Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
  • C12M33/02—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by impregnation, e.g. using swabs or loops
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/02—Burettes; Pipettes
  • B01L3/0275—Interchangeable or disposable dispensing tips
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/02—Burettes; Pipettes
  • B01L3/0275—Interchangeable or disposable dispensing tips
  • B01L3/0279—Interchangeable or disposable dispensing tips co-operating with positive ejection means
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/069—Absorbents; Gels to retain a fluid
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N2001/028—Sampling from a surface, swabbing, vaporising
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
  • G01N2001/2826—Collecting by adsorption or absorption

Definitions

• the technical field of the present invention is that of devices intended to pick up colonies of microorganisms on agar culture medium for analysis. More particularly, the present invention relates to a sampling tip having in its distal portion a sampling end of polymeric materials. The invention also relates to a sampling device comprising such a tip and a sampling method implementing them.
• these consumables do not easily allow optimal deposition of the biological material taken from analysis media such as plates for a MALDI-TOF mass spectrometry analysis. Moreover, it is also very important to be able to take a bacterial colony or a fraction of this colony, without taking the culture medium located under the colony. This can indeed distort the results of subsequent analyzes.
• the quality of the analysis results may also depend on the concentration of the biological deposit, formed from the sample taken, as well as its homogeneity on the support on which it is deposited. This is particularly the case for MALDI-TOF microorganism analyzes, in which the sample must form a thin and uniform layer to allow optimal analysis.
• the groove having been designed to calibrate a given quantity of microorganisms (generally between ⁇ ⁇ ⁇ and ⁇ ), has at its end a metal or plastic loop which has a diameter generally greater than 1mm.
• This tip may be larger than the surface on which the deposit is to be made.
• the rigid nature of the eye is not particularly suitable for spreading on a hard surface of a microbial colony in a uniform thin layer. It is also possible to use other consumables such as swabs, wooden sticks, micropipette tips.
• US 9,181,522 discloses a method and apparatus for aseptic transfer of biological material.
• the apparatus consists of a double-walled chamber for accommodating single-sized tips with a ball in the head, for the transfer of biological material from one place to another aseptically.
• Such a device has the first disadvantage of being of relatively complex design, with an integrated ultraviolet tip sterilization system, its double-walled architecture and its internal loading system and ejection tips. Such complexity undoubtedly affects the cost price and therefore sales.
• the material used to make the ball has a disposable tip head made of hard material, of the metal or polypropylene type, which is not conducive to the sampling process but especially to the deposit of biological material, such as a bacterial colony, in particular on a mass spectrometry analysis plate of the MALDI-TOF type.
• the document FR 2 668 495 describes a sterile sampling cone for bacteriological use.
• Said cone has at its distal end a full protuberance and slightly frustoconical, offset from the longitudinal axis of the cone.
• This protuberance allows the removal of biological material. It can present for this purpose, according to a particular embodiment, an optional handle.
• the cone described in this document is nonetheless constituted by a material conventionally used for this kind of product. Namely a hard and smooth plastic material that is not suitable for the collection and deposition of biological material.
• its particular shape does not make it easy to use for depositing biological material, such as a bacterial colony on a very small surface, such as a MALDI-TOF mass spectrometry analysis plate.
• the Applicant has previously solved all or some of the disadvantages mentioned above by proposing a method for taking all or part of a sample of biological material grown in contact with an agar culture medium, which uses a probe provided with a termination end.
• Said sampling method is essentially based on the cooling of the termination end of the probe, allowing all or part of the sample of biological material to be collected by contacting the termination end with the sample of material biological or by the application of a pressure exerted by the terminating end on the sample of biological material, then the release of all or part of the sample of biological material by heating the termination end of the probe . This process is described in the patent application WO 2012/004545.
• the objectives of the present invention are therefore to respond to these shortcomings by providing a simple design tip, easy to produce, allowing when placed on a biological material removal device to accurately collect and deposit this biological material, in particular on a mass spectrometry analysis plate, MALDI-TOF type.
• a tip adapted to be fitted on the body of a manual or automated sampling device of biological material of microbial origin comprising:
• a distal end comprising means for collecting biological material of microbial origin
• said tip being characterized in that all or part of the distal free end is made of a fibrous material having a porosity of at least 30%.
• biological material of microbial origin is essentially biological material consisting of bacteria, yeasts or molds.
• the tip according to the invention consists entirely of a fibrous material having a porosity of at least 30%.
• the fibrous material has a porosity greater than 50%, preferably greater than 70%.
• the fibrous material is taken from the group comprising: polyethylene, polyesters, polyethylene terephthalate (PET), PET / polyethylene copolymer, PET / PET copolymer, polyamide, cotton.
• PET polyethylene terephthalate
• PET / polyethylene copolymer PET / PET copolymer
• polyamide cotton
• the sampling tip has a substantially conical or frustoconical overall shape.
• the sampling means is advantageously of cylindrical overall shape, frustoconical or spherical.
• Another subject of the present invention relates to a biological material sampling device of microbial origin comprising:
• a proximal portion serving at least partially as a gripping area of said device and a distal portion having a free end at the end of which is fixed said tip.
• This device further comprises a nozzle ejection system.
• the ejection system comprises a rod positioned inside said body and movable in translation, so as to come to press the tip and thus eject it.
• said rod is movable in translation by means of a push button.
• Another subject of the invention relates to a method for sampling biological material of microbial origin comprising the following steps:
• Another subject of the present invention relates to a method for preparing an analysis plate for a microbiological analysis in MALDI-TOF type mass spectrometry from a sample of biological material comprising the following steps:
• Microbiological analysis essentially means any analysis that makes it possible to identify a microorganism, such as a bacterium or yeast, but also makes it possible to demonstrate any marker of resistance to an antimicrobial, any characteristic of typing or expression by said microorganism of a virulence factor.
• Another subject of the invention relates to a process for isolating biological material of microbial origin on an agar culture medium, comprising the following steps:
• all of the methods described above further comprise an ultimate step of ejection of the sampling tip.
• the sample of biological material can be obtained from a colony of biological material, according to the sampling method described above.
• the sample of biological material can be obtained from a suspension of biological material. Such a suspension is obtained traditionally by resuspension of one or more colonies of biological material in a saline solution.
• the sampling tip is soaked in a fraction of the suspension, to allow the absorption of the liquid by the sampling means, thanks to the absorbency of the fibrous material constituting said sampling means.
• the biological material is then in contact with said sampling means.
• concentration of the bacterial suspension is determined by those skilled in the art, according to the growth characteristics of the microorganism considered. This is part of this general knowledge.
• the fraction of suspension used to charge the tip of biological material is also determined on purpose. It is advantageously from a few microliters to a few tens of microliters.
• Figure 1A shows a perspective view of a sampling tip according to the present invention, according to a first embodiment.
• Figure 1B shows a side view of the tip shown in Figure 1 A.
• FIG. 1C represents a perspective view of a sampling tip according to a second embodiment
• Figure 2A shows a side view of a biological material removal device according to a first embodiment.
• Fig. 2B shows an exploded side view of the biological material removal device, shown in Fig. 2A.
• Fig. 2C shows a perspective view of the body of the biological material removal device as shown in Fig. 2A.
• Fig. 2D shows a side view of a biological material removal device as shown in Fig. 2A, in the ejection configuration of the sampling tip.
• Figure 3A shows a side view of a biological material removal device, according to a second embodiment.
• Fig. 3B shows an exploded side view of the biological material removal device, shown in Fig. 3A.
• Figure 3C is an enlarged, exploded perspective view of the pusher ring fastening system on the shank of the pickup device shown in Figure 3A.
• Fig. 4A shows a side view of a biological material removal device according to a third embodiment.
• Fig. 4B shows an exploded side view of the biological material removal device, shown in Fig. 4A.
• Fig. 4C is an enlarged view of the ejection mechanism of the tip of the pickup device shown in Fig. 4A.
• Figure 5A is a perspective view of a biological material removal device according to a fourth embodiment.
• Fig. 5B is an exploded perspective view of the biological material removal device shown in Fig. 5A.
• FIG 5C shows a perspective view of the biological material removal device, shown in Figure 5A, in the ejection configuration of the sampling tip.
• the tip 10 according to a first embodiment is shown in a perspective view. It is shown in side view, in Figure 1B.
• the tip 10 is of generally frustoconical shape. It is nevertheless quite conceivable that the tip 10 according to the invention is of different shape. It consists of three distinct parts. Firstly, a distal portion 12 of substantially frustoconical shape. This distal portion 12 constitutes the receiving part of a sampling means 14. For this purpose, the distal portion 12 has a free end 16, at the level of which is formed a blind cavity 18, in which the sampling means is positioned. 14.
• the tip 10 also comprises a part proximal 20, also of frustoconical shape but shorter and wider than the distal portion 12.
• the end 22 of the proximal portion 20 is free and has a blind cavity 24 inside which is housed the distal end of a sampling device.
• the tip 10 may be molded into the materials commonly used to mold the pipette tips.
• the material may for example be a polyolefin polymer. This type of material is generally cheap, sterilizable and adapted to use for the production of disposable product.
• the material constituting the latter is important.
• the sampling means has the double technical constraint of having to ensure a collection of biological material, such as a bacterial colony but also a release of said biological material when it is deposited on an analysis device such as a plate mass spectrometry analysis.
• the inventors have thus discovered that the porosity of the material used to manufacture the sampling means 14 was an essential characteristic to make it possible to find the right compromise between the sampling performance and the release performance of biological material.
• the biological material to be sampled is a bacterial colony
• the manner in which the bacterial colony behaves when it is removed or when its deposit may vary from one bacterial species to another. It is well known that bacterial colonies are more or less consistent, more or less viscous, more or less fungal depending on the bacterial species considered. It is therefore essential to have a sampling means that is able to handle any type of bacterial colony, regardless of its properties.
• a porosity of at least 30% is necessary to obtain the desired properties. Ideally a porosity of at least 70% gives the best results.
• Porosity of the material means all the voids (pores) of a solid material that can be filled with fluids (liquid or gas). Porosity also means the physical quantity defined as the ratio between the volume of the voids and the total volume of the porous medium studied.
• the porosity measurement of the material is carried out as follows: A sample of the dry fibrous material is taken.
• the sample is weighed a first time.
• the sample is immersed in water, the time necessary for its total impregnation.
• the impregnated sample is weighed again.
• the mass of water trapped in the material is deduced by calculating the difference between the mass of the impregnated material and that of the dry material.
• the density of the water being close to 1, it is deduced the volume of trapped water and thus the volume of the voids in the material.
• the volume of the impregnated sample is then measured.
• Materials particularly suitable for producing the sampling means may be fibrous materials.
• materials such as synthetic materials such as polyethylenes, polyesters and polyamides.
• polyesters mention may be made of polyethylene terephthalate (PET).
• PET polyethylene terephthalate
• copolymers such as polyethylene / polyester polymers, such as a polyethylene / PET copolymer or a PET / PET copolymer.
• the fibers may consist of a single-component or two-component material.
• a bi-component fiber may for example consist of a PET core and a polyethylene sheath.
• Natural fibrous materials can also be used. This is particularly the case with cotton fibers.
• FIG. 1C A second embodiment of the tip is shown in Figure 1C.
• This tip 11 consists of three parts: a proximal portion 13 substantially cylindrical or optionally frustoconical. Unlike the first embodiment, the free end 15 of the proximal portion 13 has no blind cavity and is clogged. Indeed, according to this embodiment, the distal end of the sampling device does not fit into the tip 11. It is instead the tip 11 which is inserted into the distal end of the device sampling, and more precisely its end 15. Of course, to do this, the sampling device must have a free distal end capable of receiving the tip 11. It must thus have a receiving cavity of the tip 11 whose dimensions must be slightly greater than the dimensions of the proximal portion 13.
• the tip 11 also has a flange 17 which allows the ejection of said tip after use. It can also serve as a stop when the tip 11 is inserted into the sampling device.
• the tip 11 has a distal portion 19 for receiving a sampling means 14, as in the case of the tip 10, according to the first embodiment.
• the distal portion 19 has a free end 21, at the level of which is formed a blind cavity 23, in which is positioned the sampling means 14.
• the proximal end 13 may have an orifice for generating a through hole between the distal and proximal ends. It is then possible to use a longer sampling means, positioned in the through hole and secured to the tip at the orifice of the proximal end, or even at both ends. Such a variant is simpler to design and therefore less expensive to produce.
• sampling means 14 as shown in Figures 1A to 1C is cylindrical. It is possible, however, that the sampling means 14 has a different shape. Thus, the free end of the latter may for example be tapered to further improve the accuracy of sampling and deposition of biological material. The free end of the tip 14 may also have any other form suitable for the removal of biological material.
• sampling tip which is made entirely of the porous material.
• the sampling tip would also constitute the sampling means.
• the sampling means, the sampling tip and the sampling device could be one. Indeed, it can be envisaged a disposable sampling device, which is secured to a porous sampling tip. Alternatively, the sampling device can be entirely made of porous material. To do this, the dimensions of the sampling means must allow easy handling and handling.
• sampling tip conjugated to the sampling means or the sampling means alone, if it is one with the sampling tip can be adapted for use with an automated biological material sampling system.
• an automated biological material sampling system Such a system could for example automatically carry out the collection of bacterial colonies on petri dishes and the preparation of analysis plates in mass spectrometry.
• FIG. 2A shows a sampling device 30 according to a first embodiment.
• This sampling device 30 comprises a distal portion 32, an intermediate portion 34 and a proximal portion 36.
• the distal portion 32 substantially frustoconical, has the function of carrying the sampling tip 10 according to the invention.
• the intermediate portion 34 also cylindrical, has the function of allowing the gripping device 30 to be gripped by its user.
• the proximal portion 36 has a general cup shape, the inner diameter of which is slightly greater than the outer diameter of the intermediate portion 34, so that the proximal portion may partially overlap the proximal end of the intermediate portion.
• FIG. 2B shows the sampling device 30 in an exploded view so that each constituent element of this device is shown separately. It is thus found that the distal 32 and intermediate 34 parts are in fact one and the same piece 38 constituting the body of the sampling device.
• this substantially cylindrical body 38 has three longitudinal and peripheral recesses 382 and a central recess 384.
• the recesses 382 are formed so as to each receive a blade 401 of the rod 40, as represented in Figure 2B.
• the recess 384 it is arranged to receive a helical spring 42, as shown in Figure 2B.
• the rod 40 is therefore mainly composed of three blades 401 whose base is secured to a stop ring 402.
• the rod 40 is positioned inside the body of the device 38 by introducing the free ends of the blades 401 into the recesses 382.
• the lower end of the coil spring 42 is housed in the recess 384, while its upper end is placed in contact with the inner face of the proximal portion 36 of the sampling device, which caps the body 38.
• This proximal part acts as a pusher, during the ejection of the sampling tip 10, once the latter used.
• the blades 401 have a length greater than the intermediate portion 34 so that when the rod 40 is positioned inside the body 38, the upper free end of the blades 401 emerges from the recesses 382. These free ends are secured to the pushbutton 36, once the spring in place. This bonding can be mechanical. Thus, it is conceivable to have pins radially projecting near the free end of the blades 401 which are housed in recesses formed in the inner wall of the pusher 36. Alternatively, it is possible to secure the free end blades 401 with the inner face of the pusher 36 by chemical bonding.
• the spring 42 tends to resume a rest position in which it no longer presses the rod 40. The latter then resumes its initial position by inverse translation, releasing the distal portion 32 which is then available to receive another sampling tip.
• a tip 10 is positioned on the free end 32 of the sampling device. This step is generally performed by coming positioned the device in a vertical position plumb with a sterile tip, usually positioned on a suitable rack. The end of the distal portion 32 of the sampling device is then fitted into the cavity 24 of the proximal portion 20 of the tip of 10, until it abuts the bottom of the cavity 24.
• the sampling device is positioned in such a way that the means for sampling the tip come into contact with the colony of microorganisms. All or part of the colony is then removed.
• the sampling device To carry out the deposition of all or part of the biological material taken from a surface such as the surface of a MALDI-TOF mass spectrometry analysis plate, the sampling device is approached from said surface, so that the end of the sampling means carrying the biological material comes into contact with the surface. Slight circular movements are then made with the sampling device to deposit a layer of biological material on said surface.
• FIGS. 3A to 3C A second embodiment of the sampling device is shown in FIGS. 3A to 3C.
• This sampling device 50 consists of a body 52 substantially cylindrical and hollow. This body 52 has in its distal portion a free end 521 of substantially frustoconical shape, intended to carry a sampling tip 10. This free end 521 is secured to the body 52 by three fins (not shown) defining three interstitial spaces.
• the body 52 also has a hooking system or clip 522 for hooking the sampling device to the pocket of a garment for example, as can be done with a pen.
• a push button 54 that leaves via the proximal end of the body 52.
• the push button 54 is integral with a rod 56, taking place inside the body 52.
• This rod 56 of substantially cylindrical shape terminates at its distal end by three fixing lugs 561, shown in close-up on the enlarged view 3C.
• the central space between said fixing lugs 561 is intended to receive a helical spring 58, as shown in FIG. 3B.
• the rod 56 is inserted into the body 52 by the proximal end of the latter, the coil spring 58 being inserted beforehand.
• Said helical spring 58 is positioned in abutment against the fins and the upper part of the distal end 521.
• the rod 56 once inserted, is positioned astride the spring so that the 561 fastening tabs are crossing the interstitial spaces defined between the fins and emerge at the base of the body 52 around the free end 521.
• a ring 60 is inserted around the free end 521. This ring 60 has three recesses 601 for receiving the end 561.
• the fixing lugs have at their end radial lugs 5611, which once the lugs positioned inside the ring 60, will come to lock in the recesses 601 and thus secure the ring 60 and the rod 56.
• this mechanical fastening system it is quite possible to replace this mechanical fastening system with a system comprising legs without pins that would be secured to the ring by gluing.
• the ring 60 bears against the body 52 at the level of the upper part of the free end 521, because of the action of the helical spring 58 which exerts on the rod 56 a force repulsion towards the upper part of the body 52.
• FIGS. 4A-4C A third embodiment of a sampling device is shown in FIGS. 4A-4C.
• This sampling device 70 consists of a body 72 substantially cylindrical and hollow.
• This body 72 has in its distal portion a free end 721 of substantially frustoconical shape, intended to carry a sampling tip 10.
• This free end 721 is secured to the body 72 by three fins (not shown) defining three interstitial spaces.
• This rod 76 also comprises a rod 76 which is inserted into the body 72 by its upper end.
• This rod 76 consists of a one-piece proximal portion 761 of substantially rectangular cross section and a distal portion consisting of three attachment tabs 762. At the interface with the distal portion, the proximal portion 761 has on these two opposite faces an inclined shoulder 7611, intended to cooperate with the push button 74.
• the rod 76 is inserted into the body 72 of the sampling device 70, by the proximal end thereof. Once the rod 76 inserted, the fixing lugs 762 pass through the interstitial spaces defined between the holding fins of the free end 721 and emerge at the base of the body 72 around said free end 721.
• a ring 78 is inserted around of the free end 721.
• This ring 78 is similar to the ring 60 described above and has three recesses for receiving the end of the fixing lugs 762, also provided with radial lugs 7621 and intended to cooperate with the recesses of the rings 78 for securing the rod 76 to the ring 78, as shown in Figure 4C.
• the push button 74 has an inverted U-shaped cross-section, to allow it to be positioned astride the proximal portion 761 of the stem 76, particularly at the inclined shoulders 7611.
• the push-button 74 is shown in FIG. each of these two lateral faces three lugs 741 intended to prevent it from emerging, once positioned in the housing 722.
• the push button 74 bears at its distal portion against the inclined shoulders 7611, preventing the rod 76 from emerging.
• the interaction between the push button and the inclined shoulders 7611 allows the rod 76 to move between a rest position and an ejection position of the sampling tip 10.
• the sampling device 80 shown in Figures 5A to 5C consists of a body 82 substantially cylindrical and hollow.
• This body 82 is integral and has in its distal portion a free end 821 of substantially frustoconical shape. This end 821 is pierced with a circular orifice 822. This orifice is intended to receive the proximal portion 13 of a sampling tip 11, as described in connection with Figure 1C.
• the sampling tip 11 is pressed into the body of the sampling device 80 until the flange 17 of the tip 11 bears against the end 821 of the body 82.
• the body 82 also comprises a push button 84 positioned laterally on the body 82, which is inserted into a housing 823 formed in the side wall of the body 82.
• This rod 86 which is inserted into the body 82 by its upper end.
• This rod 86 consists of a one-piece proximal portion 861 of substantially rectangular cross-section, of a one-piece distal portion 862 of substantially round cross section ending in a stud 863.
• proximal portion 861 and the distal portion 862 are formed with inclined shoulders 8611 on either side of the proximal portion 861.
• the proximal portion 13 which enters the lumen of the body 82 through the orifice 822 pushes the rod 86 in the opposite direction, causing the pushbutton 84 to rise vertical translation thanks to the inclined shoulders 8611 which cooperate with the push button 84.
• sampling device position at the distal end of the body but on its outer part, a mechanical system intended to bear against the tip to allow its ejection. It may be for example a sleeve movable in translation on the body of the sampling device which is in contact with the sampling tip and which allows to eject the latter when it is moved by the user.
• sampling device For example, it is possible to prepare a suspension of biological material in an ad hoc solution, such as a saline solution. To do this, it may be envisaged to release the sampling tip carrying the biological material, directly into a tube containing a quantity of resuspension solution. Vortex agitation of the tube containing the tip will then release the biological material retained on the fibrous material.
• the sampling device can also be used to carry out isolations on agar culture media. These isolations can be made directly from colonies taken with the sampling device according to the invention and then directly reseeded on an agar culture medium. They can also be made from suspensions of biological material, made using the sampling device, as described supra.
• the sampling means has a spherical end and is of sufficient diameter at its end, to prevent its application against the agar culture medium does not to damage the latter.
• a suitable diameter is for example between 2 and 7 millimeters (mm) and preferably between 2 and 4 mm.
• Isolation can be achieved from a bacterial suspension whose concentration is, for example, 10 7 CFU (Colony Forming Unit) / mL (milliliter). With such a concentration, a sample of 5 micro liters ( ⁇ ) of suspension using the sampling means is sufficient to allow isolation of good quality, on the agar culture medium. The suspension 5 are sucked by the material constituting the sampling means. Said removal means loaded in suspension is then able to allow the gradual release of this suspension when it is applied to the agar culture medium. This progressive release is allowed thanks to the absorbency of the material, constituting the means of sampling. The consequence is a better quality of isolation.
• Isolation can be carried out according to traditional techniques, such as the so-called “dial” technique or the so-called “spiral” technique.
• Example 1 Use of a sampling device with sampling tips for preparation of a plate for MALDI-TOF analysis.
• the sampling device used in this example is the device shown in FIGS. 2A to 2D and described above.
• the sampling tip is made of plastic material, such as those conventionally used to make the disposable pipette tips. It comprises a cylindrical sampling means with a diameter of 2 mm and a length of 6 mm. It is polyethylene terephthalate (PET) / PET copolymer fibers, material marketed by Porex under the reference PSU-832.
• PET polyethylene terephthalate
• the body of the sampling device is made of injected polypropylene. It has a length of 120 mm and a section of hexagonal shape with 8.2 mm between the opposite faces of the hexagon, which allows an easy grip as with a pencil, without risk of musculoskeletal disorders.
• the sampling device is used to collect colonies of different bacterial species, which grew on culture medium Columbia + 5% sheep blood (COS), marketed by the Applicant under the reference 43041.
• COS Columbia + 5% sheep blood
• the colonies collected are deposited on a 48-position disposable MALDI-TOF analysis plate, marketed by the Applicant under the reference 410893.
• a sampling tip is positioned on a sampling device.
• a bacterial colony is taken on a culture medium COS type, using the sampling device by contacting the sampling means of fibrous material carried on the sampling tip, with the colony.
• the bacterial sample taken is deposited on the MALDI-TOF analysis plate, in one of the 48 positions provided for this purpose, by putting the collection means carrying the bacterial sample in contact with the MALDI-TOF plate. , exerting pressure and spreading the sample.
• the pressure exerted on the fibrous material of the sampling means makes it possible to retain the excess sample on said fibrous material. It is possible to make successive deposits on different positions of the analysis plate in the case where it is desired to replicate the analysis. These other deposits can be done with the same tip.
• the sampling tip is ejected into a bin by exerting pressure on the push button, as described above.
• Steps 1 to 5 are repeated for each colony that is to be analyzed. 7.
• the deposit of a strain of E. coli ATCC 8739, used as calibrator and positive control, is also performed according to steps 1 to 5.
• the analysis plate is stored for the time necessary to dry the matrix.
• the plate is introduced into the MALDI-TOF VITEK® MS mass spectrometer, marketed by the applicant, the acquisition of the mass spectra is carried out.
• the identification of microorganisms is obtained by spectral analysis using the expert system and the VITEK® MS database.
• the method of the invention thus makes it possible to collect and deposit bacterial species forming colonies with an atypical consistency.
• the method of the invention thus makes it possible to collect and deposit bacterial species forming colonies with an atypical consistency.
• MALDI-TOF mass spectrometry for powdery and encrusting species such as B. lichenformis or N. farcinica, gooey species such as K. pneumoniae and species diffusing on agar as P. mirabilis.
• the different species were therefore analyzed in MALDI-TOF mass spectrometry in order to carry out their identification.
• the analysis protocol is that described in Example 1, including the sampling device.
• the material PSU892 is a material obtained from two-component fibers, consisting of a polyethylene terephthalate envelope and a polyester core. Such a material is marketed by the company POREX®. It is furthermore described in the patent application WO-A-03/080904.
• XA-20521-PS is a material made from polyethylene. Such a material is marketed by POREX® under the trade name NIBS.
• a colony is taken with the sampling device according to the invention and four deposits are made at four contiguous positions of the MALDI-TOF analysis plate.

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• 2016
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