The present invention belongs to the field of microbiology and culture of microorganisms on solid and semi-solid. It relates more specifically to alternative solutions to conventional solid culture media, intended especially for the culture, the detection and / or identification of microorganisms present in a sample.

In the field of clinical diagnosis or veterinary as well as that of industrial microbiological control (particularly in the food, pharmaceutical and cosmetics), solid culture media and semisolid commonly designated areas (culture) or agar agar nutrition - are valuable tools for the detection and study of potentially pathogenic microorganisms and / or infectious.

Appeared at the end of the XIX th century with the use of agar as a gelling agent, the solidified culture media were quickly and profoundly revolutionized the practice of microbiology. Thus, the microorganisms to detect, identify and / or study were apprehended, observed and manipulated macroscopic scale, as colonies, that is to say cell clusters visible to the naked eye and pushing the surface of the solid culture media. In doing so, new analytical and experimental perspectives have emerged, and existing technical improvements and simplifications have been made possible.

In this regard, particular include the isolation techniques of microorganisms on agar, which are still widely used in many microbiological analysis methods, given their ease of execution. It is basically isolation techniques, exhaustion or technical dials.

These isolation techniques on agar medium are to disperse on the surface of the solid culture medium, the cells of a depositing biological sample to be analyzed. This dispersion is carried out, for example, through means / mechanical tools is slid on the surface of the culture medium according to a particular route. After this dispersion process, the cells arrived at the end of the spreading path find themselves individualized, separated from each other. Each of these individual cells is then developed to form ultimately a pure culture of colony, that is to say, a cell mass containing microorganisms, all genetically identical. Each colony can then be morphological analysis (examining the shape of the colony, its elevation, regularity of edges ...) directly on the same culture medium or on other media. These cells can also be collected for preservation purposes and / or for subsequent further analysis.

In addition to offering a support microorganisms adapted to their growth and development, culture media gelled / solidified, conventionally based on agar, have the advantage of present levels of hardness and surface conditions that make perfectly suited for working up and spreading of cells, and in particular the pressure and frictional forces exerted by the tools and instrumentation developed for this purpose (cf. for example, EP 0242114, WO 2005/071055) .

The solid culture media, however, experiencing some major drawbacks. In this regard, we note a preparation which, when artisanal, requires time (at least an hour) and many operations (weighing ingredients, dissolutions, autoclaving, casting, distribution in Petri dishes). Also, as is the case for liquid culture media, these solid or semi-solid media are extremely sensitive to any form of biological contamination. Finally, due to the great difficulty to ensure sterility, the media agar "craft" must be prepared extemporaneously or quasi-extemporaneously.

There are also agar media produced industrially. These loans agar media employed have short expiry times, which rarely exceed a few months. This limited shelf life may notably be explained in part by the presence of water in the community. Moreover, to ensure maximum sterility and an acceptable level of performance, they are subjected to drastic measures in terms of conditioning (treatment sterilizing UV double packaging ...), transport and storage (storage between 2 ° C and 8 ° C, protected from light). These numerous constraints impact on the costs of sale and use of industrial agar media.

To overcome the above drawbacks, alternatives to media

solid culture have been proposed. This is particularly microbiological culture devices ready for use, industrial products, which have the particularity of incorporating nutrient compositions, possibly selective and / or discriminant, dried and activated by single (re) hydration. Therefore, in addition to the terms of preservation / storage very restrictive (to protect from moisture and high temperatures), their shelf life can reach several years.

As such, the 3M Company (USA), offers a range of industrial microbiological culture devices called Petrifilm ™. These devices, described in particular in EP 0,070,310, EP 0,620,844 or EP 0,832,180, are formed of two sealing films with water and affixed one to the other. At the interface, the internal face of each of the two films is coated with an adhesive composition to make to adhere a thin layer of powder (s) water soluble (s) cold.

For some devices Petrifilm ™, one of the inner faces of the film is coated with a first powder, which corresponds to a medium composition dehydrated culture and micronized. The other internal surface is itself coated with a second powder, which corresponds to a gelling agent (such as guar gum and / or xanthan gum) micronized.

For other devices Petrifilm ™, the two inner faces are coated with the same powder, formed by a mixture of a dried composition of culture medium and micronized, and a gelling agent also micronised.

Petrifilm ™ these devices are intended for the detection and / or enumeration of microorganisms present in a sample to be analyzed. To do this, the culture device is opened by lifting the top film, transparent. A sample volume to be analyzed (liquid or previously rendered liquid) of a high fluidity, optionally diluted beforehand and fluidized, is deposited in center of bottom film. The top film is repositioned on the lower film. Upon contact with water present in the sample to be analyzed, the gellant forms a hydrogel with a high water content, having a gelatinous and viscous aspect, from fouling of cells present in the sample. This same water in the sample for analysis also helps solubilize and activate the culture medium. By compressing the sample somewhat between the two films, the sample is spread over a larger culture surface. The microbiological culture device finally

incubated at the temperature and for the prescribed period before reading the results.

By design, the Petrifilm ™ devices, if possible actually a good cell attachment, the hydrogel formed creates a surface, very gelatinous and viscous not amenable to isolation operations and / or spreading cells as one might do on an agar culture medium, and is also not compatible with the tools and instrumentation developed for this purpose (see for example, EP 0242114, WO 2005/071055).

Are also known Compact Dry culture devices developed by the company NISSUI PHARMACEUTICAL (Japan), whose microbiological culture medium is formed by a sheet of absorbent fibrous material, incorporating within its mass a dehydrated nutritive composition optionally also selective and / or discriminant (see for example, EP 1179586).

To do this, an alcoholic suspension is prepared by mixing, in ethanol, a composition of culture medium, an adhesive is soluble in both water and ethanol (for example, poly [oxide ethylene] and hydroxypropylcellulose) and a gelling agent soluble in water but insoluble in ethanol (e.g., locust bean gum, guar gum, carrageenan, hydroxyethyl cellulose). The alcoholic suspension is used to impregnate the sheet of absorbent fibrous material. After drying, the sheet form dehydrated culture media, ready to use, shelf stable, activated by a simple rehydration.

Compact Dry ™ these devices are intended for the detection and / or enumeration of microorganisms present in a sample to be analyzed. To do this, a sample volume to be analyzed (liquid or previously rendered liquid) of a high fluidity, optionally previously diluted, is inoculated with a pipette on the culture medium by covering up to surface. The culture device is then incubated for the duration and temperature prescribed before reading the results.

This type of growing medium does not lend itself to isolation techniques implemented by spreading Γ samples for analysis. Indeed, because of their fibrous nature, these culture media have an irregular surface with many irregularities that impede the continuous sliding of regular and commonly used tools and instruments to spread and disperse the cells on the surface of an agar medium classic. Also, the cells tend to develop in depth in the thickness of the porous support, which can make it difficult visualization / identification of colonies formed.

In WO 2015/104501, the Applicant also offers microbiological culture devices ready for use, dedicated to the detection, identification and / or enumeration of microorganisms. These devices are also based on a culture substrate in fibrous and absorbent material incorporating in its thickness, a composition of culture medium. The incorporation of the dehydrated culture medium composition in the thickness of the culture medium is performed by dry impregnation technique. As with the Compact Dry ™ devices, because of their fibrous nature, these culture media have an irregular surface with numerous asperities; these devices are not compatible with the techniques and tools developed so far for the isolation and

Also, as for the Compact Dry ™ devices, the cells grow in depth in the thickness of the porous support.

To remedy these shortcomings, in WO 2014/013089 and WO 2015/107228, the

Applicant proposes to cover the fiber surface with a surface layer adapted to improve in the surface state.

In WO 2014/013089, it is thus proposed to use a membrane (micro) filtration to sufficiently close the pores to prevent the spread of the cells to the underlying layers, and to form at the same time a rendering surface relatively smooth . Said diaphragm (micro) filtration can be performed based on one or more materials selected from latex, polytetrafluoroethylene, poly (vinylidene) fluoride, polycarbonate, polystyrene, polyamide, polysulfone, polyethersulfone, cellulose, nitrocellulose.

Alternatively, in WO 2015/107228, there is provided a covering with a porous layer of composition comprising a mixture of kaolin pigments, talc, titanium dioxide and / or calcium carbonate and a binder Type styrene butadiene latex, styrene acrylic latex or carboxyl methyl cellulose.

Despite convincing biological outcomes, such culture media were deemed commercially unviable at present, mainly because of a still high manufacturing cost.

The present invention thus aims to provide a microbiological culture device while providing a long shelf life, including at room temperature, providing a real alternative to conventional solid culture media, both in terms of fertility that the compatibility with mechanical operations spreading and dispersion of the cells.

Another objective of the present invention is to provide a microbiological culture device that is compatible with the constraints of industrial and commercial exploitation, particularly in terms of cost of production and profitability. In particular, the present invention is to provide a microbiological culture device design and manufacturing facilities and adapted to the means of production currently used in the media industry and microbiological culture devices.

The present invention provides a microbiological culture comprising:

- a piece of absorbent material having an upper surface at least substantially flat and incorporating in its thickness, a composition of dehydrated culture medium (dry or dried)

- based on said piece of absorbent material, a hydrogel sheet polysaccharide dehydrated and rehydrated at room temperature, particularly at temperatures between 5 ° C and 40 ° C; said dehydrated hydrogel sheet is affixed directly to the upper face of said piece of absorbent material, or indirectly through an intermediate permeable membrane.

According to the invention said polysaccharide dried hydrogel sheet has been previously prepared by dewatering a layer of water-based composition of hydrogel and at least one gelling polysaccharide. This dehydrated hydrogel sheet has the particularity of being rehydrated at room temperature. It covers by simply absorbing hydrogel properties of an aqueous composition, without requiring any additional heat treatment and, while maintaining characteristics of shape, texture, hardness / stiffness and surface condition very similar to those as a constituent polysaccharide hydrogel sheet of a microbiological culture device according to the invention

would present if it had not been dehydrated after casting (or after display / sleeping) but simply hardened.

After rehydration, the hydrogel sheet dehydrated polysaccharide gives a piece of hydrogel layer, having a consistency and hardness / stiffness very comparable to those of a conventional agar culture medium ready. In particular, said piece hydrogel layer has a hardness between 500 and 2000 g. cm "2 , preferably between 700 and 1400 g. cm " 2 . The hardness of said hydrogel layer can be measured by means of a texture analyzer, e.g. TA.XTplus type society STABLE MICRO SYSTEMS LTD (UK).

In use, a microbiological culture device according to the invention must be hydrated to be activated. To do this, the part of absorbent material is soaked with a quantity of water or an aqueous composition. The composition of culture medium initially present in a dry state, is thus solubilized and activated. The same contact with the part of absorbent material, or through an intermediate permeable membrane, the polysaccharide hydrogel sheet rehydrates dehydrated in turn almost instantaneously and at room temperature. By rehydrating liquid from the piece of absorbent material, the hydrogel sheet regains flexibility and gives a thin layer of hydrogel (or hydrogel film) piece soaked in a culture medium composition and solubilized active. The seeding of the sample to be analyzed for polysaccharide hydrogel sheet can be performed both before and after rehydration it. The surface of the polysaccharide hydrogel sheet, either before or after rehydration, is sufficiently smooth and rigid / hard to lend itself to working up and spreading of cells, and in particular to withstand the pressure and frictional forces exerted by tools and instrumentation developed for this purpose.

Rehydrated in this system, the hydrogel layer provides polysaccharide to the surface of microbiological culture device a lubricating effect which facilitates the spreading mechanical operations and dispersion of the cells. Also, for consistency agar, it promotes the establishment of microorganisms closer to the nutritious ingredients and active agents of the culture medium. As for the piece of absorbent material, in addition to its role in the storage and distribution of the composition of the culture medium, the structure contributes to the rigidity and the overall strength of the surface of microbiological culture device, ensuring its compatibility with the against pressure and frictional forces exerted by the tools and instrumentation used to spread out and disperse the cells.

Before going further in the description of the invention, the following definitions are provided to facilitate understanding of the description of the invention.

The term "culture medium" refers to a nutritional composition for the growth and development of cells, especially bacteria, molds and / or yeasts. These media can meet the nutritional needs of microorganisms to grow. Schematically, we find in their composition:

water (generally distilled or deionized water) sterile,

at least one carbohydrate, as a carbon and energy source,

as well as other nutrients (in particular, amino acids, growth factors, vitamins, minerals, trace elements, iron salts, sodium citrate, sodium chloride ...) provided in the form of chemically complex compositions such as mixtures of peptones (milk, meat and / or potato starch, corn ...), yeast extracts, serum and / or tissue extracts of animal or vegetable origin .. .

also various salts, to establish adequate osmolarity medium, and to buffer the pH.

A culture medium in the sense of the present invention may optionally show some selectivity towards target microorganisms, that is to say, it promotes the development of the target microorganisms rather than the annex flora and / or to inhibit and / or slows the development of the annex flora. This selective effect may be obtained in particular through the use of agents for the inhibitory effect Annex flora or activating effect agents for the target microorganisms. Also, a culture medium in the sense of the present invention may optionally demonstrate discrimination abilities to differentiate, to visually distinguish the different types of microorganisms growing on the same culture medium. To this end,

specific metabolic activities they express.

The composition and formulation of many culture media are described in particular in HANDBOOK OF MICROBIOLOGICAL MEDIA (2010; 4 th Edition).

The term "sample" refers to a sample taken for analysis purposes or to a small part or small amount of this levy. The invention more specifically aims biological sample containing or suspected of containing microorganisms to be detected and / or analyzed. These biological samples can be of human, animal, plant or environmental. They may also have an industrial origin and come from levies made on a manufactured product or a product being manufactured, or on instruments, facilities encountered in an industrial environment. Industrial sectors covered here are more particularly the food processing, pharmaceutical, cosmetic and veterinary medical devices,

The term "microorganisms" and "cell" are used equivalently and refer to bacteria, yeasts, molds and / or amoeba.

By "resources / tools Isolation / spread" means mechanical instruments suitable for use in the implementation of isolation procedures (eg, burnout technique, technique streaks or dials) technical spreading or cell topping (eg, to a cell count or realization of antibiotic susceptibility testing), such as those commonly used in conventional solid culture media. These mechanical instruments used manually or in an automated way possible to form one or more occasional deposits of microorganisms on the surface of the culture medium, and by sliding on this surface, they spread the cells. As non-exhaustive examples include the oëses, platinum handles,

According to the invention, the proposed microbiological culture device is formed essentially of the combination between a piece of absorbent material incorporating in its thickness, a composition of dehydrated culture medium, and a hydrogel sheet polysaccharide also dehydrated, having the ability to rehydrate temperature

room. Its size, the polysaccharide hydrogel sheet dehydrated covers all or part of the upper face of the piece of absorbent material.

About the piece of absorbent material which composes the inner / deep layer of microbiological culture device according to the invention and which acts as a reservoir to a composition of dehydrated culture medium, its structure, its design and dimensions are widely described or suggested by EP 1179586, WO 2015/104501, WO 2014/013089, WO 2015/107228.

Many absorbent materials, hydrophilic and water-insoluble, can be used to make the part of absorbent material of a microbiological culture device according to the invention. These materials are primarily selected for their absorbing power, their capacity retention aqueous liquids and their ability to leave the aqueous liquid therethrough.

Advantageously and according to the invention, said piece of absorbent material is made from a short fiber substrate nonwoven, constituting an assembly having structural integrity and mechanical consistency. Particularly suitable substrates are natural cellulose fiber (such as cotton) or synthetic (eg rayon) in a modified cellulosic fiber (e.g., carboxymethyl cellulose, nitrocellulose), fibrous absorbent chemical polymers (such as polyacrylate salts copolymers, acrylate / acrylamide). According to a preferred embodiment, said piece of absorbent material is a textile non-woven fiber made of cellulose, in particular cotton.

The incorporation of a composition of culture medium into the body of a part made of fibrous material may be performed in various ways.

It can be done by techniques known to liquid phase impregnation (see for example, CN 102 337 324 or WO 2005/061013). These techniques consist in impregnating an absorbent material with a composition of culture medium formulated in solution in a volatile solvent (e.g., water, an alcohol). Once thoroughly soaked, the drying of the absorbing material is carried out by evaporating the solvent.

It can also be done by techniques known to dry impregnation. These techniques are intended to be transferred, in the thickness of a piece of absorbent material, a powdery composition, namely a culture medium prepared in the form of a powder or a powder assembly. For this purpose, the particles of said powder composition is sprinkled on the surface of the piece of absorbent material, and are made to vibrate under the action of ultrasonic waves (see for example, FR 2866578) or as the action of an alternating electric field (cf. e.g., WO 2015/044605, WO 2010/001043 or WO 99/22920). These particles penetrate and then sink gradually in the cavities of the porous body.

For the manufacture of a microbiological culture device according to the invention, impregnation techniques to dryness implementing an alternating electric field have been particularly adapted to allow incorporation of a composition of dried culture medium in thickness of an absorbent material. Therefore, the technical teaching given by WO 2015/044605, WO 2010/001043 and WO 99/22920 are an integral part of this description.

According to the invention, the amount of solution of the activated culture medium (hydrate) and the concentration of its constituents condition, first, the choice of material of the part of absorbent material (especially in view of its holding capacity water) and the dimensions of the piece of absorbent material and, on the other hand, the amount of culture medium powder to be incorporated there, and vice versa.

Advantageously, before a possible calendering, said piece of absorbent material has a basis weight between 50 gm "2 and 150 gm " 2 , preferably between 90 gm "2 and 110 gm " 2 , to a thickness advantageously still between 0.5 mm and 10 mm, more preferably between 1 mm and 4 mm.

According to a preferred embodiment, after the incorporation of the medium composition of finished dried culture, the piece of absorbent material is advantageously subjected to a calendering operation. Calendering, by pressure and the heating temperature generated, improves retention of the composition of the dried culture medium in the thickness of the piece of absorbent material, and its stability over time. The liner also has the advantage of improving the flatness of the surface of the piece of absorbent material, and increase the capillary power of the latter.

Calendering is preferably at a recommended temperature

between 30 ° C and 60 ° C. A temperature below 60 ° C makes it possible not denature the thermolabile compounds.

EP I 179 586, WO 2015/104501, WO 2014/013089 and WO 2015/107228, commented on earlier in this specification, disclose microbiological culture media which also include in their structures of the layers of porous material incorporating a medium composition dehydrated culture. These layers thus described may be integrated as such for use in the design of microbiological culture devices of the present invention.

According to a particularly preferred embodiment of the present invention, the piece of absorbent material incorporating in its thickness, a composition of dehydrated culture medium advantageously follows the technical characteristics of the porous support impregnated dry disclosed by WO 2015/104501.

Advantageously, the amount of culture medium composition formulated powder and incorporated in the thickness of the piece of absorbent material is between 0.01 g.cm "3 and 0.1 g. Cm " 3 , preferably between 0 , 02 g. cm "3 and 0.09 g. cm " 3 , more preferably between 0.03 g.cm "3 and 0.06 g.cm " 3 .

As regards the constituent dried polysaccharide hydrogel sheet of a microbiological culture device according to the invention, its composition and thickness have been selected to create a solid surface structure, of small thickness, having integrity and mechanical holding sufficient to allow a grip and easy handling. Furthermore, and intrinsically, the dehydrated hydrogel sheet polysaccharide forms a superabsorbent material and rehydrated at room temperature, particularly at temperatures between 5 ° C and 40 ° C, so that in contact with the part previously soaked in water absorbing material will throat of the composition of the liquid culture medium and reform an integral hydrogel layer (that is to say, an assembly having a certain structural integrity and mechanical consistency) the nutritional properties, possibly selective and / or discriminant. On this layer of hydrogel, the inoculated cells are thus deposited closer to the constituents of the composition of the culture medium.

While spreading operations are often traumatic to the cells, the latter being generally displaced despite their adherence to the culture medium, with a microbiological culture device according to the invention, with a nice surface quality hydrogel (optionally thixotropic properties), the cells are not abruptly unhooked from the holder but are driven in movement with the micro-fraction hydrogel surrounding them.

According to the invention, the dried polysaccharide hydrogel sheet is preferably a dehydrated hydrogel sheet gellan, xanthan, gallactomannane, starch and / or a mixture of these hydrogels. Said dehydrated hydrogel sheet is obtained by dehydrating a hydrogel layer of composition based on water and at least one gelling polysaccharide. Said gelling polysaccharide is preferably selected from gellan gum, xanthan gum, a galactomannan gum (e.g., gum locust bean or guar gum), starch and a mixture thereof.

Advantageously and according to the invention, the dried polysaccharide hydrogel sheet is obtained by dehydrating a polysaccharide hydrogel layer previously prepared by mixing one liter of water, 0.1 to 30 g of at least one gelling polysaccharide .

According to a first preferred embodiment, the dried polysaccharide hydrogel sheet is obtained by dehydrating a hydrogel layer gellan, previously prepared by mixing one liter of water, 10 to 20 g of gellan gum and, preferably 13 to 15 g of gellan gum.

According to an alternative embodiment, the dried polysaccharide hydrogel sheet is obtained by dehydrating a hydrogel layer xanthan, previously prepared by mixing one liter of water, 0.2 to 10 g of gum xanthan, preferably of the order of 0.5 g of xanthan gum.

According to a second alternative embodiment, the hydrogel sheet

dehydrated polysaccharide is obtained by dehydration of a xanthan hydrogel layer and gallactomannane, previously prepared from a mixture of xanthan gum and locust bean gum. The weight ratio [xanthan gum] / [carob bean gum] is advantageously between 1: 2 and 2: 1, preferably of the order of 1: 1.

According to a third alternative embodiment, the dried polysaccharide hydrogel sheet is obtained by dehydrating a hydrogel layer of starch, preferably potato starch, previously prepared by mixing one liter of water, 0.5 to 15 g of starch.

According to a fourth alternative embodiment, the dried polysaccharide hydrogel sheet is obtained by dehydrating a hydrogel layer gellan and starch, preferably potato starch, previously prepared from a mixture gellan gum and starch. The weight ratio [gellan gum] / [starch] is advantageously between 40: 1 and 2: 3.

From a composition of polysaccharide hydrogel dehydrated polysaccharide sheet can be prepared in many ways. For example, the hydrogel may be cast or coated in a continuous layer on a non-adherent surface. This continuous layer can also be obtained by a coating method. The hydrogel layer is then dried / dehydrated, and then cut to the shape and size desired.

The preparation of the dehydrated polysaccharide hydrogel sheet can also be made by a molding method, followed by a stage of dehydration.

According to a preferred embodiment of the invention, the dried polysaccharide hydrogel sheet is advantageously structurally reinforced chemically by means of a reinforcement additive selected from glycerol, ethylene glycol and polyethylene glycol. Uploading said reinforcing additive is carried out during the preparation of the polysaccharide hydrogel composition. Advantageously, this addition is carried out by gelling the mixture of step and water, prior to the dehydration step.

In this context, a hydrogel sheet according dehydrated polysaccharide

the invention further comprises at least one reinforcing additive selected from glycerol, ethylene glycol and polyethylene glycol.

Advantageously and according to the invention, said reinforcing additive is glycerol.

According to one embodiment of the invention particularly preferred polysaccharide said dehydrated hydrogel sheet is a gellan dehydrated hydrogel sheet further comprising, glycerol.

Such dried polysaccharide hydrogel sheet is prepared from gellan gum and glycerol. The weight ratio [gellan gum] / glycerol used in this preparation is preferably between 2: 1 and 1: 8, preferably between 2: 7 to 2: 9, and is typically of the order of 1: 4.

According to an advantageous embodiment of the invention, the dried polysaccharide hydrogel sheet further comprises at least one curing agent selected from the divalent cation salts. Preferably, the curing agent is selected from magnesium chloride (MgCl 2 ), calcium chloride (CaCl 2 ), magnesium sulfate (MgSO ^) and manganese chloride (MnCl 2 ). Advantageously and according to the invention, the curing agent is MgCl 2 .

Advantageously and according to the invention, the dried polysaccharide hydrogel sheet is a gellan dehydrated hydrogel sheet comprising, in addition, at least one curing agent selected from MgCl 2 , CaCl 2 , MgSO ^ and MnCl 2 . The curing agent is preferably MgCl 2 . The weight ratio gellan / MgCl 2 is preferably between 100: 1 and 3: 2, preferably between 30: 1 and 1: 3, and is typically of the order of 15: 1.

According to a particular embodiment, the dried polysaccharide hydrogel sheet is prepared from gellan gum and MgCl 2 . The weight ratio [gellan gum] / MgCl 2 used in this preparation is preferably between 100: 1 and 3: 2, preferably between 30: 1 and 1: 3, and is typically of the order of 15: 1.

According to another particular embodiment, the dried polysaccharide hydrogel sheet is prepared from gellan gum and MgSO The weight ratio [gellan gum] / MgS04 used in this preparation is

preferably between 100: 1 and 3: 2, preferably between 30: 1 and 1: 3, and is typically of the order of 15: 1.

According to a particular embodiment of the invention, the dried polysaccharide hydrogel sheet is a gellan dehydrated hydrogel sheet comprising, in addition, at least one plasticizer such as, for example, a silicone oil. Use of a plasticizer allows to obtain hydrogels dehydrated greater flexibility and flexibility. The advantage is thus able to prepare large dehydrated hydrogel surfaces, for example in long strips can be brought into rolls and stored until well layered cut to the appropriate dimensions to microbiological culture devices according to the invention.

The incorporation of the plasticizer is carried out during the preparation of the polysaccharide hydrogel composition. Advantageously, this incorporation is performed by gelling the mixture of step and water, prior to receiving the dehydrated polysaccharide slip.

According to a particular embodiment of the invention, the dried polysaccharide hydrogel sheet contains in its thickness chromogenic compounds and / or fluorogenic allowing visual observation of microorganisms according to particular metabolic activities they express. These compounds can be, for example, synthetic substrates for the detection of enzymatic activities defined, colored pH indicators.

The incorporation of chromogenic compounds and / or fluorogenic is carried out during the preparation of the polysaccharide hydrogel composition. Advantageously, this incorporation is performed by gelling the mixture of step and water, prior to receiving the dehydrated polysaccharide slip.

Advantageously and according to the invention, in addition to the part of absorbent material and the dried polysaccharide hydrogel sheet, a microbiological culture device according to the invention may also comprise an intermediate permeable membrane disposed between said part of absorbent material and said sheet of dehydrated polysaccharide hydrogel.

The composition, design and the thickness of this interlayer permeable membrane are chosen so that the latter least restricts the transfer of liquid between the part of absorbent material and the dried polysaccharide hydrogel sheet or the polysaccharide hydrogel layer rehydrated. In this respect, it can be made from a substrate made of natural cellulose fiber (such as cotton) or synthetic (eg rayon) in a modified cellulosic fiber (e.g., carboxymethyl cellulose, nitrocellulose), fibrous polymers chemical absorbents (such as polyacrylate salts, acrylate / acrylamide) or stable protein fibers (such as silk, wool).

In the context of the present invention, this intermediate permeable membrane, optionally, may be used for very various purposes, for example:

to provide mechanical reinforcement and / or additional rigidity to the whole system or only the dried polysaccharide hydrogel sheet and the polysaccharide hydrogel layer rehydrated,

- to improve the contact with the part of absorbent material with the dried polysaccharide hydrogel sheet and / or the hydrogel layer rehydrated polysaccharide,

- to act as a reservoir layer for the conservation of particular compounds, intended to be mixed with the culture medium composition solubilized from the piece of absorbent material prior to being routed to the hydrogel layer rehydrated polysaccharide,

- to improve contrast and observation of colonies growing on the surface of microbiological culture device.

According to a particular embodiment, said permeable membrane intermediate is used to improve the contrast and observation of colonies growing on the surface of microbiological culture device. For this purpose it is chosen to be sufficiently opaque to light and a high whiteness (e.g., a CIE whiteness of at least 65).

The design of the organic devices according to the invention and methods of use thereof allow the main components, such as in particular:

the part of absorbent material,

the polysaccharide hydrogel sheet dehydrated and

the possible membrane permeable insert,

can advantageously be manufactured, packaged and stored separately and completely independently. This is a real industrial asset, both from a business perspective as logistics. This also represents a significant advantage for the user who has the possibility to combine design the various components of microbiological culture device according to the invention, and to adapt itself microbiological culture device microorganisms which it has a special interest.

Similarly, a microbiological culture device according to the invention may be packaged and marketed in various forms, including:

- a microbiological culture device, composite, pre-mounted and assembled, wherein the piece of absorbent material is topped by a sheet hydrogel of polysaccharide dehydrated, possibly with an intermediate permeable membrane;

- a microbiological culture device kit, the user will assemble by itself; such a kit comprises at least one piece of absorbent material, at least one dehydrated hydrogel sheet, optionally at least one intermediate permeable membrane.

The microbiological culture device according to the invention is packaged or pre-assembled condition to be assembled extemporaneously by a user, to facilitate handling thereof:

- the piece of absorbent material,

the dried polysaccharide hydrogel sheet (hydrogel layer or rehydrated polysaccharide) and

the possible membrane permeable insert,

may advantageously be kept integral with each other by technical means advantageously applied on the periphery of these various factors, such as:

- a staple system, pin,

- an adhesive composition applied linearly or punctually

- a blocking system disposed within a receptacle (e.g., receptacle of a petri dish) specifically adapted to receive said microbiological culture device; the various components of microbiological culture device according to the invention are, in this case, shape and dimensions adapted to those of said receptacle, and the rim of said receptacle is provided on its (their) surface (s) internal (s) retention driving elements, type lugs, protruding from the surface.

The invention also relates to a microbiological culture device as described above, and presented as a kit to assemble. A microbiological culture device in a kit to be assembled, according to the invention thus comprises:

- at least one piece of absorbent material having an upper surface at least substantially flat and incorporating in its thickness, a composition of dehydrated culture medium,

- at least one polysaccharide dehydrated hydrogel sheet and rehydrated at room temperature, particularly at temperatures between 5 ° C and 40 ° C, and optionally

- optionally, at least one intermediate permeable membrane.

According to a particular aspect of the present invention, it also provides a hydrogel sheet polysaccharide dehydrated and rehydrated at room temperature, particularly at temperatures between 5 ° C and 40 ° C. Said hydrogel sheet dehydrated and rehydratable polysaccharide at room temperature is intended to be used as a microbiological culture medium.

Advantageously, a hydrogel sheet dehydrated and rehydratable polysaccharide according to the invention is characterized by all or part of the technical characteristics listed below:

- said dehydrated polysaccharide hydrogel sheet is a dehydrated hydrogel sheet gellan, xanthan, galactomannan, starch or a mixture thereof,

- said dehydrated polysaccharide hydrogel sheet has been prepared by dehydration of a water-based composition hydrogel layer and at least one

gelling polysaccharide selected from gellan gum, xanthan gum, a galactomannan gum, starch and a mixture thereof,

- said dehydrated polysaccharide hydrogel sheet is obtained by dehydrating a polysaccharide hydrogel layer prepared by mixing one liter of water, 0.1 to 30 g of at least one gelling polysaccharide previously stated,

- said dehydrated polysaccharide hydrogel sheet is obtained by dehydrating a hydrogel layer gellan, previously prepared by mixing one liter of water, 10 to 20 g of gellan gum and preferably from 13 to 15 g gellan gum,

- said dehydrated polysaccharide hydrogel sheet is obtained by dehydrating a hydrogel layer xanthan, previously prepared by mixing one liter of water, 0.2 to 10 g of xanthan gum,

- said sheet of dried polysaccharide hydrogel is obtainable by dehydrating a hydrogel layer of xanthan and gallactomannane, previously prepared from a mixture of xanthan gum and locust bean gum bean,

- said dehydrated polysaccharide hydrogel sheet is obtained by dehydrating a hydrogel layer of starch, previously prepared by mixing one liter of water, 0.5 to 15 g starch,

- said dehydrated polysaccharide hydrogel sheet is obtained by dehydrating a hydrogel layer gellan and starch, prepared from a rubber mixture of gellan and starch, in a weight ratio [gellan gum ] / [starch] between 40: 1 and 2: 3,

- said dehydrated polysaccharide hydrogel sheet is structurally reinforced chemically by means of a reinforcement additive selected from glycerol, ethylene glycol and polyethylene glycol,

- said dehydrated polysaccharide hydrogel sheet further comprises at least one curing agent selected from the divalent cation salts - in particular MgCl 2 , CaCl 2 , MgS0 4 and MnCl 2 -,

- said hydrogel sheet dehydrated polysaccharide further comprises at least one plasticizer such as, for example, a silicone oil.

Other objects, features and advantages of the invention will become apparent from the following description and examples discussed below, which refer to the accompanying figures and in which:

- Figure 1 is a schematic representation of a microbiological culture device according to the invention and the general principle of use;

- Figure 2 is a photograph showing an example of dehydrated polysaccharide hydrogel sheets after drying process / dehydration;

- Figures 3 to 13 together photographs of cultures of bacterial strains and isolates performed on microbiological culture devices according to the invention.

These examples are intended to facilitate understanding of the invention, its implementation and use. These examples are for illustrative purposes and should not limit the scope of the invention.

EXAMPLES

AI - A microbiological culture according to the invention and the general principle of use

As shown in Figure 1, a microbiological culture device according to the invention consists, firstly, of a piece of absorbing material 1, more or less thick and a hydrogel sheet polysaccharide dehydrated 2, d lesser thickness. In the example shown, the two elements 1 and 2 are of substantially square shape.

The piece of absorbent material 1, made of hydrophilic material and water-insoluble, incorporates in its thickness a composition of dehydrated culture medium. The hydrogel sheet polysaccharide dehydrated 2 is in turn formed by drying / dehydrating a polysaccharide hydrogel layer, and its mechanical structure may be reinforced by a fibrous reinforcement, eg a woven fabric.

The piece of absorbing material 1 and the hydrogel sheet polysaccharide dehydrated 2 may be supplied already preassembled, fastened, or else in the form of two mechanically independent elements.

The design and manufacture of this piece of absorbent material 1 and 2 of this dehydrated polysaccharide hydrogel sheet will be described in more detail in the following examples.

As a secondary feature, a receptacle 4 is associated with microbiological culture device for ease of manipulation, particularly for the rehydration step and activation of the device and for any movement (e.g., a transfer of the pallet to the incubator).

Use of a microbiological culture device according to the invention requires activation of the device due to hydration by the piece of absorbing material 1, with water 4.

To do this, the microbiological culture device according to the invention is placed inside the receptacle 4, with the hydrogel sheet polysaccharide dehydrated 2 facing upwards. The receptacle 4 is of larger area than that of the culture device, water is poured into the receptacle 4 5 taking care not to pour directly on the dried polysaccharide hydrogel sheet 2. The volume of water used 's roughly calibrated to be able to sufficiently humidify the part of absorbent material 1 and solubilize the composition of culture medium it contains.

Another way to operate is to pour into the bottom of the receptacle 4, the appropriate volume of water and then to deposit the piece of absorbing material 1, surmounted dehydrated polysaccharide hydrogel sheet 2. Care should be taken not not placing the hydrogel sheet polysaccharide dehydrated 2 directly in contact with free water, so that the device is effectively hydrated only by the piece of absorbing material 1.

When the microbiological culture device according to the invention is provided with the piece of absorbing material 1 and the hydrogel sheet polysaccharide dehydrated 2 in the form of two independent elements mechanically, the hydration step can then be carried out in a third way. The absorbent material piece 1 is then placed within the container 4. In the container 4 and optionally directly on this piece of absorbing material 1 is poured an amount of water 5 sufficient to thoroughly saturate the absorbent material piece 1 . then comes to cover the surface with the sheet of dehydrated 2 polysaccharide hydrogel.

During activation of a microbiological culture device according to the invention, hydration by the piece of absorbing material 1 is used, firstly, to solubilize the culture medium composition contained in the thickness of the piece of absorbent material 1. in a second step, this activation is followed by hydration of the dehydrated hydrogel polysaccharide sheet 2 applied on the surface of the piece of absorbent material 1. this rehydration of the dried polysaccharide hydrogel sheet 2 shows, on the surface of the piece of absorbing material 1, a layer of hydrogel rehydrated polysaccharide 2'- and inflated, not simply by theWater from the room by absorbing material 1, but rather by a culture medium solution from the absorbent material piece 1.

Thus activated, the cell culture device is ready for the test sample. Once the sample is inoculated onto the device, in particular by spreading operations and dispersion of the cells, for example, by means of a Oese 6, the whole is incubated at a temperature and for a set time before reading results.

By its consistency and very special texture, the layer / film of polysaccharide hydrogel rehydrated 2 'of a microbiological culture device according to the invention creates a culture surface, both lubricated and adherent to cells, capable receiving microorganisms and allow their isolation by operations and mechanical means usually used for spreading the cells on the surface of a conventional agar medium. Moreover, because of its greater exposure to gas exchange and drainage phenomena and its hyper-absorbent properties, this layer / film polysaccharide hydrogel 2 's will to self -régénérer throughout the period incubation and continuously,

As also shown in Figure 1, the microbiological culture device may also include a tab from permeable membrane 3 is interposed between the piece of absorbing material 1 and the polysaccharide hydrogel sheet dehydrated 2. Made in a permeable material, said membrane permeable

tab 3, optional, can be used for a variety of purposes such as: to provide mechanical reinforcement and / or additional rigidity to the entire system, or else only the dried polysaccharide hydrogel sheet 2 and rehydrated polysaccharide hydrogel layer 2 ',

to improve the contact with the part of absorbent material 1 with the hydrogel sheet polysaccharide dehydrated 2 and / or the hydrogel layer polysaccharide rehydrated 2 ',

- to reservoir layer office for the conservation of particular compounds to be mixed in the culture medium composition solubilized from the piece of absorbing material 1 before reaching the polysaccharide rehydrated hydrogel layer 2 ',

- to improve contrast and observation of colonies growing on the surface of microbiological culture device.

B / - Fabrication of the various components of a microbiological culture device according to the invention

Bl / - The piece of absorbent material incorporating in its thickness, a composition of dehydrated culture medium

With regard to the part of absorbent material 1 used in the construction of a microbiological culture device according to the invention, it is formed from a three-dimensional support, open, porous structure, able to receive in its in both a liquid (particularly aqueous liquid) as adapted particle size of solid particles.

In the examples and tests which follow, the tested microbiological culture devices comprise a piece of absorbent material incorporating a composition of culture medium, dried or dehydrated, made from non-woven airlaid SCA95 N81, CAS company ( Sweden). This nonwoven bicomponent PET / CoPET (Polyester / copolyester) has been treated to be adhesive-coated by simple hot-pressing (calendering), without addition of adhesive. It is also characterized by a basis weight, prior to calendering (or uncalendered), of the order of 95 gm "2 to a thickness of 2 mm. From this nonwoven, parts of approximately 6 cm square were cut.

The incorporation of a composition of dehydrated culture medium, in the same mass of these pieces of fibrous material, was carried out with a dry impregnation technique. To this end, about 0.2 g of a culture medium composition formulated in powder form is sprinkled on each piece of nonwoven cut. The assembly is placed between two electrodes applying a voltage of 3200 V.mm "1 for 15 seconds, with a relative humidity between 35% and 45%.

Once the dry impregnation completed, the non-woven pieces are calendered at 60 ° C by applying a pressure of 3.10 5 Pa.cm "2 .

B2 / - Culture media formulated powder

In order to test the microbiological culture devices according to the invention, different dehydrated culture medium compositions were used. They resumed the composition of solid culture media distributed by bioMérieux (France), with the exception of agar agar and other texturing agents. It was especially media range chromID ® (eg chromID ® Elite SPC chromID ® S. aureus chromID ® P. aeruginosa chromID ® ERV chromID ® Salmonella Elite).

Microbiological culture devices according to the invention, thus produced, could then be tested for the detection and isolation of bacteria such as Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Clostridium freundii, Streptococcus agalactiae and Serratia marcescens.

A selection of results is presented in Figures 3 to 13, in the form of photographs.

B3 / - The sheets of dehydrated polysaccharide hydrogel

The hydrogel sheets polysaccharide dehydrated 2, which enter into the composition of the microbiological culture devices according to the invention are primarily designed such that, once rehydrated, they can provide a holder for microorganism growth and development . The composition and consistency of these two dehydrated polysaccharide hydrogel sheets were also specifically designed for surfaces adapted to working up and spreading of the cells, these polysaccharide hydrogel sheets are in a dehydrated state or rehydrated .

In the examples and tests which follow, the hydrogel sheets polysaccharide dehydrated 2 constituent microbiological culture devices according to the invention were prepared by proceeding according to the general procedure given below.

a) Preparation of Polysaccharide hydrogels:

Heat 500 mL of sterile distilled water in a glass vial on a hot plate.

Add the (s) gelling agent (s) in defined quantity and wait until the mixture is homogeneous and translucent continuing to heat (boil or near Pébulition).

- After complete dissolution, add the glycerin, mix and add the (s) salt (s) of divalent cation binder and curing agent, mix and bring to boil.

Divide 5-15 ml (depending on the desired thickness of the sheet) of the still hot mixture into petri dishes of 47 mm diameter.

Allow to cool and harden on the bench until recovery by weight of gelling agent. Turn the hydrogel pieces using a scalpel if necessary.

b) Dehydration polysaccharide hydrogels:

Hydrogels grip parts between two sheets of absorbent paper. Placing the assembly in the oven, at a temperature of about 40 ° C, for 1 to 6 hours; the operation can also be performed for at least one night in a dry heat oven, brought to 32 ° C.

Figure 2 is a photograph showing the hydrogel sheets dehydrated out of the oven. As an indication, with 7 mL of preparing hydrogel poured into Petri dishes, the thickness of the hydrogel parts is of the order of 3 mm, before processing baking or in an oven. After drying, the leaves are less than 1 mm thick.

The hydrogel sheets polysaccharide dehydrated 2 of a microbiological culture device according to the invention were laid out in different versions by varying, in particular:

the nature of the gelling polysaccharide (e.g., gellan gum, xanthan gum, galactomannan gums, starch, sodium alginate, pectin, methylcellulose and hydroxymethylcellulose),

the proportion between the water and the gelling agent used in the step of preparing polysaccharide hydrogels,

the nature and quantity of any curing agents used in preparing polysaccharide hydrogels,

the nature and amount of the additives optionally used to improve the physicochemical properties of the polysaccharide hydrogels (hydrated) and / or dried polysaccharide hydrogel sheets.

B4 / - The intermediate permeable membrane (optional)

In order to improve the contrast and observation of colonies growing on the surface of microbiological culture device, a membrane is opaque to light and a high whiteness (e.g., a CIE whiteness of at least 65) can be interposed between the part of absorbent material and the dried polysaccharide hydrogel sheet. This interposed permeable membrane may consist of a sheet of absorbent paper, cellulosic composition.

Cl - Evaluation of microbiological culture devices according to the invention

For dehydrated polysaccharide hydrogel sheets which have a solid surface, structural integrity and satisfactory mechanical strength, tests were conducted to assess their ability to be able to establish microbiological culture media both effective and consistent with operations and spreading mechanical means and dispersion of the cells.

In this context, bacterial cultures were carried out with particular strains of Escherichia coli, à'Enterococcus faecalis, Proteus mirabilis, Staphylococcus aureus, Serratia marcescens, Pseudomonas aeruginosa, Enterobacter cloacae, Clostridium freundii and Cronobacter sakazaki . Depending on the bacteria of interest to grow, the polysaccharide hydrogel sheets dehydrated test are associated with pieces of absorbent material incorporating in a thickness

particular dehydrated culture medium composition. The peculiarity of this composition of dehydrated culture medium is that it has been selected to be adapted to the growth and development of bacteria of interest, and they include chromogenic components to facilitate the visual identification of these bacteria of interest.

To do this, the pieces of absorbent material, which incorporate in their composition a thickness of dehydrated culture medium are placed in Petri dishes 90 mm in diameter, then moistened with 6-7 ml of sterile distilled water. Then overcomes these parts by absorbing a dried polysaccharide hydrogel sheet material. Once the hydration of the absorbent material piece produced, the culture medium enabled and the regenerated polysaccharide hydrogel layer, microbiological culture device is seeded with 10 μΐ ^ a calibrated solution to a theoretical load of 10 bacterial 7cfu / mL. The cell sample is deposited by means of a first Oese on the first face of the hydrogel surface. The second dial is inoculated with a loopful new stretching several streaks from the first dial. The third dial is seeded as the second without changing Oese. The fourth face is seeded with unstretched streaks from the second dial.

The dishes are placed in a jar with a little water so that the microbiological culture devices do not dry out. The whole is then incubated at 37 ° C for 24 hours.

Some of the results have been compiled and presented in Figures 3 to 13, in the form of photographs.

In Figure 3, are exposed photographs of cultures and I isolates. coli conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from hydrogels gellan 13 g / L (that is to say, 13 g of gellan gum for 1 liter of water ), reinforced structure to glycerol at the rate of 43 mL / L (that is to say, 43 mL of glycerol per liter of water used in the preparation of the hydrogel), and cured with:

3A: MgCl 2 at a rate of 1 g / L (that is to say, 1 g of MgCl 2 per liter of water used in the preparation of the hydrogel), or

3B: the MgS0 4 , at 1 g / L.

Gellan gum used here to prepare the dehydrated polysaccharide hydrogel sheets is Gelrite ® , distributed by the company CARL ROTH GmbH, Germany.

That the curing agent is MgCl 2 or MgS0 4 , the results are very similar. The colonies of E. coli grow on the surface of the hydrogel sheet. They have very good size and a beautiful coloring. Their morphological type matches that of the colonies of E. coli growing on chromogenic agar reference such as the chromID ® Elite CPS.

In Figure 4, are exposed photographs of a culture and isolation of E. coli conducted on a microbiological culture device whose dried polysaccharide hydrogel sheet was obtained from a hydrogel gellan 15 g / L, reinforced structure to glycerol at the rate of 43 mL / L, and cured with CaCl 2 at a rate of 1 g / L.

Gellan gum used here is Gelrite ® .

Compared to the previous examples, the I colonies. coli appear smaller, with a slight diffusion of the periphery coloring.

In Figure 5, a photograph is exposed of cultures and isolates of E. coli conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from hydrogels gellan 15 g / L, reinforced structure to glycerol at the rate of 43 mL / L, and cured with:

- 5A: MgCl 2 at a rate of 1 g / L, or

5B: MgCl 2 at a rate of 5 g / L.

Gellan gum used here is the Gelzan ™, distributed by the company CP Kelco, USA.

With 1 g / L MgCl 2 , colonies of E. coli growing on the surface of the hydrogel sheet, have a very nice size and beautiful coloring. Their morphological type matches that of the colonies of E. coli growing on chromogenic agar reference such as the chromID ® Elite CPS (Figure 5, Part 5C).

With 5 g / L MgCl 2 , colonies are smaller.

Without MgCl 2 , the colonies are very diffuse (Figure 5, part 5D).

In Figure 6, are exposed photographs of cultures and I isolates. faecalis conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from hydrogels gellan 15 g / L, reinforced structure to glycerol at the rate of 43 mL / L, and cured with:

6A: MgCl 2 at a rate of 1 g / L,

6B: MgCl 2 , at 2 g / L, or

- 6C: MgCl 2 at a rate of 5 g / L.

Gellan gum used here is the Gelzan ™.

The colonies of E. faecalis pushing to the surface of the hydrogel sheet, are identical in size, color and morphological type, the colonies of E. faecalis growing on chromogenic agar reference such as the chromID ® Elite SPC.

In Figure 7, are exposed photographs of cultures and P. mirabilis isolates conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from hydrogels gellan 15 g / L, reinforced structure to glycerol at the rate of 43 mL / L, and cured with:

- 7A: MgCl 2 at a rate of 1 g / L,

7B: MgCl 2 , at 2 g / L, or

7C: MgCl 2 at a rate of 5 g / L.

Gellan gum used here is Gelrite ® .

The colonies of P. mirabilis pushing to the surface of the hydrogel sheet are identical in size, color and morphological type, the colonies of P. mirabilis growing on chromogenic agar reference such as the chromID ® Elite SPC.

In Figure 8, a photograph is exposed of cultures and isolates of E. coli conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from gellan hydrogels at 13 g / L or 15 g / L, reinforced structure to glycerol at the rate of 43 mL / L and cured with CaCl 2 used at 1 g / L or 2 g / L:

- 8 A, 8 A ': gellan gum 13 g / L, CaCl 2 1 g / L,

- 8B, 8B ': gellan gum 13 g / L, CaCl 2 2 g / L,

- 8C, 8C: gellan gum 15 g / L, CaCl 2 1 g / L,

- 8D, 8D ': gellan gum 15 g / L, CaCl 2 2 g / L

Gellan gum used here is the Gelzan ™.

The four microbiological culture devices tested here give very similar results. Compared to the examples of Figure 5, wherein MgCl 2 is used to cure hydrogels, E. coli form here slightly smaller size of colonies.

In Figure 9, is exposed and a photograph of cultures of E. faecalis isolates conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from hydrogels gellan 15 g / L, reinforced structure to glycerol at the rate of 43 mL / L, and cured with:

- 9A: CaCl 2 1 g / L

- 9B: you CaCl 2 2 g / L

- 9C: CaCl 2 3 g / L.

Gellan gum used here is the Gelzan ™.

While the above examples corresponding to Figure 8 suggest an advantage to use MgCl 2 instead of CaCl 2 to the I culture. coli, this finding is less obvious to the culture of E. faecalis.

In Figure 10, are exposed photographs of a culture and isolation of E. coli conducted on a microbiological culture device whose dried polysaccharide hydrogel sheet was obtained from a hydrogel gellan 15 g / L, reinforced structure to glycerol at the rate of 43 mL / L, and cured with MnCl 2 at a rate of 1 g / L.

Gellan gum used here is Gelrite ® .

Compared to a culture of E. coli on the reference medium chromID Elite SPC, the I colonies. coli appear here smaller, but their color is substantially intensified by the CaCl 2 .

In Figure 11, are exposed photographs of cultures and I isolates. coli conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from polysaccharide hydrogels of various compositions:

11A, gellan gum 15 g / L (more specifically the Phytagel, distributed by Sigma Aldrich, USA), Glycerol 43ml / L, MgCl 2 1 g / L,

- 11 A 2 : gellan gum 30 g / L (Phytagel ™), Glycerol 43ml / L, MgCl 2 1 g / L,

- 1 lbi: gellan gum 25 g / L (Gelrite ® ), Glycerol 43ml / L, MgCl 2 1 g / L,

- 11B 2 : gellan gum 20 g / L (Gelrite ® ), Glycerol 43ml / L, MgCl 2 1 g / L,

- 11B 3 : gellan gum 8 g / L (Gelrite ® ), Glycerol 43ml / L, MgCl 2 1 g / L,

- 11B 4 : gellan gum 6 g / L (Gelrite ® ), Glycerol 43ml / L, MgCl 2 1 g / L,

- 1 LCI: gellan gum 20 g / L (Gelzan ™), Glycerol 43ml / L, MgCl 2 1 g / L,

- 11C 2 : gellan gum 8 g / L (Gelzan ™), Glycerol 43ml / L, MgCl 2 1 g / L,

- the LDI: xanthan gum 0.5 g / L, distributed by SIGMA ALDRICH,

France,

- 11D 2 : xanthan gum 10 g / L

- 1 Lei, Lei 1 ': potato starch 0.5 g / L, distributed by the company CARL ROTH

GmbH, Germany.

- 11E 2 , 11E 2 ': potato starch soil 15 g / L

In Figure 12, are exposed photographs of cultures and isolations E. coli conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from hydrogels gellan 15 g / L (in this case, Gelrite ® ), cured with MgCl 2 at 1 g / L, and whose structure has been strengthened glycerol at various concentrations:

- 12A : 32 mL/L

- 12B : 52 mL/L

- 12C : 62 mL/L

By increasing the concentration of glycerol hydrogels, E.coli form larger colonies, which nevertheless seem less curved and spread over hydrogels.

In Figure 13, are exposed photographs of cultures and microorganisms isolates conducted on microbiological culture devices including dried polysaccharide hydrogel sheets were obtained from a hydrogel Gelzan ™ 15 g / L, cured with MgCl 2 at a rate of 1 g / L.

These microbiological culture devices were also tested successfully for cultivation and detection of Streptococcus agalactiae (13 A), Serratia marcescens (13B), and for the co-culture and co-detection of S. marcescens and S . aureus (13C).

WE CLAIMS

1. A microbiological culture comprising:

- a piece of absorbent material (1) having an upper surface at least substantially flat and incorporating in its thickness, a composition of dehydrated culture medium,

- based on said piece of absorbent material (1), a hydrogel sheet polysaccharide dried (2) and rehydrated at temperatures between 5 ° C and 40 ° C; said dehydrated hydrogel sheet (2) being affixed directly on the upper face of said piece of absorbent material (1), or indirectly through an intermediate permeable membrane (3).

2. A microbiological culture according to claim 1, wherein said dehydrated polysaccharide hydrogel sheet (2) is a dehydrated hydrogel sheet gellan, xanthan, galactomannan, starch and / or a mixture of them.

3. A microbiological culture according to claim 1 or 2, wherein said dehydrated polysaccharide hydrogel sheet (2) was prepared by dehydration of a water-based composition hydrogel layer and at least one gelling polysaccharide selected from gellan gum, xanthan gum, a galactomannan gum, starch and a mixture thereof.

4. A microbiological culture according to claim 3, wherein said polysaccharide dried hydrogel sheet (2) is obtained by dehydrating a polysaccharide hydrogel layer prepared by mixing one liter of water, 0.1 to 30 g of at least one gelling polysaccharide.

5. A microbiological culture according to any one of claims 1 to 4, wherein said dehydrated polysaccharide hydrogel sheet (2) is obtained by dehydrating a hydrogel layer gellan, previously prepared by mixing one liter of water, 10 to 20 g of gellan gum and preferably from 13 to 15 g of gellan gum.

6. A microbiological culture according to any one of claims 1 to 4, wherein said dehydrated polysaccharide hydrogel sheet (2) is obtained by dehydrating a hydrogel layer xanthan, previously prepared by mixing one liter water, 0.2 to 10 g of xanthan gum.

7. A microbiological culture according to any one of claims 1 to 4, wherein said dehydrated polysaccharide hydrogel sheet (2) is obtained by dehydrating a hydrogel layer of xanthan and gallactomannane, previously prepared from a mixture of xanthan gum and locust bean gum.

8. A microbiological culture according to any one of claims 1 to 4, wherein said dehydrated polysaccharide hydrogel sheet (2) is obtained by dehydrating a hydrogel layer of starch, previously prepared by mixing in a liter of water, 0.5 to 15 g of starch.

9. A microbiological culture according to any one of claims 1 to 4, wherein said dehydrated polysaccharide hydrogel sheet (2) is obtained by dehydrating a hydrogel layer gellan and starch prepared from a mixture of gellan gum and starch in a weight ratio [gellan gum] / [starch] between 40: 1 and 2: 3.

10. A microbiological culture according to claim 4, wherein said dehydrated polysaccharide hydrogel sheet (2) is structurally reinforced chemically by means of a reinforcement additive selected from glycerol, ethylene glycol and polyethylene glycol .

11. A microbiological culture according to any preceding claim, wherein said polysaccharide dried hydrogel sheet (2) further comprises at least one curing agent selected from the divalent cation salts.

12. A microbiological culture according to any preceding claim, wherein said polysaccharide dried hydrogel sheet (2) further comprises at least one curing agent selected from magnesium chloride (MgCl 2 ), chloride calcium (CaCl 2 ), magnesium sulfate (MgSO ^) and manganese chloride (MnCl 2 ).

13. A microbiological culture according to any preceding claim, wherein said polysaccharide dried hydrogel sheet (2) further comprises at least one plasticizer.

14. A microbiological culture according to any one of the preceding claims, characterized in that, non-calendered, said piece of absorbent material (1) has a basis weight between 50 gm "2 and 150 gm " 2 , for a thickness of between 0.5 mm and 10 mm.

15. A microbiological culture kit to be assembled, comprising:

- at least one piece of absorbent material (1) having an upper surface at least substantially flat and incorporating in its thickness, a composition of dehydrated culture medium,

- at least one polysaccharide dehydrated hydrogel sheet (2) and rehydrated at temperatures between 5 ° C and 40 ° C, and

- optionally, at least one intermediate permeable membrane (3).

16. A microbiological culture kit for assembling according to claim 15, for mounting a microbiological culture device as defined by any one of claims 1 to 14.