EXTRACTION SYSTEM AND METHOD FOR EXTRACTING MICROORGANISMS CONTAINED IN A SAMPLE

Abstract

An extraction system includes a support bar including a sample well, a reagent well, at least one washing well, and at least one protection and retention housing; a pipetting element removably mounted on the support bar and extending at least partially into the at least one protection and retention housing; and a filtering and transfer device configured to capture and accumulate microorganisms contained in the sample and transfer the captured and accumulated microorganisms, the filtering and transfer device being removably mounted on the support bar and extending at least partially into the at least one protection and retention housing. The filtering and transfer device includes a hollow body having an elongated shape and including a proximal end portion and a distal end portion; and a filtering element fixed to the distal end portion of the hollow body.

Claims

1. An extraction system for extracting microorganisms contained in a sample, said extraction system comprising: a support bar comprising: a sample well configured to receive a sample containing microorganisms, a reagent well configured to contain a selective lysis buffer, at least one washing well configured to contain a washing reagent, and at least one protection and retention housing, a pipetting element removably mounted on the support bar and extending at least partially into the at least one protection and retention housing, a filtering and transfer device configured to capture and accumulate microorganisms contained in the sample and transfer the captured and accumulated microorganisms, the filtering and transfer device being removably mounted on the support bar and extending at least partially into the at least one protection and retention housing, said filtering and transfer device comprising: a hollow body having an elongated shape and comprising a proximal end portion and a distal end portion, and a filtering element fixed to the distal end portion of the hollow body.

2. The extraction system according to claim 1, in which the distal end portion of the hollow body and the filtering element extend into the at least one protection and retention housing.

3. The extraction system according to claim 1, in which the pipetting element comprises a distal end portion extending at least partially into the at least one protection and retention housing.

4. The extraction system according to claim 1, in which the filtering and transfer device comprises an absorbent material arranged in the hollow body.

5. The extraction system according to claim 1, in which the support bar further comprises a release well.

6. The extraction system according to claim 1, in which the at least one washing well contains a washing reagent and the reagent well contains a selective lysis buffer.

7. The extraction system according to claim 6, in which the reagent well and the at least one washing well are each sealed by a sealing element.

8. The extraction system according to claim 1, which further comprises a sealing device movable between a sealing position in which the sealing device seals the sample well and a release position in which the sealing device releases the sample well at least partly.

9. A method for identifying and/or characterizing microorganisms contained in a sample, said identifying and/or characterizing method comprising the following steps: providing an extraction system according to claim 1, in which the reagent well contains a selective lysis buffer and the at least one washing well contains a washing reagent, introducing a predetermined amount of a sample containing microorganisms in the sample well, withdrawing a predetermined amount of the selective lysis buffer contained in the reagent well by the pipetting element, placing the withdrawn selective lysis buffer in the sample well by the pipetting element, lysing cells and/or particles that are not micro-organic contained in the sample and/or lysing predetermined microorganisms contained in the sample so as to supply a lysed sample, at least partially immersing the filtering and transfer device in the lysed sample contained in the sample well, filtering at least part of the lysed sample through the filtering element under aspiration, capturing and accumulating microorganisms in the filtering element, at least partially immersing the filtering and transfer device in the washing reagent contained in the at least one washing well, washing the filtering element and the captured and accumulated microorganisms in the washing reagent contained in the at least one washing well, by the filtering and transfer device, transferring the washed microorganisms onto an analysis support or into a receiver, analyzing the transferred microorganisms so as to identify them and/or characterize them.

10. The method according to claim 9, in which the transfer step comprises a step consisting of resuspending the washed microorganisms in a saline solution contained in a release well.

11. The method according to claim 10, in which the resuspending step comprises the following steps: at least partially immersing the filtering and transfer device in the saline solution contained in the release well, repeatedly contacting the filtering element with an inner layer of crosslinked silicone at least partly covering the inside surface of the release well for a predetermined time.

12. The method according to claim 11, which further comprises the following steps: measuring the optical density of the saline solution contained in the release well and containing the washed microorganisms, adjusting the concentration of microorganisms in the saline solution contained in the release well by adding saline solution to the release well, withdrawing, from the release well, a predetermined amount of the saline solution containing the microorganisms, introducing the withdrawn saline solution into an analysis container containing a predetermined amount of saline solution.

13. The method according to claim 12, in which the analysis step comprises the following steps: transferring a predetermined amount of the consumable contained in the analysis container and containing the transferred microorganisms, into an antibiogram analyser, and performing an antibiogram analysis on the consumable transferred.

14. The method according to claim 9, in which the transfer step comprises a step consisting of depositing, by the filtering and transfer device, at least one part of the washed microorganisms onto one or more predetermined spots of a MALDI-TOF MS analysis plate, and in which the analysis step comprises a step consisting of performing a mass spectrometry analysis of the MALDI-TOF type on the microorganisms deposited on said MALDI-TOF MS plate.

Description

[0162] FIG. 1 is a side view of an extraction system according to a first embodiment of the invention.

[0163] FIG. 2 is a sectional view of the extraction system in FIG. 1.

[0164] FIG. 3 is a partial sectional view of a distal end portion of a filtering and transfer device of the extraction system in FIG. 1.

[0165] FIG. 4 is a perspective view of a rack equipped with a plurality of extraction systems from FIG. 1.

[0166] FIG. 5 is a perspective view of an automatic machine equipped with the rack in FIG. 4.

[0167] FIG. 6 is a perspective partial view of an extraction system according to a second embodiment of the invention.

[0168] FIG. 7 is a perspective view of a rack equipped with a plurality of extraction systems from FIG. 6 and moreover with additional containers.

[0169] FIG. 8 is a sectional view of the rack in FIG. 7, but with addition of sealing elements configured for sealing a reagent well and with washing wells of an extraction system provided on the rack.

[0170] FIGS. 9 and 10 are perspective partial views of an extraction system according to a third embodiment of the invention.

[0171] FIG. 11 is a perspective view of a MALDI-TOF MS analysis plate.

[0172] FIGS. 1 to 3 show an extraction system 2, for extracting microorganisms contained in a sample, according to a first embodiment of the invention. The sample may for example be a microbiological culture, such as a positive blood culture. The sample may moreover be a body fluid, such as cerebrospinal fluid, synovial fluid, urine or pus that has been cultured beforehand in a suitable liquid culture medium, in order to increase the concentration of microorganisms per mL.

[0173] The extraction system 2 comprises a support bar 3 made for example of plastic, and advantageously provided with an identifying code, such as a barcode.

[0174] As shown more particularly in FIG. 2, the support bar 3 notably comprises a sample well 4 configured to receive a sample containing microorganisms, a reagent well 5 containing a selective lysis buffer L, and one or more washing wells each containing a washing reagent W. According to the embodiment shown in FIGS. 1 to 3, the sample well 4, the reagent well 5 and the washing well or wells are mounted on the support bar 3. However, according to another embodiment of the extraction system 2, the support bar 3, the sample well 4, the reagent well 5 and the washing well or wells could be produced directly by molding, and notably by injection molding (see FIG. 6).

[0175] According to the embodiment shown in FIGS. 1 to 3, the support bar 3 comprises a washing well 6.1 containing a washing reagent having a formulation identical to the formulations described in document WO2013/016211, a washing well 6.2 containing a washing reagent having a formulation identical to the formulations described in document WO2013/016211, and a washing well 6.3 containing for example sterile deionized water. Each of the washing wells may for example contain 1400 L of the respective washing reagent.

[0176] According to one embodiment of the invention, the selective lysis buffer L contained in the reagent well 5 has a formulation identical to the formulations described in document WO2013/016211.

[0177] The sample well 4, the reagent well 5 and the washing well or wells 6.1, 6.2, 6.3 are advantageously aligned in the direction of extension of the support bar 3. Advantageously, the reagent well 5 and the washing well or wells 6.1, 6.2, 6.3 are sealed with respective sealing covers 7, consisting for example of an aluminum/polyethylene composite heat-sealed to the surface of the reagent and washing wells.

[0178] The support bar 3 also comprises a gripping part 3.1 facilitating manipulation thereof by an operator.

[0179] The support bar 3 further comprises a protecting portion 8 delimiting a protection and retention housing 9 which is open at the top and whose function will be stated below. According to the embodiment shown in FIGS. 1 to 3, the protecting portion 8 is arranged between the sample well 4 and the reagent well 5. This position is provided in order to minimize the risks of soiling, during movement of the pipetting element above the support bar 3.

[0180] The extraction system 2 further comprises a pipetting element 10, for example made of plastic, removably mounted on the support bar 3, and extending at least partially into the protection and retention housing 9. According to the embodiment shown in FIGS. 1 to 3, the pipetting element 10 comprises a tapered distal end portion 10.1 extending into the protection and retention housing 9, and a proximal end portion 10.2 configured for being connected to a vacuum source or a volume displacement device, such as an electronically controlled syringe. Advantageously, the protection and retention housing 9 is configured for retaining drops of liquid, such as drops of sample or of reagents, able to flow by gravity from the tapered distal end portion 10.1 of the pipetting element 10. Once the pipetting operations have been carried out, the pipetting element 10 is repositioned in its housing in the extraction bar 3.

[0181] The extraction system 2 also comprises a filtering and transfer device 11 configured to capture and accumulate microorganisms contained in the sample and to transfer the captured and accumulated microorganisms notably to an analysis support or receiver. The filtering and transfer device 11 is removably mounted on the support bar 3, and extends at least partially into the protection and retention housing 9.

[0182] The filtering and transfer device 11 more particularly comprises a hollow body 12 having an elongated shape and comprising a proximal end portion 12.1 and a distal end portion 12.2, and a filtering element 13 fixed to the distal end portion 12.2 of the hollow body 12, for example by welding and notably by heat-sealing or by ultrasonic welding, or else with solvent. The distal end portion 12.2 of the hollow body 12 and the filtering element 13 extend into the protection and retention housing 9, and the protection and retention housing 9 is configured for retaining drops of sample or of reagents able to flow by gravity from the filtering and transfer device 11. However, according to another embodiment of the extraction system 2 (see FIGS. 6 to 8), the protecting portion 8 could comprise two protection and retention housings 9.1, 9.2, in which the distal end portion 10.1 of the pipetting element 10 and the distal end portion 12.2 of the hollow body 12 of the filtering and transfer device 11, respectively, would be mounted.

[0183] The hollow body 12 of the filtering and transfer device 11 is advantageously rigid or semirigid, and may be made of plastic, and for example of injection-molded crystalline polystyrene or of injection-molded polypropylene.

[0184] According to the embodiment shown in FIGS. 1 to 3, the hollow body 12 comprises a tapered intermediate portion 12.3 converging toward the distal end portion 12.2 of the hollow body 12, and the distal end portion 12.2 is cylindrical. However, the hollow body 12 could have a different shape, and the distal end portion 12.2 could notably be tapered. For example, according to the embodiment shown in FIG. 8, the hollow body 12 has a cross section that decreases gradually from the proximal end portion 12.1 to the distal end portion 12.2.

[0185] It should be noted that the proximal end portion 12.1 of the hollow body 12 is configured for being connected to the source of partial vacuum, and may for example be provided with a retaining flange 14. This retaining flange 14 also serves as a reference plane for accurately positioning the filtering and transfer device in Cartesian coordinates during the steps of transfer onto the MALDI-TOF MS analysis plate.

[0186] As shown more particularly in FIG. 3, the filtering element 13 extends at least partially outside the hollow body 12. For example, the filtering element 13 projects from the distal end portion 12.2 of the hollow body 12. According to the embodiment shown in FIGS. 1 to 3, the filtering element 13 is of convex shape, and more particularly is dome-shaped. However, the filtering element 13 could have some other shape, and for example be flat and disk-shaped. The filtering element 13 could also be of cylindrical shape and have a flat or hemispherical distal portion. The filtering element 13 could moreover have a diameter adapted to the diameter of a spot of a MALDI-TOF MS analysis plate. The filtering element 13 could moreover be made in situ in the hollow body 12.

[0187] The filtering element 13 advantageously has a pore size between 0.1 and 10 m, and for example of about 0.45 m, and is more particularly configured for retaining at least one portion of the microorganisms contained in the sample and for allowing other elements contained in the sample to pass through, such as elements that are not micro-organic, for example cellular debris and the formed elements of the blood previously lysed with the lysis reagent.

[0188] The filtering and transfer device 11 may further comprise an absorbent material 15 arranged in the hollow body 12 and at least partly in contact with the filtering element 13. The absorbent material 15 may for example be selected from the group comprising cotton, cellulose fiber, aggregate of polyethylene beads, polyester, an absorbent resin, a silica gel, a hydrogel, a molecular sieve, a zeolite or other absorbent materials.

[0189] A first method for identifying and/or characterizing microorganisms contained in a sample using an extraction system 2 according to the first embodiment of the invention will now be described.

[0190] The first method of identification and/or characterization comprises the following steps: [0191] detecting the identifying code that is provided on the container containing the sample initially (for example a vial of positive blood culture), [0192] loading the support bar 3 on a rack 16 (see FIG. 4), [0193] detecting the identifying code that is provided on the extraction bar 3, [0194] placing a predetermined amount, for example 1 mL, of a sample containing microorganisms, for example a positive blood culture, in the sample well 4 of the extraction system 2, [0195] loading the rack 16 equipped with the support bar 3 in an automatic machine 17 (see FIG. 5) comprising a manipulating part 18 configured for grasping/releasing and moving the pipetting element 10 mounted on the support bar 3 according to Cartesian movements and for grasping/releasing and moving the filtering and transfer device 11 mounted on the support bar 3 according to Cartesian movements, the manipulating part 18 being configured for example for moving the pipetting element 10 and the filtering and transfer device 11 of the or of each support bar 3 loaded on the rack 16, along the Y axis and along the Z axis of a Cartesian system, and comprising for example several first manipulating elements 18.1 each configured for grasping/releasing a respective pipetting element 10 and several second manipulating elements 18.2 each configured for grasping/releasing a respective filtering and transfer device 11, the first manipulating elements 18.1 being for example aligned according to a first direction of alignment and the second manipulating elements 18.2 being for example aligned according to a second direction of alignment approximately parallel to the first direction of alignment, [0196] optionally, by a camera 19 belonging to the automatic machine 17, taking images of predetermined spots 21 of a MALDI-TOF MS mass spectrometry analysis plate 22 arranged on a support module 23 of the automatic machine 17, before making deposits on these predetermined spots 21, the support module 23 being for example movable along the X axis of a Cartesian system, [0197] grasping the pipetting element 10 mounted on the support bar 3 by the manipulating part 18, [0198] piercing the sealing films 7 of the washing wells 6.1, 6.2, 6.3 and of the reagent well 5 on progressing from the washing well 6.3 to the reagent well, for example by the pipetting element 10, [0199] withdrawing a predetermined amount, for example 500 L, of the selective lysis buffer L contained in the reagent well 5 by the pipetting element 10, [0200] placing, by the pipetting element 10, the withdrawn selective lysis buffer in the sample well 4, [0201] mixing the sample and the selective lysis buffer contained in the sample well 4 by the pipetting element 10, [0202] repositioning the pipetting element 10 on the support bar 3, [0203] incubating the mixture for a predetermined time, for example between 1 and 5 minutes, preferably for 2 minutes, at room temperature so as to lyse cells and/or particles that are not micro-organic contained in the sample and/or to lyse predetermined microorganisms contained in the sample in order to supply a lysed sample, also called lysate, [0204] grasping the filtering and transfer device 11 mounted on the support bar 3 by the manipulating part 18, [0205] applying and maintaining a partial vacuum between 50 mbar and 980 mbar relative to atmospheric pressure, preferably at about 600 mbar relative to atmospheric pressure, in the hollow body 12 of the filtering and transfer device 11, the partial vacuum being generated by a pump 20 belonging to the automatic machine 17 and connected fluidically to the manipulating part 18, the pump 20 being for example a vacuum and booster pump, and which may for example be connected to a vacuum ballast and a pressure ballast, [0206] at least partially immersing the distal end portion 12.2 of the filtering and transfer device 11 in the lysed sample contained in the sample well 4, [0207] filtering with aspiration, for example for 2 minutes, at least part of the lysed sample through the filtering element 13, [0208] capturing and accumulating microorganisms in the filtering element 13, [0209] at least partially immersing the distal end portion 12.2 of the filtering and transfer device 11 in the washing reagent contained in the washing well 6.1, [0210] washing the filtering element 13 and the captured and accumulated microorganisms, in the washing reagent contained in the washing well 6.1, for example by a cycle of reciprocating vertical movement for 30 s to 2 min, preferably 1 min, [0211] at least partially immersing the distal end portion 12.2 of the filtering and transfer device 11 in the washing reagent contained in the washing well 6.2, [0212] washing the filtering element 13 and the captured and accumulated microorganisms in the washing reagent contained in the washing well 6.2, for example by a cycle of reciprocating vertical movement for 30 s to 2 min, and then statically for 30 s to 5 min, preferably 3 min, [0213] at least partially immersing the distal end portion 12.2 of the filtering and transfer device 11 in the washing reagent contained in the washing well 6.3, [0214] washing the filtering element 13 and the captured and accumulated microorganisms in the washing reagent contained in the washing well 6.3, for example by a cycle of reciprocating vertical movement for 30 s to 2 min, and then statically for 30 s to 5 min, preferably 3 min, [0215] retracting the distal portion of the filtering and transfer device 11 out of the washing reagent contained in the washing well 6.3 and moving the distal end portion 12.2 of the hollow body 12 transversely in the direction of extension of the hollow body 12 in such a way that the distal end portion of the hollow body 12 comes into contact with the inside wall of the washing well 6.3 and so that the residual washing drops that are present at the level of the distal end portion 12.2 of the hollow body 12 are collected in the washing well 6.3, [0216] holding the distal end portion 12.2 of the hollow body 12 against the inside wall of the washing well 6.3 for a predetermined time, for example for 5 to 15 seconds, while maintaining the level of partial vacuum, [0217] returning the hollow body 12 of the filtering and transfer device 11 to atmospheric pressure or slight overpressure (50 to 200 mbar) for the time required for the next step, [0218] depositing, by the filtering and transfer device 11, at least one part of the washed microorganisms on one or more predetermined locations 21, also called spots, of the MALDI-TOF MS analysis plate 22 (see FIG. 11), [0219] repositioning the filtering and transfer device 11 on the support bar 3, [0220] incubating the MALDI-TOF MS analysis plate 22 at between 40 and 80 C., for example at 45 C. or at 55 C., for a predetermined time, for example between 2 and 6 minutes, preferably 4 minutes, so as to dry or evaporate any residual washing reagent present on the or each predetermined spot 21 of the MALDI-TOF MS analysis plate 22, for which purpose the support module 23 may be provided with at least one heating element 24, [0221] optionally, taking an image of the predetermined spots 21 by the camera 19, [0222] optionally, adding a predetermined amount of formic acid, for example from 0.5 to 1 L, preferably 0.5 L, on each spot 21 of the MALDI-TOF MS analysis plate 22 where microorganisms have been deposited, for example using a pipetting device (not shown in the figures) belonging to the automatic machine 17 and movable for example along the Y axis and along the Z axis of a Cartesian system, [0223] incubating the MALDI-TOF MS analysis plate at between 40 and 80 C., for example at 45 C. or at 55 C., for a predetermined time, for example between 5 and 7 minutes, preferably 6 minutes so as to dry each predetermined spot 21 of the MALDI-TOF MS analysis plate 22, [0224] optionally, taking an image of the spots 21 by the camera 19, [0225] adding a predetermined amount of matrix, for example from 0.5 to 2 L, preferably 1 L of HCCA matrix, on each spot 21 of the MALDI-TOF MS analysis plate 22 where microorganisms have been deposited, for example by the pipetting device, [0226] incubating the MALDI-TOF MS analysis plate 22 at between 40 and 80 C. for a predetermined time, for example between 1 and 3 minutes, preferably 2 minutes, [0227] optionally, taking an image of the spots 21 by the camera 19, [0228] inserting the MALDI-TOF MS analysis plate 22 in a mass spectrometer, and [0229] performing a mass spectrometry analysis of the microorganisms transferred onto each predetermined spot 21 of the MALDI-TOF MS analysis plate 22 so as to identify and/or characterize the microorganisms.

[0230] According to one embodiment of the first extraction method, each washing step comprises a step consisting of moving, for a predetermined time, for example between 30 seconds and 5 minutes, preferably for 2 minutes, the filtering and transfer device 11 in the respective washing well, for example with a reciprocating movement in Z. Advantageously, each step of moving the filtering and transfer device 11 consists of performing several cycles of vertical displacement of the filtering and transfer device 11 in the respective washing well. Advantageously, the filtering and transfer device 11 is withdrawn from the respective washing well completely at each cycle of displacement, for example at a frequency of about 0.5 to 2 Hz, and preferably 1 Hz.

[0231] According to one embodiment of the first extraction method, the step of immersing the filtering and transfer device 11 in the washing reagent contained in the washing well 6.1 consists of plunging the filtering and transfer device 11 more deeply, for example from 1 to 3 mm deeper, than in the lysed sample contained in the sample well 4, the step of immersing the filtering and transfer device 11 in the washing reagent contained in the washing well 6.2 consists of plunging the filtering and transfer device 11 more deeply, for example from 1 to 3 mm deeper, than in the washing reagent contained in the washing well 6.1, and the step of immersing the filtering and transfer device 11 in the washing reagent contained in the washing well 6.3 consists of plunging the filtering and transfer device 11 more deeply, for example from 1 to 3 mm deeper, than in the washing reagent contained in the washing well 6.2.

[0232] According to one embodiment of the first extraction method, the step of depositing the washed microorganisms on one or more predetermined spots 21 of the MALDI-TOF MS analysis plate 22 comprises a step consisting of dabbing the filtering element 13 several times, for example between 3 and 7 times, on the or each predetermined spot 21 of the MALDI-TOF MS analysis plate 22 with a force of application between 100 grams and 1 kg, preferably 600 grams, and according to a frequency of dabbing between 0.5 and 10 Hz. These arrangements make it possible to deposit a sufficient amount of microorganisms on each predetermined spot of the MALDI-TOF MS analysis plate 22.

[0233] According to one embodiment of the first extraction method, the step of depositing the microorganisms on the predetermined spot or spots 21 of the MALDI-TOF MS analysis plate 22 comprises a step consisting of vibrating the filtering and transfer device 11 at least during contact of the filtering element 13 on the or each predetermined spot of the MALDI-TOF MS analysis plate. These arrangements make it possible to improve the transfer of the microorganisms (quantity and reproducibility) from the filtering element 13 to the MALDI-TOF MS analysis plate, notably for the microorganisms that are difficult to detach and identify or are known to generate low concentrations while they grow in a blood culture flask.

[0234] According to one embodiment of the first extraction method, the step of vibrating the filtering and transfer device 11 consists of applying a vibration of between 0.1 and 200 Hz, for example between 10 and 100 Hz, preferably 50 Hz, to the filtering and transfer device 11 for a predetermined time, and for example for 2 seconds.

[0235] According to one embodiment of the first extraction method, the step of depositing the microorganisms on the predetermined spot or spots 21 of the MALDI-TOF MS analysis plate 22 comprises a step consisting of applying a cycle of increased pressure (adjustment of the pressure, of the pulse number, of the cyclic ratio or of the duration of the pressure pulse cycle), relative to atmospheric pressure, in the hollow body 12 of the filtering and transfer device 11 during contact of the filtering element 13 on the or each predetermined spot 21 of the MALDI-TOF MS analysis plate 22 or between two contacts of the filtering element 13 on the MALDI-TOF MS analysis plate 22. These arrangements make it possible to improve the transfer of the microorganisms from the filtering element 13 to the MALDI-TOF MS analysis plate 22.

[0236] According to one embodiment of the first extraction method, the step of mixing the sample and the selective lysis buffer consists of performing several mixing cycles, each mixing cycle consisting of aspirating and discharging a predetermined amount of the mixture contained in the sample well 4, for example 500 L, by the pipetting element.

[0237] According to one embodiment of the first extraction method, the latter comprises a step carried out prior to the step of adding the matrix and consisting of adding a predetermined amount of formic acid, for example from 0.5 to 2 L, preferably 0.5 to 1 L, on each spot of the MALDI-TOF MS analysis plate where microorganisms have been deposited. These arrangements notably make it possible to improve the results of identification for the Gram-positive bacteria and the yeasts.

[0238] According to one embodiment of the first extraction method, the latter comprises a step carried out after the step of adding formic acid and consisting of incubating the MALDI-TOF MS analysis plate 22 at between 40 and 80 C., for example at 45 C. or at 55 C., for a predetermined time, for example between 5 and 7 minutes, preferably 6 minutes, so as to speed up the evaporation of the drops of formic acid, while ensuring sufficient contact of the formic acid with the microorganisms present on the MALDI-TOF MS analysis plate to guarantee effective lysis.

[0239] According to one embodiment of the first extraction method, the latter comprises a step of taking an image, for example under two different types of illumination, of the predetermined spots at different steps of the method and preferably after each drying step (vacuum drying, microorganisms, formic acid, matrix) for process control at each step (notably the following points: correct transfer of the microorganisms, localization and correct mixing of the formic acid and of the matrix with the deposited microorganisms, satisfactory level of crystallization of the matrix with the microorganisms).

[0240] According to one embodiment of the first extraction method, the latter comprises a step of taking an image of the rack after insertion in the automatic machine using an inexpensive camera (webcam) incorporated in the automatic machine. This taking of an image makes it possible, with suitable image processing, to verify the position of the rack, the number and the proper insertion of the bars, and proper presence of the pipetting and filtering elements before starting the extraction cycle.

[0241] A second method for identifying and/or characterizing microorganisms contained in a sample using an extraction system 2 according to the first embodiment of the invention will now be described.

[0242] The second method of identification and/or characterization comprises the following steps: [0243] loading the support bar 3 and a release well 26 on a rack 16, [0244] detecting the identifying code that is provided on the extraction bar 3 and detecting the identifying code that is provided on the container containing the sample (for example the blood culture flask), [0245] placing a predetermined amount, for example 1 mL, of a sample containing microorganisms, for example a positive blood culture, in the sample well 4 of the extraction system 2, [0246] loading the rack 16 equipped with the support bar 3 in an automatic machine, [0247] grasping the pipetting element 10 mounted on the support bar 3 by the manipulating part 18, [0248] withdrawing a predetermined amount, for example 500 L, of the selective lysis buffer L contained in the reagent well 5 by the pipetting element 10, [0249] placing the withdrawn selective lysis buffer in the sample well 4, by the pipetting element 10, [0250] mixing the sample and the selective lysis buffer contained in the sample well 4 by the pipetting element 10, [0251] repositioning the pipetting element 10 on the support bar 3, [0252] incubating the mixture for a predetermined time, for example between 1 and 5 minutes, preferably for 2 minutes, at room temperature so as to lyse cells and/or particles that are not micro-organic contained in the sample and/or to lyse predetermined microorganisms contained in the sample in order to supply a lysed sample, also called lysate, [0253] grasping the filtering and transfer device 11 mounted on the support bar 3 by the manipulating part 18, [0254] applying and maintaining a partial vacuum between 50 mbar and 980 mbar relative to atmospheric pressure, preferably at about 600 mbar relative to atmospheric pressure, in the hollow body 12 of the filtering and transfer device 11, [0255] at least partially immersing the distal end portion 12.2 of the filtering and transfer device 11 in the lysed sample contained in the sample well 4, [0256] filtering with aspiration, for example for 2 minutes, at least part of the lysed sample through the filtering element 13, [0257] capturing and accumulating microorganisms in the filtering element 13, [0258] immersing under aspiration, at least partially, the distal end portion 12.2 of the filtering and transfer device 11 in the washing reagent contained in the washing well 6.1, [0259] washing the filtering element 13 and the captured and accumulated microorganisms under aspiration, in the washing reagent contained in the washing well 6.1, [0260] immersing under aspiration, at least partially, the distal end portion 12.2 of the filtering and transfer device 11 in the washing reagent contained in the washing well 6.2, [0261] washing the filtering element 13 and the captured and accumulated microorganisms under aspiration, in the washing reagent contained in the washing well 6.2, [0262] returning the hollow body 12 of the filtering and transfer device 11 to atmospheric pressure or slight overpressure, [0263] resuspending the microorganisms in a saline solution S1, for example at 0.45%, contained in the release well 26 for example arranged on the rack 16 near the support bar 3, the resuspending step comprising the following steps: [0264] immersing, at atmospheric pressure or at slight overpressure (50 to 200 mbar), at least partially, the distal end portion 12.2 of the filtering and transfer device 11 in the saline solution S1 contained in the release well 26, [0265] repeatedly contacting, for example by a reciprocating vertical movement of the release well 26 and/or of the filtering and transfer device 11, the filtering element 13 with an inner layer 26.1 of crosslinked silicone, at least partly covering the inside surface of the release well 26, for a predetermined time so as to resuspend the microorganisms accumulated by the filtering element 13 in the saline solution S1 contained in the release well 26, wherein the contacting step may for example consist of applying a vibration between 0.1 and 200 Hz, for example between 10 and 100 Hz, preferably 50 Hz, to the filtering and transfer device 11 for a predetermined time, and for example for 30 seconds, [0266] optionally, performing this contacting repeatedly, [0267] measuring the optical density (OD), calculating the dilution required and adjusting the OD by pipetting a volume of saline solution present in reserve in the washing well 6.3 as a function of the calculation carried out, [0268] withdrawing, from the release well 26, a predetermined amount of the saline solution S1 containing the microorganisms, preferably between 100 and 300 L and for example 145 or 285 L, [0269] introducing the withdrawn saline solution into an analysis container 27, for example arranged on the rack 16 near the release well 26, containing a predetermined amount, for example between 2 and 4 mL, preferably 3 mL, of saline solution S2, for example at 0.45%, [0270] transferring the consumable contained in the analysis container 27, into an antibiogram analyzer, [0271] performing an antibiogram analysis on the consumable that has been transferred.

[0272] According to one embodiment of the second extraction method, the latter comprises a step consisting of measuring the optical density of the saline solution S1 contained in the release well 26.

[0273] According to one embodiment of the second extraction method, the latter comprises a step consisting of adjusting to 0.5 F the concentration of microorganisms in the saline solution S1 contained in the release well 26 by adding saline solution withdrawn from the well 6.3 containing a reserve of 0.45% saline solution.

[0274] According to one embodiment of the second extraction method, the analysis container 27 could be mounted, for example removably, directly on the support bar 3.

[0275] According to a variant embodiment of the second extraction method, the step of resuspending the microorganisms could consist of rubbing the filtering element 13 on a nylon grating wetted beforehand with a saline solution at 0.45%.

[0276] According to yet another variant embodiment of the second extraction method, the resuspending step could comprise the following steps: [0277] introducing, at least partially, the filtering and transfer device 11 into the release well 26, [0278] cutting out the filtering element 13, for example by a cutting component inserted in the release well 26, [0279] releasing the filtering element 13 in the saline solution S1 contained in the release well 26, [0280] causing movement of the release well 26 for a predetermined time, for example for 30 seconds to 1 minute, so as to release the microorganisms accumulated in the filtering element 13 and resuspend them in the saline solution S1 contained in the release well 26.

[0281] FIGS. 9 and 10 show an extraction system 2 according to a third embodiment, which differs from the first embodiment essentially in that the extraction system 2 comprises a sealing device 28 movable between a sealing position in which the sealing device 28 seals the sample well 4 and a release position in which the sealing device 28 releases the sample well 4 at least partly. The sealing device 28 may for example be mounted pivoting or sliding on the support bar 3. Furthermore, the rack 16 could be configured for automatically moving the sealing device 28 into the sealing position during positioning of the support bar on the rack 16.

[0282] The support bar 3 could additionally comprise a locking device configured for interacting with a rack 16 so as to lock the support bar 3 on the rack 16. The locking device could for example comprise (see FIG. 2) a locking element, such as a locking lug, configured for interacting with the rack so as to immobilize the support bar 3 in a Y direction of a Cartesian system, and a retaining element, such as a steel ball, configured for interacting with a permanent magnet provided on the rack 16 so as to immobilize the support bar 3 in a Z direction of the Cartesian system during insertion of the bar in the rack. The steel ball could however be replaced for example with a retaining recess configured for receiving a retaining lug provided on the rack.

[0283] It goes without saying that the invention is not limited to just the embodiment of this extraction system described above as an example, on the contrary it covers all the variant embodiments.