Device and process for the microbiological testing of pressurized liquids

Abstract: The object of the invention is a device and a process for the microbiological testing of pressurized liquids. A completely closed container ( 1 ), at the base of which a filter membrane ( 4 ) is sealed, is fitted, upstream from the membrane, with a built-in reading window ( 3 ) and, downstream from the membrane, with a flange ( 7 ) comprising a lip seal ( 10 ) and a female threaded part ( 8 ). The process consists of screwing to this container ( 1 ) a filtration support, in taking and filtering the sample to be analyzed, and then in substituting for this support an incubation cartridge for the subsequent development, in an incubator, of the colonies collected on the membrane ( 4 ) (Figure 1).

Description: This invention concerns a device and a process for the microbiological testing of pressurized liquids, which serves to count the living microorganisms contained in these liquids. In many industries, including the food, pharmaceutical and electronics industries, it is important to be able to check the microbiological quality of the pressurized fluids, whether during transfer through lines or whether kept in storage tanks. So far, the standard method used was to sample, in aseptic conditions, a certain volume of liquid in a receptacle with the aid of a small sample tap (small valve) and then to test this sample by the usual filtration method or the method described in Patent No.FR-B-2.558.847 filed on 31 January 1984 by the company Millipore SA. It is easy to imagine the risks of 'false positives' incurred by these samples and various transfers of samples. Moreover, these different operations waste time and cause delays in receiving the results of the analyses. The invention described below eliminates these risks, because the filtration, necessary for the analysis, as well as the placing of the filter in contact with the nutrient medium, necessary for the development of the microorganisms that are retained on the membrane, can be carried out on the spot. This accordingly simplifies the operations hitherto necessary for the microbiological testing of these liquids. The devices for the microbiological testing of a pressurized liquid sample according this this invention is a device, preferably pre-sterilized and for single use only, which makes use of a filter membrane. This device is characterized by a completely closed container of transparent plastic, at the base of which a filter membrane is sealed, the said container being fitted, upstream from the membrane, with a built-in reading window and, downstream from the membrane, a flange with a lip seal and a female threaded part. A filtration support or an incubation cartridge, both of plastic, can be fitted to the female threaded part of the container. The filtration support has a chamber fitted externally with a male threaded part cooperating with the female threaded part of the container and an upper plate fitted with a system of drainage channels terminating in an axial outlet orifice, the said filtration support also comprising a sealing cone cooperating with the lip seal of the container to prevent any infiltration or contamination from the exterior on the periphery of the support, during the filtration of the liquid sample to be analyzed. The incubation cartridge comprises a chamber containing a suitable liquid or dry microorganism culture medium, fitted externally with a male threaded part cooperating with the female threaded part of the container, the said cartridge possibly exhibiting, at its upper end, a conical part designed to cooperate with the lip seal of the container, to guarantee the seal on the downstream periphery of the filter membrane and thus to prevent any dehydration of the culture medium during incubation. The device according to the invention offers the advantage of maintaining the filter membrane in a perfectly closed container, hence sheltered from any exogenous contamination liable to distort the results of the microbiological analysis to be performed. It also serves to take the liquid sample to be analyzed, under pressures up to 6·10 5 Pa, which was not the case of any previously known device. And it also serves to carry out the incubation of any microorganisms collected on the filtration membrane directly and rapidly, without any handling of the membrane. The present invention is also designed for a process of microbiological testing of a pressurized liquid sample using a filter membrane, which comprises in succession the introduction of the sample to be analyzed in a device comprising the container defined above screwed to a filtration support, the positive or negative pressurization of the container to terminate the filtration, the replacement of the filtration support by an incubation cartridge screwed into the container, and incubation of the assembly consisting of the container and the cartridge, in the reversed position, in an incubator. It should be noted that such a process, independent of its simplicity and its speed, serves to check the microbiological quality of a pressured liquid with maximum reliability, because it avoids any handling of the liquid sample or of the membrane collecting any germs present and to be detected. The invention will now be described with the aid of the enclosed drawings, where: Figure 1 shows an elevation section of the container of the device according to the invention, Figure 2 is a plan view of the container shown in Figure 1, Figure 3 is an elevation section of the filtration support designed to cooperate with the container of Figures 1 and 2, Figure 4 is a plan view of the filtration support shown in Figure 3, Figure 5 is an elevation section of an incubation cartridge, for liquid culture medium, designed to cooperate with the container in Figures 1 and 2, Figure 6 is an elevation section of another incubation cartridge, for dry culture medium, designed to cooperate with the container in Figures 1 and 2, Figure 7 is a cross-section of a first embodiment of the lip seal of the device according to the invention, Figure 8 is a cross-section of a second embodiment of the lip seal of the device according to the invention, Figure 9 is a view of a liquid sampling to be tested with the device according to the invention, during filtration, Figure 10 is a view showing the end of filtration of a sample by aseptic pressurization of the device according to the invention using a syringe, Figure 11 is a view showing the end of filtration of a sample by pumping of the remnant of the liquid using a syringe, Figures 12 and 13 are views, partially sectional, of the device according to the invention in the reversed position in an incubator, and comprising an incubation cartridge for liquid or dry culture medium respectively, Figures 14 and 15 are views showing how the container of the device according to the invention can be perforated to reach the colonies for their sampling and their identification, Figure 16 is a view showing the sampling of a colony on the membrane of the device according to the invention, Figure 17 is a view showing how the incubation of the device according to the invention can be continued after the sampling of a colony. The reservoir ( 1 ), shown in Figures 1 and 2, comprises a body ( 2 ), generally cylindrical, closed at its upper part by a reading window ( 3 ) and, at its lower part, by a filter membrane ( 4 ), located in a plane parallel to that of the reading window. The cylindrical body ( 2 ) is fitted with a side inlet orifice ( 5 ) of the female Luer type, which can be closed by a male Luer plug ( 6 ) and whose axis is parallel to the plane of the membrane ( 4 ). A tubular flange ( 7 ), with a larger diameter than that of the cylindrical body ( 2 ), prolongs this body downstream from the filter membrane and is fitted internally with a female threaded part ( 8 ), which can consist in particular of three female thread initiations to guarantee rapid clamping on a removable element fitted with corresponding male threads. The connecting part between the cylindrical body ( 2 ) and the tubular flange ( 7 ) comprises in succession, from upstream to downstream, a shoulder ( 9 ), a lip seal ( 10 ) and a locking stop ( 11 ). The shoulder ( 9 ), in the form of a circular ring with the same axis as that of the body ( 2 ), delimits the base of the container ( 1 ) to which to filter membrane ( 4 ) is sealed, in a plane perpendicular to the common axis of the body ( 2 ) and the flange ( 7 ). The lip seal ( 10 ) in the embodiment shown in Figure 1 is in the form of a circular tab, with an intermediate diameter between those of the body ( 2 ) and of the flange ( 7 ), and with the same axis as the latter two, extending axially with the internal and external faces. The locking stop ( 11 ), also in the form of a circular ring with the same axis as the cylindrical body ( 2 ), is intended to prevent the crushing of the circular end of the filter membrane during the screwing of the removable element to the threaded part ( 8 ) of the flange. As shown by Figure 1, the reading window ( 3 ) is cleared of any obstruction to avoid masking the view of the filter membrane ( 4 ). The container ( 1 ) is made of plastic that must be transparent, pressure-resistant, and susceptible to perforation without fracture preferably laterally, if the need arises to sample a microbe colony that has developed, after incubation, on the filter membrane. The Applicant has found that a suitable plastic for the container was a copolymer of methyl methacrylate, butadiene and styrene. The filter membrane ( 4 ) is known and in general of plastic of the cellulose ester type, with a pore diameter that varies according to the size of the microorganisms to be retained. For commonly determined microorganisms, the pore diameter generally used is 0·45 µm. Other filter materials, hydrophilic or hydrophobic, can be used for the retention of other microorganisms. The filtration support ( 12 ), which completes the device according to the invention, is shown in Figures 3 and 4. It comprises a chamber ( 13 ), generally cylindrical, fitted externally with a male threaded part ( 14 ), which may consist for example of three male threads, cooperating with the female threaded part ( 8 ) of the flange ( 7 ) of the container ( 1 ), and, if necessary, gripping components ( 30 ). The chamber ( 13 ) is closed at its upper part by a plate ( 14 ), comprising a circular peripheral ledge ( 16 ) and a system of circular ( 17 ) and axial ( 18 ) drainage channels terminating in an axial outlet orifice ( 19 ). The peripheral ledge ( 16 ) is axially prolonged slightly beyond the upper face of the plate ( 15 ) as shown by Figure 3 in particular, in order to form a central circular cavity serving for the reception of a disc-shaped removable porous aseptic insert ( 20 ) which rests on the top of the drainage channels. The upper face of this insert ( 20 ), designed to enter into contact with the entire lower filtering surface of the membrane ( 4 ) to support it, when the support ( 12 ) is screwed into the container ( 1 ), lies at the same level as the upper face of the peripheral ledge ( 16 ), which has a number of fine radial slits ( 21 ) to facilitate the subsequent removal of any liquid that has infiltrated between the downstream periphery of the membrane and the surface ( 16 ), through the porous aseptic insert ( 20 ). The connecting part between the threaded chamber ( 13 ) and the plate ( 15 ) of the filtration support ( 12 ) comprises, in the embodiment shown in Figure 1, and shown more clearly in Figure 7, an outer shoulder ( 22 ) a circular gutter ( 23 ) and a sealing cone ( 24 ) delimiting the peripheral ledge ( 16 ). When the filtration support ( 12 ) is screwed inside the flange of the container ( 1 ), the shoulder ( 22 ) enters into contact with the stop ( 11 ) to prevent the crushing of the circular membrane (not shown in Figure 7), and the sealing cone ( 24 ) deforms the inner face of the lip seal ( 10 ) to guarantee perfect tightness between the support ( 12 ) and the container ( 1 ). In another embodiment shown in Figure 8, the outer shoulder ( 22 ) of the filtration support ( 12 ), not only enters into contact with the stop ( 11 ) of the container ( 1 ), but it also has a sealing cone ( 24 ) which deforms the outer face, and not the inner face, of the lip seal ( 10 ), to guarantee perfect tightness during the screwing of the support ( 12 ) into the container ( 1 ). The embodiment shown in Figure 8 serves to perform samplings of liquids to be analyzed at pressures higher than those that can be used with the embodiment in Figure 7, and which can be as high as 6·10 5 Pa, since any increase in the pressure of the liquid to be filtered tends to increase the tightness of the container on the sealing cone of the filtration support. The plastic of the filtration support ( 12 ) may, for example, be a styrene polymer such as acrylonitrile/butadiene/styrene resin, and the insert ( 20 ) may, for example, be of sintered polyethylene. The incubation cartridge ( 25 ), designed to complete the device according to the invention, may be a plastic cartridge for liquid (Figure 5) or dry (Figure 6) culture medium. This cartridge ( 25 ) comprises a chamber ( 26 ), generally cylindrical, containing a culture medium ( 27 ) appropriate to the microorganism to be detected, and which is externally fitted with a male threaded part ( 28 ), which can cooperate with the female threaded part ( 8 ) of the flange ( 7 ) of the container, and consisting, for example, of three male thread initiations for one, and females for the other, to guarantee rapid locking. The chamber ( 26 ) may also be provided with gripping components ( 29 ) and, at its top, a shoulder ( 37 ) designed to enter into contact with the stop ( 11 ) during the screwing of the cartridge ( 25 ) into the container ( 1 ). In the cartridge for liquid culture medium shown in Figure 5, the chamber ( 26 ) has, at its upper part, a support that is convexed externally ( 31 ) and made of one piece with the chamber. This convex support ( 31 ) is fitted with a system of feed channels connected to an axial feed orifice ( 32 ), closed by a removable plug ( 36 ). The convex support ( 31 ) is slightly recessed with respect to its peripheral ledge to form a central circular cavity, where an absorbent pad ( 33 ) impregnated with appropriate liquid culture medium rests, and which can be protected by a strippable protective paper ( 34 ) glued to the said peripheral ledge. The convex support ( 31 ) is connected to the upper end of the chamber ( 26 ) by a conical part ( 35 ), designed to cooperate with the lip seal ( 10 ) of the container ( 1 ), to prevent drying of the periphery of the liquid culture medium of the cartridge, screwed into the reservoir, during incubation. The incubation cartridge for dry culture medium, shown in Figure 6, does not have a conical sealing part, but its chamber ( 26 ) is closed at each end by a removable lid ( 38 and 39 ). The lid ( 38 ), located opposite the shoulder ( 37 ), serves to fill the cartridge, and the lid ( 39 ), which is convex towards the outside, is removed during the screwing of the cartridge ( 25 ) into the container ( 1 ). The plastic used to make the cartridge body may be polystyrene and that for the lids may be polypropylene. The process according to the invention consists, after filling the cartridges with the suitable culture medium, of the operations required for microbiological testing directly at the sampling location of the liquid sample to be tested. The process begins by screwing a container ( 1 ) to a filtration support ( 12 ) up to the sealing stop. The container can then withstand a filtration pressure of the liquid to be analyzed that is higher than 3·10 5 Pa, in the embodiment shown in Figures 1, 3 and 7, and up to 6·10 5 Pa in the embodiment shown in Figure 8. The small male Luer plug ( 6 ) is then removed and the container ( 1 ) connected to the sample tap. The valve of the sample tap is opened, and the desired volume of liquid to be tested is allowed to filter through, and the sample volume is collected in a beaker located downstream from the filtration support (Figure 9). The sample tap valve is closed at the end of sampling, and the filtration unit is removed. Filtration is terminated either by aseptically pressurizing the container by means of a syringe ( 40 ) fitted with an air filter, as shown in Figure 10, which has the advantage of expelling all the liquid present upstream, downstream and in the pores of the membrane, and which avoids any risk of dilution of the culture medium downstream from the filter membrane, or by causing a negative pressure to prevail by pumping the remnant of liquid with the aid of a syringe ( 40 ) as shown in Figure 11. The taking of the sample having been terminated, the filtration support ( 12 ) is unscrewed and one of the cartridges ( 25 ) containing the desired culture medium is screwed in its place. It then suffices to place the overall unit consisting of the container ( 1 ) and the cartridge ( 25 ) in the reversed position, in an incubator at the temperature and during the time required for the development of the colonies resulting from any microorganisms collected on the filter membrane ( 4 ), as shown in Figures 12 and 13. It should be noted that the device according to the present invention also serves to take a sample of product to be analyzed by means of a vacuum source connected to the filtration support ( 12 ), such as a vacuum syringe or flask. This alternative was not available with previously known devices. After development of the colonies, they can either be counted, as in a Petri dish, since the reading window ( 3 ) is perfectly cleared, or one of them can be identified by sampling, by perforating the container by means of an apparatus ( 41 ) designed for the purpose in order to be able to sample the desired colony with a platinum loop ( 42 ). With the container placed in a support, a cutting point with a triangular section penetrates parallel to the filter membrane into the walls of the cylindrical body ( 2 ) of the container ( 1 ), thus creating a completely clean hole without removal of any plastic (Figures 14 and 15). If so desired, it is possible to make several holes to make the colonies located on the surface of the filter membrane more accessible. In this case, it suffices to introduce the platinum loop ( 42 ) through the best-positioned hole to make the sampling (Figure 16). If it is desired to continue incubation, it suffices to envelop the test unit consisting of the container and the incubation cartridge in a suitable film ( 43 ) in order to isolate the container from the outside air (Figure 17).

Claims: Device for the microbiological testing of a pressurized liquid sample using a filter membrane, preferably pre-sterilized and for single use only, and comprising, upstream from the filter membrane (4), a liquid inlet in the form of an orifice (5), which can be closed by a removable plug (6), characterized in that it comprises a completely closed container (1) of transparent plastic, at the base of which a filter membrane (4) is sealed, the said container being fitted , upstream from the membrane, with a built-in reading window (13) and, downstream from the membrane, with a flange (7) comprising a lip seal (10) and a female threaded part (8). Device according to claim 1, characterized in that the liquid inlet in the form of an orifice (5) is provided with an axis parallel to the plane of the membrane (4). Device according to any one of the previous claims, characterized in that the container ( 1 ) comprises a locking stop (11) located between the lip seal (10) and the female threaded part (8). Device according to any one of claims 1 to 3, characterized in that the lip seal (10) consists of a circular tab whose inside face guarantees tightness by cooperating with a conical part of a removable element that can be screwed into the female threaded part of the container. Device according to any one of claims 1 to 3, characterized in that the lip seal (10) consists of a circular tab whose outer faces guarantees tightness by cooperating with a conical part of a removable element than can be screwed into the female threaded part of the container. Device according to any one of the previous claims, characterized in that the threaded part (8) of the flange (7) of the container ( 1 ) has three female thread initiations to guarantee rapid locking. Device according to any one of the previous claims, characterized in that the container ( 1 ), of which the built-in reading window ( 3 ) constituting the upper face and located in a plane parallel to that of the filter membrane ( 4 ), is cleared of any obstruction to avoid masking the view of the membrane, and can be perforated, preferably laterally, by penetration, thus making accessible the top of the filter membrane for any sampling of microbe colonies by means of a platinum loop ( 42 ). Device according to any one of the previous claims, characterized in that the transparent plastic of the container ( 1 ) is a copolymer of methyl methacrylate, butadiene and styrene. Device according to any one of the previous claims, characterized in that it comprises a filtration support ( 12 ) made of plastic comprising a chamber ( 13 ) fitted externally with a male threaded part ( 14 ) cooperating with the female threaded part ( 8 ) of the container ( 1 ), and an upper plate ( 15 ) fitted with a system of drainage channels ( 17 , 18 ) terminating in an axial outlet orifice ( 19 ), the said filtration support ( 12 ) also comprising a sealing cone ( 24 ) cooperating with the lip seal ( 10 ) of the container ( 1 ) to prevent any infiltration or contamination from the outside on the periphery of the support during the filtration of a liquid sample to be analyzed. Device according to Claim 9, characterized in that the peripheral ledge ( 16 ) of the upper plate ( 15 ) of the filtration support ( 12 ) is axially prolonged beyond the upper face of the plate ( 15 ) to form a central circular cavity serving for the insertion of a removable porous aseptic insert ( 20 ), resting on the top of the drainage channels ( 17 , 18 ) and designed to enter into contact with the entire filtering surface of the membrane ( 4 ) of the container ( 1 ). Device according to any one of Claims 9 and 10, taken in combination with Claim 6, characterized in that the threaded part ( 14 ) of the filtration support ( 12 ) has three male thread initiations to guarantee rapid locking on the three corresponding female threads ( 8 ) of the container ( 1 ). Device according to any one of Claims 9 to 11, characterized in that the plastic of the filtration support ( 12 ) is a styrene polymer and, in particular, an acrylonitrile/butadiene/styrene resin. Device according to any one of Claims 1 to 8, characterized in that it comprises an incubation cartridge made of plastic ( 25 ) comprising a chamber ( 26 ) containing a suitable culture medium of microorganisms ( 27 ) and fitted externally with a male threaded part ( 28 ) cooperating with the female threaded part ( 8 ) of the container ( 1 ). Device according to Claim 13, characterized in that the incubation cartridge ( 25 ) comprises a support convexed outward ( 31 ) receiving an absorbent pad ( 33 ) impregnated with liquid culture medium, which can be protected by a strippable protective paper ( 34 ), and in that the upper end of the chamber ( 26 ) of the cartridge displays a conical part ( 35 ) designed to cooperate with the lip seal ( 10 ) of the container ( 1 ) to guarantee tightness on the downstream periphery of the filter membrane ( 4 ) and thus to prevent any dehydration of the culture medium ( 27 ) during incubation. Device according to any one of Claims 13 to 14, characterized in that the plastic of the incubation cartridge is polystyrene. Process for the microbiological testing of a pressurized liquid sample by means of a filter membrane ( 4 ), characterized in that it comprises in succession the introduction of the sample to be analyzed into a device forming the subject of any one of Claims 1 to 8, in which the container ( 1 ) is screwed tight on a filtration support ( 12 ), the positive or negative pressurization of the container ( 1 ) to terminate filtration, the replacement of the filtration support ( 12 ) by an incubation cartridge ( 25 ) screwed tight into the container ( 1 ), and incubation of the combination consisting of the container and the cartridge, in the reversed position, in an incubator.

Inventors: LEMONNIER JEAN, LEMONNIER, JEAN
Applicants: MILLIPORE SA, MILLIPORE S.A.
Application year: 1992
Main classification: C12M1
IPC codes: C12Q1, C12M3, C12M1