Device and method for treating a filtration medium

Abstract

A device (1) and a method are provided for treating a porous filtration medium (37) having a receiving unit (2) with of a receiving part (5) and a base part (6). The porous filtration medium (37) can be lifted by the receiving part (5) from a lower part (33) of a filtration device (32), and the receiving part (5) with the porous filtration medium (37) can be mounted on the base part (6). The receiving part (5) is latchable to the base part (6). The base part (6), towards the filtration medium (37) has an incubation chamber (17) connected to a base part (6) outlet (3) that faces away from the receiving part (5), and the outlet (3) has a projection onto which a receiving vessel (4) containing a solvent (28) for dissolving the porous filtration medium (37) can be detachably pushed on.

Claims

1. A device (1) for treating a porous filtration medium (37), the device comprising: a receiving unit (2) that includes: a receiving part (5) configured to have the porous filtration medium (37 mounted thereon, and a base part (6) releasably engaged with the receiving part (5), the base part (6) having opposite first and second ends, the first end facing towards the receiving part (5); and the base part (6) defines conical incubation chamber (17) that widens toward the receiving part (5); the second end of the base part (6) defining an outlet (3) facing away from the receiving part (5); the outlet (3) comprises a projection (24) with an outlet channel (21) extending therethrough; and the outlet channel (21) is fluidically connected to the incubation chamber (17); and a receiving vessel (4) detachably push-fit on the projection (24) of the outlet (3), the receiving vessel (4) containing a solvent (28) for dissolving the porous filtration medium (37) and grinding balls (29) that support cell disruption, wherein the outlet channel (21) of the outlet (3) is an oblong slot with a long direction of the oblong slot being aligned at a right angle to a longitudinal axis (22) of the base part (6), and the oblong slot (22) has a clear width (23) transverse to the long direction thereof that is smaller than an outside diameter of the grinding balls (29).

2. The device of claim 1, wherein the solvent (28) for dissolving the filtration medium (37) is an organic solvent.

3. The device of claim 2, wherein the solvent (28) is chloroform or methylene chloride.

4. The device of claim 1, wherein the receiving vessel (4) to be push-fitted to the base part (6) contains both the solvent (28) and a lysis buffer that supports cell disruption.

5. The device of claim 1, wherein when the receiving vessel (4) is detached from the projection (24) of the outlet (3), an open end (26) of the receiving vessel (4) is configured to be sealed with a cover to prevent fluids from leaking out of the receiving vessel (4).

6. The device of claim 1, further comprising the porous filtration medium mounted on the receiving part (5), wherein the receiving part (5) has an inner wall (8) positioned outside a surface of the filtration medium (37) that can be used for filtration, and the inner wall (8) has a fixing edge (11) arranged in the receiving part (5) and positioned on an edge (38) of the filtration medium (37), and at least one of the fixing edge (11) of the receiving part (5) and the edge (38) of the filtration medium (37) has an adhesive thereon forming an adhesive bond between the fixing edge (11) of the receiving part (5) and the edge (38) of the filtration medium (37).

7. The device of claim 1, further comprising the porous filtration medium mounted on the receiving part (5), wherein the filtration medium (37) is made of polycarbonate or polyethersulfone.

8. The device of claim 1, further comprising a centrifuge adapter (39), and wherein the receiving unit (2) is attachable to the receiving vessel (4) arranged vertically at a bottom in the centrifuge adapter (39) so that the receiving unit (2) and the receiving vessel (4) can be centrifuged with the centrifuge adapter (39) in a centrifuge, whereby the filtration medium (37) dissolved in the solvent (28), including retained microorganisms, can be completely transferred into the receiving vessel (4).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a side view in cross-section of a receiving part of a device for treating a porous filtration medium.

(2) FIG. 2 is a side view in cross-section of a base part of a device for treating a porous filtration medium.

(3) FIG. 3 is a view from below of the base part of FIG. 2.

(4) FIG. 4 is a side view in cross-section of a receiving unit with received porous filtration medium, the receiving part and base part of said unit being latched together, and with a push-fitted receiving container containing solvent and grinding balls.

(5) FIG. 5 is an enlarged view of detail V (latching arrangement) of FIG. 4.

(6) FIG. 6 is an enlarged view of a further latching arrangement corresponding to detail V of FIG. 4.

(7) FIG. 7 is a side view in cross-section of a further receiving unit with received a porous filtration medium, the receiving part and base part of said unit being latched together, and with a push-fitted receiving container containing solvent and grinding balls.

(8) FIG. 8 is a side view in cross-section of the centrifuge adapter of FIG. 10 along the line VIII-VIII.

(9) FIG. 9 is a side view of the centrifuge adapter of FIG. 10 viewed from direction IX.

(10) FIG. 10 is a top view of the centrifuge adapter of FIGS. 8 and 9.

(11) FIG. 11 is a side view in cross-section of a filtration device according to the prior art with a filtration medium arranged on a lower part.

(12) FIG. 12 is curve plots of the samples of B. subtilis processed according to a preferred embodiment of the invention.

(13) FIG. 13 is curve plots of the spore samples of B. subtilis processed according to the preferred embodiment of the invention compared to the prior art.

DETAILED DESCRIPTION

(14) A device 1 consists essentially of a receiving unit 2 with an outlet 3 and a receiving vessel 4.

(15) The receiving unit 2 comprises two parts and consists of a receiving part 5 and a base part 6.

(16) The receiving part 5 forms a circumferential contour with an outer wall 7 and an inner wall running parallel to it. The receiving part 5 is sealed in a vertical direction at the top by a top wall 9. On its inner surface 10 of the receiving part, facing the base part 6, the top wall 9 has the inner wall 8, the free end face of which forms a fixing edge 11. In the exemplary embodiments the fixing edge 11 has an adhesive layer 12 made of an appropriate adhesive.

(17) The adhesive layer 12 could, for example, be made of a PSA dispersion adhesive or of acrylate-copolymer microspheres. Appropriate adhesives are those that are based on organic solvent(s) and that are soluble in organic solvents that are used in the context of dissolving a filtration medium. Furthermore, the adhesives must demonstrate permanent adhesive strength (from the date of production of the device until it is used by the user). The adhesive must be sterilizable using ETO (ethylene oxide). In addition, adhesives that demonstrate no non-specific reactions or signals with reagents and reaction methods used in subsequent analyses are used. In particular it is preferred that the adhesive be free of DNA and that it contain no substances which might interfere with the subsequent analyses through coloration, fluorescence or chemical reaction.

(18) The outer wall 7 has an inside outer wall surface 13 with a ridge 14 running around its circumference.

(19) The base part 6 has a circumferential outer wall 15 with an outer surface 16 that corresponds with, i.e. interacts with, the inner wall 13 of the receiving part 5. On the side facing the receiving part 5, the base part 6 has a funnel-shaped incubation chamber 17 with a conical drainage surface 18 that is inclined to the horizontal at an angle 20 of, for example, 25° sloping down to the outlet 3. The outlet 3 has an outlet channel 21 designed as an oblong slot with a narrow clear width arranged at a right angle to the longitudinal axis 22 of the base part 6.

(20) The exterior lateral surface of the outlet 3 forms a slightly conical projection 24 onto which the receiving vessel 4 can be detachably push-fitted. At its open end 26, the receiving vessel 4 has an outside thread 27 and can be tightly sealed by screwing on a cover (not shown) having an inside thread. The receiving vessel 4 contains a solvent 28 and grinding balls 29.

(21) Alternatively, instead of or in addition to the grinding balls 29, the receiving vessel 4 can contain a lysis buffer for the microorganisms retained by a filtration medium 37, wherein the lysis buffer, as an aqueous liquid, forms a two-phase system with the solvent 28.

(22) The base part 6 has a ring-shaped indentation 30 running around its outer surface 16 that corresponds to the ridge 14 on the receiving part 5 and forms a latching arrangement 31 (see FIGS. 4, 5, 7). The latching arrangement 31 may also be designed to be irreversible, as depicted in FIG. 6.

(23) A known filtration device 32 according to FIG. 11 consists of a lower part 33 with a receiving shoulder 34, on which a funnel-shaped attachment 35 can be mounted. The preferably disc-shaped filtration medium 37, which is designed, for instance, as a porous filter membrane, is arranged between the attachment 35 and a filter-supporting surface 36 of the lower part 33.

(24) After a filtering process, the attachment 35 can be removed from the lower part 33 and the receiving part 5 of the device 1 can be placed on the lower part 33 in place of the attachment 35. In the process, the receiving part 5 with its fixing edge 11 is placed on an edge 38 of the disc-shaped filtration medium 37 so that the disc-shaped filtration medium 37 adheres to the adhesive layer 12 of the fixing edge 11 and can be lifted off the lower part 33.

(25) The device 1 with the receiving vessel 4 can be placed in a centrifuge adapter 39, which has an appropriately adapted recess 40.

(26) Treatment of the porous filtration medium 37 with the receiving unit 2, which consists of the receiving part 5 and the base part 6, of the device 1 is carried out according to the following steps: the receiving part 5 is placed on the filtration medium 37, which is arranged in the lower part 33 of the filtration device 32 and is exposed to a liquid sample, with a fixing edge 11 arranged in the receiving part 5 being connected with an edge 38 of the filtration medium 37, the receiving part 5 with the attached filtration medium 37 is lifted off the lower part 33 and placed on the base part 6, whereby the receiving part 5 and the base part 6 are latched together by means of the latching arrangement 31, 31′, a receiving vessel 4 containing a solvent 28 for dissolving the porous filtration medium 37 and grinding balls 29 is detachably connected to the outlet 3 arranged on the base part 6, the receiving unit 2 with the receiving vessel 4 is inverted and gently shaken, whereby the solvent 28 is added to the filtration medium 37 via the outlet 3 of the base part 6, and dissolves the filtration medium 37.

(27) The following steps can then be carried out: the receiving unit 2, with the receiving vessel 4 arranged vertically at the bottom, is mounted in a centrifuge adapter 39 and centrifuged in a centrifuge, whereby the filtration medium 37 dissolved in the solvent 28, including retained microorganisms, is completely transferred into the receiving vessel 4, the receiving vessel 4 is removed from the receiving unit 2 and sealed by screwing on a cover, the sealed receiving vessel 4 is processed in a homogenizer, and the cell disruption of the microorganisms is facilitated by the grinding balls 29.

(28) If a lysis buffer is used instead of or in addition to the grinding balls 29 for cell disruption, the following steps can be carried out: the receiving unit 2, with the receiving vessel 4 arranged vertically at the bottom, is mounted in a centrifuge adapter 39 and centrifuged in a centrifuge, whereby the filtration medium 37 dissolved in the solvent 28, including retained microorganisms, is completely transferred into the receiving vessel 4, the receiving vessel 4 is removed from the receiving unit 2 and is filled with lysis buffer before being sealed with a screw-on a cover, the sealed receiving vessel 4 is processed in a homogenizer, and the cell disruption of the microorganisms is facilitated by the grinding balls 29 and/or lysis buffer.

(29) If cell disruption is carried out without using grinding balls and only with a lysis buffer, cell disruption can alternatively also take place in an agitation incubator instead of in a homogenizer.

(30) The following experiments were performed:

Example 1

(31) Determination of Sensitivity for Detecting Bacillus subtilis Using the Device 1 Including the Receiving Vessel 4.

(32) A dilution series of an exponential phase culture of Bacillus subtilis in a 0.9% NaCl solution was incubated, using double determination after filtration, on Sartorius nutrient agar (47-mm cellulose-nitrate membrane with a pore diameter of 0.45 μm; enumeration of the colonies after 24 h), and at the same time one sample per dilution stage was processed according to a preferred embodiment of the invention.

(33) A preferred embodiment of the invention comprises the following process steps: 1. Membrane filtration of an aqueous sample (membrane diameter 47 mm, track-etched polycarbonate membrane, pore diameter 0.4 μm, membrane thickness 6 to 11 μm) using a lower part 33 (ideally made of plastic; ETO sterile) as depicted in FIG. 11. 2. Removal of the attachment 35 and lifting up of the membrane filter/filtration medium 37 with the help of the adhesive bond on the fixing edge 11 of the receiving part 5 (made of polypropylene; ETO sterile). 3. Connecting of the receiving part 5 and base part 6 of the receiving unit 2 by means of the latching arrangement 31. 4. The receiving vessel 4 (made of polypropylene; ETO sterile) with attached screw-on cover with a capacity of 2 ml contains 15 steel balls/grinding balls 29 (diameter 3 mm) and 750 μl chloroform (molecular biology grade; above all free of DNA and DNase). After the receiving vessel 4 is opened, it should be attached to the outlet 3 of the base part 6 by means of a plug-in connection (downward tapering projection 24 is inserted into the open end 26 of the receiving vessel 4). 5. The receiving unit 2 with receiving vessel 4 is inverted and gently shaken to transfer the chloroform completely from the receiving vessel 4 to the receiving unit 2. The receiving unit 2 with the receiving vessel 4 is then inverted and gently swirled for several seconds to ensure that the membrane filter/filtration medium 37 dissolves completely. 6. The receiving unit 2 with attached receiving vessel 4 is turned upright (receiving part 5 faces upwards, receiving vessel 4 faces downward) and placed in the special centrifuge adapter 39. The adapter is a swing-out centrifuge adapter to ensure the quantitatively complete transfer of the dissolved membrane/filter medium 37, including the particles retained by the membrane 37, and of the solvent 28. Furthermore, the centrifuge adapter 39 is constructed such that the receiving vessel 4 cannot become loose or fall off during the centrifugation step. The centrifuge adapter 39 is mounted on a suitable centrifuge and centrifuged for one minute at at least 3,000×g in order to completely transfer the membrane 37 dissolved in the chloroform, including retained microorganisms, to the receiving vessel 4 with the steel grinding balls 29. 7. The receiving vessel 4 is removed from the receiving unit 2 and tightly sealed by screwing on the attached cover. 8. To disrupt the microorganisms and make their DNA accessible, the receiving vessel 4 is processed for 2 min at 6.5 m/s in a homogenizer (FastPrep-24 Instrument from MP Biomedicals). (Alternatively, other comparably performing homogenizers can be used.) 9. The receiving vessel 4 is removed from the homogenizer, and 500 μl of 1×TE buffer (tris-EDTA) with 0.01% SDS (sodium dodecyl sulfate) are added (molecular biology grade). 10. A 10-minute extraction (extraction of DNA from the organic to the aqueous phase) at room temperature follows. To do this, the receiving vessel 4 is attached either horizontally on a vortexer or horizontally on a thermomixer at 750 RPM. 11. Add a spatula tip of DNA- and DNase-free silicone paste (e.g. Phase Lock Gel from 5 PRIME or GE Bayer Silicones, high viscosity) through the opening in the receiving vessel 4. 12. Centrifuge the receiving vessel 4 for 3 min at 16,000×g. 13. Because of the different densities, three phases separated: The top, aqueous phase including DNA, the middle, silicone-gel phase as a barrier layer, and the bottom, organic phase. The entire top, aqueous phase is transferred to a new, empty receiving vessel 4 using a pipette (a 1.5-ml reaction vessel is sufficient). 14. Add 600 μl isopropanol (molecular biology grade) and 2 μl glycogen as a DNA carrier (molecular biology grade) and invert the reaction vessel 4 50 times. 15. Centrifuge the reaction vessel 4 for 3 min at 16,000×g, so that a small, white DNA-glycogen pellet forms on the floor of the reaction vessel 4. 16. Discard the isopropanol; the DNA-glycogen pellet remains in the reaction vessel 4. 17. Use a pipette to add 600 μl of 70% ethanol to the DNA-glycogen pellet and invert the reaction vessel 4 20 times. 18. Centrifuge the reaction vessel 4 for 1 min at 16,000×g. 19. Discard the 70% ethanol (remove with a pipette); the DNA-glycogen pellet remains in the reaction vessel 4. 20. Dry the DNA-glycogen pellet in the opened reaction vessel 25 either for 10 min at 37° C. in a sealed thermoblock or for 15 to 20 min under the sterile bench. 21. Dissolve the pellet in 50 to 100 μl of rehydration buffer (10 mM tris, 1 mM EDTA, pH 7-8, free of DNA and DNase) for 1 h at 65° C. in a thermoblock (in the closed reaction vessel). 22. Analysis/detection with quantitative real-time PCR, e.g. with universal or specific bacterial primers.
Reaction Conditions, Example 1:
25 μl PCR reaction volume (12.5 μl MAXIMA SYBR Green qPCR Master Mix from Fermentas, 10 nM ROX,
0.3 μM Forward Primer SEQ ID NO. 1: 5″-AAGTCGAGCGGACAGATGG-3″,
0.3 μM Reverse Primer SEQ ID NO. 2: 5″-TGCGGTTCAAACAACCATCCG-3″,
10 μl DNA (obtained according to the preferred embodiment of the invention),
add water (PCR grade) for a total of 25 μl.
Temperature profile: 10 min at 95° C.; 40 cycles of 15 seconds at 95° C., 30 seconds at 60° C., 30 seconds at 72° C. (fluorescence detection at 72° C.); melting curve with 1 min at 95° C., 30 seconds at 55° C., temperature ramp up to 95° C. with fluorescence measurement, 30 seconds at 95° C.
Results of Exemplary Embodiment 1:

(34) TABLE-US-00001 TABLE 1 Ct (cycle threshold) values and melting points of Exemplary Embodiment 1 Cycle Melting point of the Sample designation Threshold amplicon [° C.] 2 × 10.sup.2 CFU/ml* 34.22 83.80 2 × 10.sup.2 CFU/ml* 34.37 83.80 2 × 10.sup.3 CFU/ml* 33.48 83.80 2 × 10.sup.3 CFU/ml* 33.90 83.80 2 × 10.sup.4 CFU/ml* 32.18 83.80 2 × 10.sup.4 CFU/ml* 32.21 83.80 2 × 10.sup.5 CFU/ml* 29.05 83.80 2 × 10.sup.5 CFU/ml* 28.91 83.80 PCR negative control No Ct 69.38 PCR negative control No Ct 69.38 PCR negative control No Ct 69.38 *CFU (colony-forming unit) concentrations determined by plating.

(35) FIG. 12 shows the curve plots of the samples of B. subtilis processed according to the preferred embodiment of the invention: diamonds (2×10.sup.5 CFU/ml), triangles (2×10.sup.4 CFU/ml), squares (2×10.sup.3 CFU/ml), circles (2×10.sup.2 CFU/ml), stars (0 CFU/ml, extraction negative controls). Using the preferred embodiment of the invention, Bacillus subtilis can be detected in a sample volume of any size with a sensitivity of at least 2×10.sup.2 CFU/ml (using primer with Sequences SEQ 1 and SEQ 2).

Example 2

(36) Invention with a preferred embodiment vs. prior art (L. J. DiMichele, Am. Soc. Brew. Chem., 1993, Vol. 51 No. 2, pp. 63-66, and K. Stärk, Applied and Environmental Microbiology, 1998, Vol. 64, No. 2, pp. 543-548; Further treatment of dissolved filtration medium 37 without a cell lysis step). Sensitivity comparison for the detection of B. subtilis spores using device 1 incl. receiving vessel 4 and filtration device 32.

(37) Two membrane filters/filtration medium 37 were processed according to a preferred embodiment of the invention (i.e. cell disruption using grinding balls 29 in a homogenizer). Two membrane filters/filtration medium 37 were processed according to this preferred embodiment of the invention, however without a cell lysis step (corresponds to the prior art according to K. Stark and L. J. DiMichele). Two membrane filters/filtration medium 37 were processed as extraction negative controls according to the preferred embodiment of the invention, however without the application of microorganisms. 106 B. subtilis spores were applied to each membrane filter/filtration medium 37, and the two extraction negative controls were brought into contact only with sterile water (PCR grade). The six samples were processed according to the preferred embodiment of the invention as described in Example 1 (Steps 1 to 22). In the case of the prior art samples (according to K. Stark and L. J. DiMichele), receiving vessels 4 without grinding balls 29 were used and the cell-lysis step in the homogenizer was omitted.

(38) Results of Exemplary Embodiment 2:

(39) TABLE-US-00002 TABLE 2 Ct (cycle threshold) values and melting points of Exemplary Embodiment 2 Cycle Melting point of the Sample designation Threshold amplicon [° C.] PCR negative control No Ct 56.92 PCR negative control No Ct 56.91 Extraction negative No Ct 56.91 control Extraction negative No Ct 56.46 control Prior art 33.23 83.97 Prior art 33.55 83.97 Invention (preferred 29.96 83.97 embodiment) Invention (preferred 29.86 83.97 embodiment)

(40) FIG. 13 shows the curve plots of the spore samples of B. subtilis processed according to the preferred embodiment of the invention compared to the prior art: diamonds (preferred embodiment of the invention), triangles (prior art), squares (PCR negative controls and extraction negative controls).

(41) Example 2 demonstrates that the invention in the preferred embodiment is superior to the prior art because an increase in sensitivity of more than 3 Ct units was achieved, which corresponds to a factor of approximately ten genome units/B. subtilis spores.

LIST OF REFERENCE NUMBERS

(42) 1 device 2 receiving unit 3 outlet of 2 4 receiving vessel 5 receiving part of 2 6 base part of 2 7 outer wall of 5 8 inner wall of 5 9 top wall 10 inner surface of receiving part 11 fixing edge 12 adhesive layer 13 inside surface of outer wall 14 ridge of 5 15 outer wall of 6 16 outer surface of 15 17 incubation chamber 18 drainage surface 20 angle 21 outlet channel 22 longitudinal axis of 6 23 clear width of 21 24 projection of 3 26 open end of 25 27 outside thread of 25 28 solvent 29 grinding balls 30 indentation of 6 31, 31′ latching arrangement 32 filtration device 33 lower part of 32 34 receiving shoulder of 33 35 attachment 36 filter supporting surface 37 filtration medium 38 edge of 37 39 centrifuge adapter 40 recess