume V and the conical interior dead space 50 of the filter assembly 10 may occur via a plurality of smaller diameter holes 24 in the reservoir plate 22, the smaller diameter holes 24 being in fluid communication with the filter support plate. That is, fluid communication between the reservoir volume V and the tapered interior 50 of the filter assembly 10 may occur via a plurality of smaller diameter holes 24 (shown in an enlarged perspective view) in the reservoir plate 22.

The filter support plate 26 itself may preferably include a plurality of concentric grooves 27 to facilitate further distribution of the flow of fluid within the filter unit. The filter support plate 26 preferably includes radial scalloped grooves 25, the radial scalloped grooves 25 intersecting a plurality of concentric grooves 27 to provide a fluid flow path between the support plate 26 and the abutting filter media 28, preferably the size of the holes 24 in the reservoir plate may vary relative to the volume of the filter unit and the fluid being filtered, but may preferably be about 0.020 ±.002 inches in diameter. Accordingly, the diameter of each bore 24 itself may preferably be in the range of about 0.015 inches to 0.025 inches. The filter unit includes an inlet 34 and an outlet 36, and the aperture size of the inlet 34 and the outlet 36 may preferably be about 0.625 inches. It will be appreciated that the inlet and outlet are preferably of any size such that they are small enough that fluid exchange into and out of the reservoir does not occur within the same well; if the diameter is too large, this exchange will occur.

To better ensure that the flow paths of the preservative and the filtrate remain separated during operation, the two apertures 24 may preferably be arranged radially at an angle of about 90-180 ° to each other. It is contemplated that there may be more than 2 holes. For example, 3 to 10 holes may be utilized, which may be arranged to provide two separate flow paths. In terms of the number of pores, a larger number of pores can increase the flow rate of the preservative, and it was also observed that the loss of the preservative during filtration increased. Accordingly, a balance between flow rate and preservative loss was found, which balance has been observed to be best achieved by means of using 2 holes (as mentioned above).

Referring now to fig. 5B, in order for laminar convective flow to occur in the filter unit 10, the densities of the two fluids, preservative P and filtrate F, must be different. The density difference between P and F can be plus or minus 0.01g/cc, which is sufficient to maintain laminar convective flow. A greater density difference between the fluids may provide faster flow. For example, the density differential can be plus or minus 0.05g/cc, or plus or minus 0.01g/cc up to 0.50 g/cc. Accordingly, the density difference between the preservative P and the filtrate F can be in the range of plus or minus 0.01g/cc up to 0.50 g/cc. Convection flow then begins when one of the wells is exposed to filtrate with the indicated density difference. Convective flow is particularly preferred in such filtration applications disclosed herein because diffusion of the preservative through the filtration medium is a relatively slow process, while convective flow is relatively fast.

Fig. 5A-5D schematically illustrate the function of the filter assembly, using a grey mould (greydie) F to represent the flow path of the filtrate. The filter assembly 10 in its deployed configuration, ready for use in, for example, a sea water body, is shown in fig. 5A. The interior volume V of the filter assembly 10 is hermetically filled with a preservative P (shown in blank form in fig. 5A-5D). At the beginning of filtration or sample acquisition as shown in fig. 5B, the conical stagnant space 50 within filter assembly 10 is flushed free of preservative P due to the flow of heavier (higher density) filtrate F (shown as gray seawater in fig. 5A-5D), which flows into filter assembly 10 and displaces the lighter (less density) preservative in conical stagnant space 50. Preservative P is swept from above the filter media 28 so that the sample to be collected is not exposed to preservative during filtration (fig. 5B).

Because the reservoir remains filled at all times, any preservative "I" convected through one or more of the holes 24 must be balanced against an equal volume of filtered media "O" entering the other holes 24 (the holes 24 may be radially displaced from each other). The density difference between the preservative P and the sample filtrate F (e.g. seawater) causes laminar convective flow to be initiated and the less dense preservative fluid P to flow to a relative position above the more dense fluid F. The flow rate may be controlled by the size of the two orifices 24. During filtration, the filtration media 28 is not exposed to the preservative P because the filtrate is continuously passing through the media 28 to the outlet 36. It is also noteworthy that during filtration, fig. 5B, preservative exiting the reservoir from conical stagnant space 50 is immediately flushed out of the assembly along with filtrate that has been filtered.

When filtration is stopped, the preservative P undergoes a laminar convective flow regime below the filter support plate 26 (see fig. 4) in the conical stagnant space 50 and also passes through the filter media 28. This is then used to chemically preserve the particulate matter accumulated on the surface of the filter (fig. 5C). Such preservation may preferably occur over a period of less than or equal to 30 minutes, depending on the density difference between the preservative and the filtrate. The preservative delivery rate may depend linearly on the density difference between the preservative and the filtrate. For example, when the density difference is about 0.03g/cc, the shelf life may occur over a period of 15-20 minutes without any mechanical manipulation. Finally, the filter assembly 10 is again equilibrated with respect to the different density fluids, maintaining preservation of the sample (fig. 5D). It will be appreciated that the relatively low density preservative eventually covers the filter media, thereby displacing the relatively high density seawater that may flow to the bottom of the reservoir 20.

The examples shown in fig. 5A-5D are applicable when the preservative/fixative P has a density lower than the density of the medium F being filtered. When the preservative P has a density higher than that of the medium F being filtered, the filter assembly 10 can be inverted to function as described. In this example, the preservative P may flow in a downward direction (as opposed to an upward direction when the preservative has a low density) during laminar convective flow. The filter can then be isolated again and the sample can be collected and analyzed. The sample may comprise, for example, biological and inanimate particle samples.

Fig. 6 is a schematic over time of a laminar convective flow process looking down on the filter media, wherein a dye is used in place of a preservative and has a density less than that of the media being filtered. As can be seen, the filter at the beginning of the laminar convective process (time zero or OT) is clean because no dye flows into or through the filter. As time goes on (5, 10, 15, 20 minutes), the filter appears to change color (more and more grey) as the dye flows through the filter and stains the filter. Accordingly, images over time demonstrate that the dye permeates the filter media in a laminar convective manner. Depending on the value of the density difference between the fluids, the duration may be less than or greater than 20 minutes.

More specifically, in the example in FIG. 6, the density difference between the filtrate (D-1.03 g/cc) and the preservative density (1.00 g/cc) is about 3.00%. If the density difference between the filtrate and the preservative is large (e.g. as in the case of a largeWith a density of 1.22g/cc, 1.03g/cc for seawater, and a density differential of 19.0%), the preservative delivery rate will be several times faster, which is beneficial, preserving the sample in about 7-8 minutes. On the other hand, the loss of preservative during filtration will also be relatively large, which is disadvantageous.

The filter assembly of the present disclosure may have a wide range of potential uses, namely, the use of collection and preservation of filtered samples collected by a robotic time sequential water sampler for subsequent molecular analysis. The filter assembly of the present disclosure can operate effectively in deep water environments where it would be desirable to minimize moving parts and maximize compatibility with automatic timing instrumentation. Since the assembly is a stand-alone module with no moving parts, there is now no need for any other connections than the inlet and outlet for filtrate (e.g. seawater). It would also eliminate the need for connections to electromechanical devices for active delivery of preservatives to the filtered sample. This ensures the following features: the filter assembly herein may be adapted to accommodate various filtration applications where minimization of mechanical steps is desirable or critical.

While particular embodiments of the present disclosure have been shown and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the disclosure. Accordingly, all changes and modifications that come within the scope of the disclosure are intended to be covered by the appended claims.

Claims (23)

1. A filter assembly for filtrate flow and collection of samples on the surface of a filter medium and which chemically preserves the collected samples by means of a physical process that does not require moving parts, the filter assembly comprising:

a. for having a density D₁And comprises a flow having a density D₂A reservoir of a second fluid;

b. an interior space for a filter media;

c. a reservoir plate separating the reservoir from the interior space, wherein the reservoir plate comprises at least two openings in communication with the reservoir and the interior space, wherein a second fluid in the reservoir is exchangeable with the first fluid in the interior space due to a density difference of the first and second fluids, wherein:

(1) when the filtrate has a density D₁Greater than D₂While the filtrate flows out of the interior space into the reservoir, and the second fluid displaces the filtrate in the interior space; or

(2) When the filtrate has a density D₁Is less than D₂While the second fluid flows from the reservoir into the interior space, and the second fluid displaces the filtrate in the interior space; and is

Wherein the second fluid is a preservative for a biological sample or an inanimate particle sample.

2. The filter assembly of claim 1, further comprising an inlet plate having a surface, and wherein the reservoir plate has a surface, and wherein the surface of the inlet plate and the surface of the reservoir plate form the interior space.

3. The filter assembly of claim 2, wherein the surface of the reservoir plate and the surface of the inlet plate have a conical taper.

4. The filter assembly of claim 2, wherein the surface of the reservoir plate and the surface of the inlet plate are concave.

5. The filter assembly of claim 1, wherein the filter media is at least partially supported in the space by a filter support plate.

6. The filter assembly of claim 1, wherein the filtrate exiting the interior space into the reservoir undergoes laminar convective flow.

7. The filter assembly of claim 1, wherein the second fluid flowing from the reservoir into the interior space undergoes laminar flow.

8. The filter assembly of claim 1, wherein the first fluid is seawater, fresh water, or a brine solution.

9. The filter assembly of claim 1, wherein D₁And D₂The densities differ by + -0.01 g/cc to 0.50 g/cc.

10. The filter assembly of claim 1, wherein the preservative is to attenuate RNA/DNA/protein degradation of a sample contained in the filtrate.

11. The filter assembly of claim 1, wherein the filter media is one of fiberglass, cellulose acetate fibers, or polycarbonate membranes.

12. One for obtaining a first density D₁The method for collecting and immobilizing microorganisms in the filtrate of (1), which comprises the steps of:

providing a filter assembly comprising: an inlet and an outlet and a reservoir comprising a second density D₂A second fluid of (a); a filter media within the filter assembly between the filter assembly inlet and outlet, and wherein the filter media is positioned away from the reservoir and within an interior space separated from the reservoir by a reservoir plate; the reservoir plate comprises an opening for the flow of the filtrate andat least two openings in communication with the reservoir and the interior space;

passing the filtrate through the filter assembly and collecting microorganisms and/or particulate material on the filter media;

exposing the microorganisms and/or particulate material to the second fluid under conditions such that:

(1) when the filtrate has a density D₁Greater than D₂While the filtrate flows out of the interior space into the reservoir, and the second fluid displaces the filtrate in the interior space; or

(2) When the filtrate has a density D₁Is less than D₂While the second fluid flows from the reservoir into the interior space, and the second fluid displaces the filtrate in the interior space.

13. The method of claim 12, further comprising an inlet plate having a surface, and wherein the reservoir plate has a surface, and wherein the surface of the inlet plate and the surface of the reservoir plate form the interior space.

14. The method of claim 13, wherein the surface of the reservoir plate and the surface of the inlet plate have a conical taper.

15. The method of claim 13, wherein the surface of the reservoir plate and the surface of the inlet plate are concave.

16. The method of claim 12, wherein the filter media is at least partially supported in the space by a filter support plate.

17. The method of claim 12, wherein the filtrate flowing out of the interior space into the reservoir undergoes laminar convective flow.

18. The method of claim 12, wherein the second fluid flowing from the reservoir into the interior space undergoes laminar flow.

19. The method of claim 12, wherein the second fluid is a preservative for a biological sample or an inanimate particulate sample.

20. The method of claim 12, wherein the first fluid is seawater, fresh water, or a brine solution.

21. The method of claim 12, wherein D₁And D₂The densities differ by + -0.01 g/cc to 0.50 g/cc.

22. The method of claim 19, wherein the preservative is used to attenuate RNA degradation of the sample contained in the filtrate.

23. The method of claim 12, wherein the filter media is one of glass fibers, cellulose acetate fibers, or polycarbonate membranes.