WO2014197831A1

WO2014197831A1 - Closed system for sterility testing

Info

Publication number: WO2014197831A1
Application number: PCT/US2014/041353
Authority: WO
Inventors: Stefan Koelzer; Herbert Engels
Original assignee: Graphic Controls Acquisition Corp.
Priority date: 2013-06-06
Filing date: 2014-06-06
Publication date: 2014-12-11

Abstract

A closed system for testing sterility of a sample including a first closed volume, a second closed volume and a third closed volume. Each of the first, second and third closed volumes includes an enclosed reservoir having a first end and a second end, the first end having a fluid inlet and the second end including a first securing feature, and a filter unit having a first end and a second end, the first end having a second securing feature adapted to complimentarily engage the first securing feature and the second end adapted to receive and releasably secure a filter therein, wherein the sample passes through the closed volumes and the filters.

Description

CLOSED SYSTEM FOR STERILITY TESTING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit under 35 U.S.C. § 1 19(e) and Article

4 of the Stockholm Act of the Paris Convention for the Protection of Industrial Property of United States Provisional Patent Application No. 61/831,914, filed June 6, 2013, which application is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a method and apparatus used for testing solutions, such as pharmaceutical solutions, in a closed system in which the solutions flow through to determine the presence of microorganisms. The testing apparatus comprises a distribution system to manage the flow of solution to multiple sampling cups/reservoirs each having a micro-porous membrane filter, and various other components including a sampling cup cap that allows for use of solid culture media during the micro assay process.

BACKGROUND OF THE INVENTION

[0003] In the production of pharmacological products federal regulations of various nations require sterility testing to ensure that these products are free of microorganisms such as bacteria and fungi. To prevent contamination of the samples, it is prescribed to use closed test systems to perform these tests.

[0004] Presently, for different contamination sources, various test methods are established. These test methods require specialized liquid or solid culture media for incubation of the various relevant microorganism types that could harm the pharmacological products. After incubation the microorganisms can be detected in various ways. Closed test systems for liquid culture media incubation are currently available on the market. For incubation with solid culture media so far no sufficient closed system is available. Presently open funnels are used wherein the sample liquid is filled in and the dissolving process of solid products is a multi-step procedure of adding liquid, dissolving product, and obtaining the sample. The process in general is performed in isolator chambers to prevent an external contamination of the process but there is a high risk of cross contamination between the analyzed samples due to the open equipment. A closed system is required that minimizes the risks of contamination thereby optimizing the analyzing and sampling processes and that meets federal regulations, e.g., detection limits. BRIEF SUMMARY OF THE INVENTION

[0005] With the present invention closed system for solid culture media, an embodiment combines the advantages of the closed systems for liquid culture media (preventing cross contamination) with the handling advantages of an open system while improving the sampling process for liquid and solid samples in various sample containing systems. In an embodiment, the present invention closed sterility test system includes novel components such as a unique sample distribution system having a three way distributor that allows for distribution of a dissolved liquid sample in three equal flow streams independent of the overall flow rate.

[0006] In an embodiment, the present invention includes a novel reversible cover for a culture media cartridge module (culture media cartridge, membrane holder and cover) which allows incubating a cartridge module either under anaerobic or aerobic conditions, depending on the installation position of the cover. Moreover, the cover facilitates stacking the cartridge module, improves the handling of the cartridge and minimizes the required space in the incubator.

[0007] In an embodiment, the present invention comprises a closed system for testing sterility of a sample including a first closed volume, a second closed volume and a third closed volume. Each of the first, second and third closed volumes includes an enclosed reservoir having a first end and a second end, the first end having a fluid inlet and the second end having a first securing feature, and a filter unit having a first end and a second end, the first end having a second securing feature adapted to complimentarily engage the first securing feature and the second end adapted to receive and releasably secure a filter therein, wherein the sample passes through the closed volumes and the filters.

[0008] In some embodiments, each of the enclosed reservoirs further includes an air inlet adapted to prevent overpressurization of the respective closed volume during use. In some embodiments, the closed system further includes a three-way distributor having a sample inlet, a first sample outlet, a second sample outlet and a third sample outlet, wherein the first, second and third sample outlets are in fluid communication with the first, second and third closed volumes, respectively, via each respective fluid inlet. In some embodiments, the sample inlet includes a first diameter and each of the first, second and third sample outlets includes a second diameter smaller than the first diameter. In some embodiments, the sample inlet is arranged perpendicularly relative to each of the first, second and third sample outlets. In some embodiments, each of the first, second and third sample outlets is arranged at one hundred twenty degrees relative to each of the other of the first, second and third sample outlets. In some embodiments, the three-way distributor is arranged elevationally below the first, second and third closed volumes, the sample inlet includes a longitudinal axis and the longitudinal axis is arranged in a substantially vertical orientation.

[0009] In an embodiment, the present invention comprises a cover for an incubation chamber including a first side having a first plurality of arcuate extensions arranged in a first circular formation and having a first height, a second plurality of arcuate extensions arranged in a second circular formation and having a second height, and a third plurality of arcuate extensions arranged in a third circular formation having a third height, wherein the second plurality of arcuate extensions is positioned between the first and third pluralities of arcuate extensions and the second height is less than the first and third heights. The cover further includes a second side having a first extension arranged in a closed circular formation, and a second plurality of arcuate extensions arranged in a circular formation. In accordance with the foregoing, positioning the first side in contact with the incubation chamber forms an aerobic incubation chamber and positioning the second side in contact with the incubation chamber forms an anaerobic incubation chamber.

[0010] In some embodiments, the incubation chamber includes a rim and the second plurality of arcuate extensions on the first side are arranged to contact the rim when forming the aerobic incubation chamber. In some embodiments, the incubation chamber includes a filter unit disposed on a container having a culturing media therein, the filter unit having a rim and the second plurality of arcuate extensions on the first side are arranged to contact the rim when forming the aerobic incubation chamber.

[0011] In an embodiment, the present invention comprises a method of testing a sample for the presence of microorganisms including: a) obtaining the sample in a liquid form for microorganism quantification; b) passing the sample through a three-way distributor having a sample inlet, a first sample outlet, a second sample outlet and a third sample outlet to form a first sample portion, a second sample portion and a third sample portion; c) passing the first, second and third sample portions through first, second and third closed volumes, respectively, wherein each of the first, second and third closed volumes includes: an enclosed reservoir having a first end and a second end, the first end having a fluid inlet and the second end having a first securing feature; and, a filter unit having a first end and a second end, the first end having a second securing feature adapted to complimentarily engage the first securing feature and the second end adapted to receive and releasably secure a filter therein, wherein the first, second and third sample outlets are in fluid communication with the first, second and third closed volumes, respectively, via each respective fluid inlet; d) transferring each respective filter to a respective incubation chamber each comprising a growth media; e) covering each respective incubation chamber with a cover adapted for either aerobic incubation or anaerobic incubation; f) incubating each respective incubation chamber for a time based on the microorganisms being observed; and, g) observing each respective incubation chamber for indications of microorganism content.

[0012] In some embodiments, the sample inlet includes a first diameter and each of the first, second and third sample outlets includes a second diameter smaller than the first diameter. In some embodiments, the three-way distributor is arranged elevationally below the first, second and third closed volumes, the sample inlet includes a longitudinal axis and the longitudinal axis is arranged in a substantially vertical orientation. In some embodiments, each of the covers includes a first side having a first plurality of arcuate extensions arranged in a first circular formation and having a first height, a second plurality of arcuate extensions arranged in a second circular formation and having a second height, and a third plurality of arcuate extensions arranged in a third circular formation having a third height, wherein the second plurality of arcuate extensions is positioned between the first and third pluralities of arcuate extensions and the second height is less than the first and third heights, and a second side having a first extension arranged in a closed circular formation, and a second plurality of arcuate extensions arranged in a circular formation, wherein positioning the first side in contact with one of the incubation chambers forms an aerobic incubation chamber and positioning the second side in contact with one of the incubation chambers forms an anaerobic incubation chamber.

[0013] In some embodiments, each of the incubation chambers includes a rim and the second plurality of arcuate extensions are arranged to contact the rim when forming the aerobic incubation chamber. In some embodiments, each of the incubation chambers includes the filter unit disposed on a container comprising a culturing media therein, the filter unit comprising a rim and the second plurality of arcuate extensions on the first side are arranged to contact the rim when forming the aerobic incubation chamber. In some embodiments, the step of transferring each respective filter includes positioning each respective filter unit on a container having a culturing media therein. In some embodiments, the step of transferring each respective filter comprises contacting each respective filter on a culturing media held in a container.

[0014] In an embodiment, the present invention comprises a fluid distribution apparatus including a sample inlet, a first sample outlet, a second sample outlet, and a third sample outlet. The sample inlet has a first diameter and each of the first, second and third sample outlets have a second diameter smaller than the first diameter.

[0015] These and other objects and advantages of the present invention will be readily appreciable from the following description of preferred embodiments of the invention and from the accompanying drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The nature and mode of operation of the present invention will now be more fully described in the following detailed description of the invention taken with the accompanying drawing figures, in which:

Figure 1 is a schematic drawing of a first embodiment of a closed system for sterility testing;

Figure 2 is a schematic drawing of a second embodiment of a closed system for sterility testing;

Figure 3 is a schematic drawing of a third embodiment of a closed system for sterility testing;

Figure 4 is a schematic drawing of a fourth embodiment of a closed system for sterility testing;

Figure 5A is a top plan view of an embodiment of a three way tubing splitter or distribution article;

Figure 5B is a side elevational view of the three way tubing splitter or distribution article depicted in Figure 5A;

Figure 5C is a perspective view of the three way tubing splitter or distribution article depicted in Figure 5A;

Figure 6A is a cross sectional view of an embodiment of a filter reservoir taken generally along line 6A-6A in Figure 6D;

Figure 6B is a cross sectional view of an embodiment of a filter reservoir with a filter unit secured thereto taken generally along line 6B-6B in Figure 6D; Figure 6C is a side elevational view of an embodiment of a filter reservoir; Figure 6D is a top plan view of an embodiment of a filter reservoir with a filter unit secured thereto;

Figure 6E is an enlarged cross sectional view of the encircled region 6E of Figure 6A;

Figure 7A is a cross sectional view of an embodiment of a membrane holder taken generally along line 7A-7A in Figure 7B;

Figure 7B is a top plan view of an embodiment of a membrane holder;

Figure 8A is a cross sectional view of an embodiment of a membrane holder having a filter membrane disposed therein taken generally along line 8A-8A in Figure 8B;

Figure 8B is a top plan view of an embodiment of a membrane holder having a filter membrane disposed therein;

Figure 9A is a top plan view of an embodiment of a cover showing the side used to form an air permeable seal with a culture dish for aerobic incubation;

Figure 9B is a cross sectional view of the cover depicted in Figure 9A taken generally along line 9B-9B;

Figure 9C is a bottom plan view of an embodiment of a cover showing the side used to form an air impermeable seal with a culture dish for anaerobic incubation;

Figure 9D is an enlarged cross sectional view of the encircled region 9D of Figure 9B;

Figure 1 OA is a top plan view of an embodiment of a hand protection;

Figure 10B is a perspective view of the hand protection depicted in Figure

10A;

Figure IOC is a cross sectional view of an embodiment of a hand protection taken generally along line IOC- IOC in Figure 10A;

Figure 10D is a cross sectional view of an embodiment of a hand protection taken generally along line 10D-10D in Figure 10A;

Figure 10E is an enlarged cross sectional view of the encircled region 10E of

Figure 10D;

Figure 1 1 A is a top plan view of an embodiment of a protection plate;

Figure 11B is a cross sectional view of an embodiment of a protection plate taken generally along line 1 IB- 1 IB in Figure 1 1A; Figure 12A is a partial cross sectional view of an embodiment of a present cover arranged on a culture system for anaerobic incubation;

Figure 12B is a partial cross sectional view of an embodiment of a present cover arranged on a culture system for aerobic incubation; and,

Figure 12C is a partial cross sectional view of an embodiment of a present cover arranged on a culture system for anaerobic incubation.

DETAILED DESCRIPTION OF THE INVENTION

[0017] At the outset, it should be appreciated that like drawing numbers on different drawing views identify identical, or functionally similar, structural elements of the invention. While the present invention is described with respect to what is presently considered to be the preferred aspects, it is to be understood that the invention as claimed is not limited to the disclosed aspects.

[0018] Furthermore, it is understood that this invention is not limited to the particular methodology, materials and modifications described and as such may, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices or materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices, and materials are now described.

[0020] An embodiment of the present closed sterility test system generally comprises three portions: a) an inlet portion which provides different options to withdraw samples from various sample containers, e.g., syringes, vials with liquids, vials with a solid phase product and ampoules; b) a distribution portion adapted to equalize and distribute the sample liquid to direct it to a single filtration unit, or three or more filtration units; and c) filtration units adapted to filter the samples and retain any bacteria/fungi present in the samples.

[0021] Portion A

[0022] Embodiments of the present inlet portion are arranged depending on the sterile test version for different components, e.g., Embodiments A, B, C and D. It should be appreciated that these example embodiments are not limiting, and that other embodiments are also possible.

[0023] Embodiment A or system 50, depicted in Figure 1, is arranged for lyophilized or solid samples. Embodiment A comprises tube system 52 with vented inlet spike 54 for introduction into a container (not shown) with a liquid that is necessary to rinse system 50 and to dissolve and transport the sample. From spike 54, tube 58 is fitted with roll clamp 60 to adjust the flow rate to valve system 62 with another inlet spike 64 for introduction into the sample containers. Valve system 62 comprising hydrophobic filter 66 and two way distributor 68, where distributor 68 is necessary to reduce liquid consumption and to ease handling. During the rinse process, filter-valve 62 is closed by screw cap 72 and the sample spike 64 is covered by an empty sample container to allow continuous flow. During the sampling process, the flow can be stopped and started by cap 72 or by tube clamp 60, e.g., closing the vent (cap 72) causes liquid to flow, while opening the vent (cap 72) causes liquid to stop flowing. When the liquid stops, the sample container connected to valve system 62 can be easily changed and the dissolving/mixing process in the container can be initiated by restarting the liquid flow. Subsequently, the liquid flows through the sample container to dissolve and transport the solid phase through system 50. To enable this process, a slight vacuum is created in tube system 52 by connecting a peristaltic pump (not shown) via connection tube 82 inserted into the next station in system 50. The pump creates a slight vacuum in the first section, i.e., inlet and valve/sample spikes 54 and 62, respectively, of connection tube system 52 and behind the pump a slight overpressure is created to push the liquid to distribution system 84.

[0024] Embodiment B, depicted in Figure 2, may be used for liquid samples. System

90 comprises connection portion 92 which includes vented inlet spike 94 and tube 96, which tube passes through a peristaltic pump (not shown). In this embodiment, the sample is included in the liquid, or alternatively a sample container and the liquid uses the same single inlet spike, i.e., inlet spike 94.

[0025] Embodiment C, depicted in Figure 3, may be used for liquid bags, e.g., infusion bags and ampoules. Connection portion 100 comprises non-vented inlet 102 using hollow-needle 104 with a specially designed hand shield 106 to prevent injuries and connection tube 108 leading through a peristaltic pump (not shown). To allow venting of the liquid bags, system 110 comprises a separate hollow-needle, i.e., needle 112, with hydrophobic filter 114 that can be plugged into the top of a bag during the period the sample is obtained..

[0026] Embodiment D, depicted in Figure 4, may be used for sampling from syringes.

The construction of connection portion 120 is similar to the construction of connection portion 52 of Embodiment A described above, however roll clamp 60 is not present and spike valve system 62 is replaced by valve/injection port system 122 with membrane 124 that allows injecting a liquid into connection tube system 120. During sample introduction, a user's hand/fingers are protected from accidental injury, e.g., needle injury, by shield 125. The rinse and stop process is similar to the process described above relative to Embodiment A, however a roll clamp is not necessary. From injection port 126, connection tube 128 leads through a peristaltic pump (not shown) to distribution portion 84 of system 130.

[0027] Portion B

[0028] The distribution portion enables the separation of liquid flow, independent of the flow rate, into a plurality of partial flows, e.g., three equal partial flows, feeding into the various filtration/sampling units. It has been found that the incoming flow can be equally distributed by careful design of the flow distribution means. In a preferred embodiment, the novel distributor comprises three way distributor 84 which includes one inlet, i.e., inlet 150, and three outlets, i.e., outlets 152. In this embodiment, inlet axis 154, i.e., a line parallel and coaxial with the longitudinal axis of inlet 150, and each outlet axis 156, i.e., lines parallel and coaxial with the longitudinal axes of outlet 152, are positioned ninety degrees relative to each other, while each outlet axis 156 is positioned one hundred twenty degrees relative to each other outlet axis. Distributor 84 is connected to the connection tube that comes from the inlet portion described above relative to the various embodiments of Portion A. Outlets 152 of distributor 84 are connected to inlets 158 of the upper part of the three filtration/sampling units, i.e., reservoir 160. It has been found that distributor 84 should be positioned under the inlets of the filter units and ideally inlet 150 of distributor 84 should be directed downwards, i.e., vertically, to optimize the flow separation. The tube that connects the distribution portion (Portion B) and the filter portion (Portion C) may comprise tube clamps, e.g., clamps 162, that allow closing the tube and, if desired for incubation purposes, disconnecting the closed filter units from the distribution portion after the filtration process.

[0029] Distributor 84 may also include other features to ensure that even distribution of flow to outlets 152 occurs. For example, the inner diameters of outlets 152 are each equal to the other outlet inner diameters and all outlet inner diameters are smaller than the inner diameter of inlet 150. The foregoing arrangement creates a backpressure within the system thereby ensuring that equal distribution of fluid through outlets 152.

[0030] Portion C

[0031] The filtration units comprise an upper portion, also known as a reservoir, e.g., reservoir 160, and a lower portion, also known as a membrane holder or filter unit, e.g., membrane holder 164. The upper and the lower portions are tightly connected by a bayonet connector system to tighten the system, prevent an accidental disconnection of the upper and lower part, and to allow an easy intentional disconnection if necessary. The bayonet connector system, in the embodiments depicted, comprises receivers 161 and extensions 162. The upper portion comprises inlet connection 158 at the top of reservoir 160 for the sample liquid from the inlet portion (Portion A) and distribution portion (Portion B), and hydrophobic venting filter unit 166 at the top to prevent an overpressure in the system. Reservoir 160 is generally cylindrical having, in some embodiments, a conical reduced portion at its top, and is able to contain up to 150 ml of liquid; however, other reservoir sizes are also possible, e.g., greater than or less than 150 ml, and such sizes are within the spirit and scope of the claimed invention. Reservoir 160 may also include ring markers on the outside of the reservoir that shows the filling volume, e.g., 50 ml and 100 ml. One half of the bayonet connector that connects the upper part with the lower part is located at the bottom of reservoir 160.

[0032] Membrane holder 164 comprises a circular plastic portion, i.e., circular portion 168, that includes at its top the second part of the above described bayonet connector and at its bottom a connection area, i.e., connection area 170, that includes a 57 mm PVDF filter membrane circle having a 0.45μιη pore size, for example. Connection area 170 is constructed to fit vacuum bars or vacuum pumps (not shown) and culture media cartridges, e.g., Millipore equipment, but use with other vacuum and culture media equipment is also possible.

[0033] In an initial state, the upper and the lower portions are connected. To start the filtration process, filter unit 164 has to be connected to a vacuum bar or a vacuum pump that creates a vacuum to draw the sample liquid through filter 172, while the connection tube, e.g., tube 82, 96, 108 and/or 128, has to be connected with the peristaltic pump. The sample liquid enters the system, passes the distributing portion as described above, and enters filter unit 164 through inlet 158 adjacent to the inner closed volume of reservoir 160 and flows towards filter unit 164 and filter 172. Here the liquid passes through filter membrane 172 where any potentially existing fungi and/or bacteria, i.e., microorganisms, are retained on the surface of filter membrane 172. The filtered liquid passes through vacuum bar/vacuum pump and is disposed.

[0034] Filter unit 164 is disconnected from the vacuum bar / vacuum pump and plugged onto culture media cartridge 174. Here it may be necessary to create a slight overpressure to bring membrane 172 into direct contact with solid culture media 176, e.g., by using an air filled syringe to press the air through the hydrophobic filter in the closed system. After connecting filter unit 164 with culture media cartridge 174 either the complete module could be transferred into an incubator to incubate membrane 172 to identify fungi/bacteria growth, or the upper portion of container 178, e.g., rim 180, holding culture media 176 could be disconnected and a specially developed cap, i.e., cap 182, could be plugged onto the membrane holder. Cap 182 allows incubating the sample under aerobic or anaerobic conditions and makes the samples more easily stackable. After incubation cap 182 and/or upper part of the module can be removed and the sample can be analyzed. It should be appreciated that the culture media system in which incubation occurs may comprise the container and cap (See Figures 12A and 12B), or alternatively, may comprise the container, the filter unit and cap (See Figure 12C).

[0035] The orientation of cap 182 permits either aerobic or anaerobic incubation of samples. More specifically, aerobic incubation occurs when side 184 of cap 182 is in contact with rim 180, e.g., a rim of a petri dish having agar held therein or a rim of filter unit 164. In this orientation, outer wall segments 186 and/or inner wall segments 188 engage wall 190 adjacent to rim 180, while standoffs 192 create a slight separation between side 184 and rim 180. The partial open arrangements of outer wall segments 186, inner wall segments 188 and standoffs 192 permit air to flow into and out of culture media system 194, i.e., aerobic incubation. Contrarily, anaerobic incubation occurs when side 196 of cap 182 is in contact with rim 180. In this orientation, outer wall 198 and/or inner wall 200 engage walls 190 thereby forming a seal. The sealing arrangement of outer wall 198 and/or inner wall 200 relative to walls 190 prevents air from moving into or out of culture media system 202, i.e., anaerobic incubation. It should be appreciated that although outer wall 198 and inner wall 200 are both depicted as continuous walls, one of the two walls may be formed as a plurality of arcuate segments, i.e., only a single wall must be continuous.

[0036] If a vacuum bar is used for creating the vacuum it is possible to use the same peristaltic pump that feeds the sample liquid in the system to create the vacuum that draws the liquid out of the system. For this reason an additional piece of equipment is added to the sterile test set, i.e., a drainage tube. This drainage tube comprises a "pump tube" that is equal to the tube that is used in the connection system and fits into the peristaltic pump, and connection tubes that connect the pump tube with the vacuum bar and a drain. The pump and the drainage tubes are connected with standard plastic tube adapters.

[0037] Thus, it is seen that the objects of the present invention are efficiently obtained, although modifications and changes to the invention should be readily apparent to those having ordinary skill in the art, which modifications are intended to be within the spirit and scope of the invention as claimed. It also is understood that the foregoing description is illustrative of the present invention and should not be considered as limiting. Therefore, other embodiments of the present invention are possible without departing from the spirit and scope of the present invention.

Claims

What Is Claimed Is:

1. A closed system for testing sterility of a sample comprising:

a first closed volume, a second closed volume and a third closed volume, wherein each of the first, second and third closed volumes comprises:

an enclosed reservoir comprising a first end and a second end, the first end comprising a fluid inlet and the second end comprising a first securing feature; and, a filter unit comprising a first end and a second end, the first end comprising a second securing feature adapted to complimentarily engage the first securing feature and the second end adapted to receive and releasably secure a filter therein,

wherein the sample passes through the closed volumes and the filters.

2. The closed system of Claim 1 wherein each of the enclosed reservoirs further comprises an air inlet adapted to prevent overpressurization of the respective closed volume during use.

3. The closed system of Claim 1 further comprising a three-way distributor comprising a sample inlet, a first sample outlet, a second sample outlet and a third sample outlet, wherein the first, second and third sample outlets are in fluid communication with the first, second and third closed volumes, respectively, via each respective fluid inlet.

4. The closed system of Claim 3 wherein the sample inlet comprises a first diameter and each of the first, second and third sample outlets comprises a second diameter smaller than the first diameter.

5. The closed system of Claim 3 wherein the sample inlet is arranged perpendicularly relative to each of the first, second and third sample outlets.

6. The closed system of Claim 5 wherein each of the first, second and third sample outlets is arranged at one hundred twenty degrees relative to each of the other of the first, second and third sample outlets.

7. The closed system of Claim 3 wherein each of the first, second and third sample outlets is arranged at one hundred twenty degrees relative to each of the other of the first, second and third sample outlets.

8. The closed system of Claim 3 wherein the three-way distributor is arranged elevationally below the first, second and third closed volumes, the sample inlet comprises a longitudinal axis and the longitudinal axis is arranged in a substantially vertical orientation.

9. A cover for an incubation chamber comprising:

a first side comprising a first plurality of arcuate extensions arranged in a first circular formation and comprising a first height, a second plurality of arcuate extensions arranged in a second circular formation and comprising a second height, and a third plurality of arcuate extensions arranged in a third circular formation comprising a third height, wherein the second plurality of arcuate extensions is positioned between the first and third pluralities of arcuate extensions and the second height is less than the first and third heights; and,

a second side comprising a first extension arranged in a closed circular formation, and a second plurality of arcuate extensions arranged in a circular formation, wherein positioning the first side in contact with the incubation chamber forms an aerobic incubation chamber and positioning the second side in contact with the incubation chamber forms an anaerobic incubation chamber.

10. The cover of Claim 9 wherein the incubation chamber comprises a rim and the second plurality of arcuate extensions on the first side are arranged to contact the rim when forming the aerobic incubation chamber.

11. The cover of Claim 9 wherein the incubation chamber comprises a filter unit disposed on a container comprising a culturing media therein, the filter unit comprising a rim and the second plurality of arcuate extensions on the first side are arranged to contact the rim when forming the aerobic incubation chamber.

12. A method of testing a sample for the presence of microorganisms comprising:

a) obtaining the sample in a liquid form for microorganism quantification;

b) passing the sample through a three-way distributor comprising a sample inlet, a first sample outlet, a second sample outlet and a third sample outlet to form a first sample portion, a second sample portion and a third sample portion;

c) passing the first, second and third sample portions through first, second and third closed volumes, respectively, wherein each of the first, second and third closed volumes comprises:

an enclosed reservoir comprising a first end and a second end, the first end comprising a fluid inlet and the second end comprising a first securing feature; and,

a filter unit comprising a first end and a second end, the first end comprising a second securing feature adapted to complimentarily engage the first securing feature and the second end adapted to receive and releasably secure a filter therein, wherein the first, second and third sample outlets are in fluid communication with the first, second and third closed volumes, respectively, via each respective fluid inlet;

d) transferring each respective filter to a respective incubation chamber each comprising a growth media;

e) covering each respective incubation chamber with a cover adapted for either aerobic incubation or anaerobic incubation;

f) incubating each respective incubation chamber for a time based on the microorganisms being observed; and,

g) observing each respective incubation chamber for indications of microorganism content.

13. The method of testing of Claim 12 wherein the sample inlet comprises a first diameter and each of the first, second and third sample outlets comprises a second diameter smaller than the first diameter.

14. The method of testing of Claim 12 wherein the three-way distributor is arranged elevationally below the first, second and third closed volumes, the sample inlet comprises a longitudinal axis and the longitudinal axis is arranged in a substantially vertical orientation.

15. The method of testing of Claim 12 wherein each of the covers comprises:

a second side comprising a first plurality of arcuate extensions arranged in a first circular formation, and a second plurality of arcuate extensions arranged in a second circular formation,

wherein positioning the first side in contact with one of the incubation chambers forms an aerobic incubation chamber and positioning the second side in contact with one of the incubation chambers forms an anaerobic incubation chamber.

16. The method of testing of Claim 15 wherein each of the incubation chambers comprises a rim and the second plurality of arcuate extensions are arranged to contact the rim when forming the aerobic incubation chamber.

17. The method of testing of Claim 15 wherein each of the incubation chambers comprises the filter unit disposed on a container comprising a culturing media therein, the filter unit comprising a rim and the second plurality of arcuate extensions on the first side are arranged to contact the rim when forming the aerobic incubation chamber.

18. The method of testing of Claim 12 wherein the step of transferring each respective filter comprises positioning each respective filter unit on a container comprising a culturing media therein.

19. The method of testing of Claim 12 wherein the step of transferring each respective filter comprises contacting each respective filter on a culturing media held in a container.

20. A fluid distribution apparatus comprising:

a sample inlet;

a first sample outlet;

a second sample outlet; and,

a third sample outlet, wherein the sample inlet comprises a first diameter and each of the first, second and third sample outlets comprises a second diameter smaller than the first diameter.