Culture medium for detecting microorganisms comprising a mixture of agar and kappa-carrageenan as gelling agent

Abstract

The invention relates to the field of molecular biology and more particularly that of clinical and industrial microbiology. More specifically, the present invention relates to a culture medium for detecting microorganisms, comprising, as a gelling agent, a mixture of agar and kappa-carrageenan.

Description

[0001] Description

[0002] TITLE OF THE INVENTION: Culture medium for detecting microorganisms comprising a mixture of agar and kappa-carrageenan as a gelling agent.

[0003] TECHNICAL FIELD

[0004] The present invention relates to the field of microbiology and more particularly to that of industrial and clinical microbiology. More specifically, the present invention relates to a novel culture medium for detecting microorganisms comprising a mixture of agar and K-carrageenan as a gelling agent.

[0005] PRIOR TECHNIQUE

[0006] The detection and identification of microorganisms is very important in clinical and industrial fields. In the pharmaceutical and food industries, culture media are still the reference for detecting and enumerating microorganisms. The food industry uses these products for microbiological controls of products intended for human and animal consumption. In the pharmaceutical industry, media are mainly used to carry out environmental controls (air, surface, operator, etc.) and to verify that there is no bacterial contamination in the products during manufacturing. Very often, the controls carried out are taken into consideration for the release of batches of finished or semi-finished products. Many tests and operating procedures depend on the ability of culture media to give consistent and reproducible results.Media requirements can be specific to both the sample and the strains to be tested. Culture media meeting established performance criteria are therefore a prerequisite for any reliable microbiological analysis. Ready-to-use media are manufactured under standardized production conditions and solve many problems in microbiology laboratories where time, equipment, or trained personnel are often lacking. Thus, ready-to-use culture media of recognized quality enable reliable microbiological analysis.

[0007] They can be in the form of liquid, solid or semi-solid media supplied in boxes, bottles, tubes or other containers.

[0008] A major component of solid or semi-solid culture media is the gelling agent, particularly agar. The latter is extracted from red algae of the Rhodophyta family. Agar has the disadvantage of being a limited natural resource. Thus, Lines (Applied and Environmental Microbiology, Dec 1977, p 637-639) describes the use of K-carrageenans as a substitute for agar in a culture medium. The disadvantage of a culture medium whose agar has been substituted by K-carrageenan is that it begins to solidify at high temperatures. While agar is dispensed at a temperature of approximately 45-55°C, a culture medium based on K-carrageenans requires, to be dispensed into the dishes, a temperature above 60°C.This results in problems with the industrialization of agar culture media, with the maintenance of a high temperature throughout the manufacturing process, or the risk of burns to technicians if the Petri dishes are poured at the point of use. In addition, a high gelation temperature is not compatible with the production of culture media containing heat-sensitive compounds such as antibiotics or blood.

[0009] On the other hand, the gelling agent is also the key compound for improving the shelf life of culture media. Indeed, agar-based culture media, although widely used, have disadvantages such as syneresis (water leaving the gel network) and shrinkage that should be avoided. Thus, document WO 2004050675 describes the use of a mixture of agar and iota-carrageenan in an agar culture medium. This mixture makes it possible to limit the syneresis of the gel, thus improving its stability and shelf life.

[0010] However, there is still a need to develop an agar culture medium offering physicochemical parameters and resistance to shrinkage to improve its lifespan while maintaining optimal quality and remaining compatible with industrialization.

[0011] SUMMARY OF THE INVENTION

[0012] An objective of the present invention is to provide a culture medium resistant to dehydration due to its capacity not to shrink, or to shrink very little.

[0013] Another objective of the invention is to provide a culture medium having a gelling temperature compatible with industrialization or the use of heat-sensitive compounds.

[0014] These objectives among others are achieved by the present invention which relates to a culture medium for the detection of microorganisms comprising - agar or agarose,

[0015] - K-carrageenan, the weight ratio [agar, K-carrageenan]: [other gelling agent] being greater than 70:30.

[0016] Advantageously, the weight ratio of agar: K-carrageenan in the culture medium is between 80:20 and 30:70.

[0017] In a particular embodiment of the invention, when the concentration of salts present in the medium is less than 5 g / l, the weight ratio of agar: K-carrageenan is between 80:20 and 60:40.

[0018] In another particular embodiment of the invention, when the concentration of salts present in the medium is between 5 g / l and 20 g / l, the weight ratio of agar: K-carrageenan is between 80:20 and 30:70.

[0019] In another particular embodiment of the invention, when the concentration of salt present in the medium is greater than 20 g / l and the weight ratio of agar: K-carrageenan is between 80:20 and 60:40.

[0020] Advantageously, the culture medium further comprises a heat-sensitive compound, such as an antibiotic and / or blood.

[0021] Advantageously, the culture medium is gelled in a Petri dish.

[0022] Another subject of the invention relates to a method for obtaining a medium according to the invention, characterized in that it essentially consists of:

[0023] - supercool the compounds in the culture medium in order to mix them

[0024] - cool to a first temperature above the gelation temperature of the culture medium

[0025] - fill the Petri dishes with this culture medium in solution

[0026] - allow to cool to reach the gelling temperature and allow the formation of a gelled culture medium, the gelling temperature being between 30°C and 45°C.

[0027] Another subject of the invention relates to a culture medium comprising a mixture of agar: K-carrageenan having a weight ratio of between 80:20 and 30:70 and a gelling temperature of between 30°C and 45°C. Another subject of the invention relates to a method of in vitro microbiological culture, in which microorganisms likely to be present in a sample are inoculated in or onto a culture medium according to the invention.

[0028] Another subject of the invention relates to a method for detecting a target microorganism in a sample likely to contain it, comprising the following steps:

[0029] - bringing said sample into contact with a gelled culture medium according to the invention

[0030] - incubate

[0031] - detect the presence of said microorganism.

[0032] DESCRIPTION OF FIGURES

[0033] Figure 1 is a diagram showing the percentage of intact (unshrunk) culture medium after the Extreme Dehydration Test. Media in series "a" comprise 100% agar (or agarose for medium 1). Media in series "b" comprise a 50:50 agar:K-carrageenan ratio (or a 50:50 agarose:K-carrageenan ratio for medium 1).

[0034] Origin of T agar (or agarose for medium 1):

[0035] Medium 1a and medium 1b: Sigma Aldrich agarose

[0036] Medium 2a and medium 2b: Sigma Aldrich agar

[0037] Medium 3a and medium 3b: Roko American Agar

[0038] Medium 4a and medium 4b: Roko European Agar

[0039] Medium 5a and medium 5b: Setexam European Agar

[0040] Medium 6a and medium 6b: Roko’s Gracilaria agar

[0041] Figure 2 shows photos of the culture media after the Extreme Dehydration Test. Media in series "a" consist of 100% agar (or agarose for medium 1). Media in series "b" consist of a 50:50 agar:K-carrageenan ratio (or a 50:50 agarose:K-carrageenan ratio for medium 1). Source of agar (or agarose for medium 1):

[0042] Medium 1a and medium 1b: Sigma Aldrich agarose

[0043] Medium 2a and medium 2b: Sigma Aldrich agar

[0044] Medium 3a and medium 3b: Roko American Agar

[0045] Medium 4a and medium 4b: Roko European Agar Medium 5a and medium 5b: Setexam European Agar

[0046] Medium 6a and medium 6b: Roko’s Gracilaria agar

[0047] Figure 3 shows pictures of the agar mix culture media with different carrageenans after the Extreme Dehydration Test.

[0048] Medium 1 includes 100% European agar (Setexam)

[0049] Medium 2 includes a 50:50 ratio of agar: K-carrageenan (Sigma Aldrich)

[0050] Medium 3 includes a 50:50 ratio of agar: K-carrageenan (Setexam)

[0051] Medium 4 includes a 50:50 ratio of agar: K-carrageenan (Roko)

[0052] Medium 5 includes a 50:50 ratio of agar:k-carrageenan (Sigma Aldrich)

[0053] Medium 6 includes a 50:50 ratio of agar: i-carrageenan (Sigma Aldrich).

[0054] Figure 4 represents the percentage of unshrunk culture media for different agar:K-carrageenan ratios after the extreme dehydration test.

[0055] Medium 1 includes 100% European agar

[0056] Medium 2 includes a 70:30 agar:K-carrageenan ratio

[0057] Medium 3 includes a 60:40 agar:K-carrageenan ratio

[0058] Medium 4 includes a 50:50 ratio of agar:K-carrageenan

[0059] Medium 5 includes a 30:70 agar:K-carrageenan ratio

[0060] Medium 6 includes a ratio of agar:K-carrageenan 0:100

[0061] Figure 5(a) represents the gelation temperature of TSA media modified with different agar:K-carrageenan ratios.

[0062] Medium 1 consists of 100% agar

[0063] Medium 2 includes a 70:30 agar:K-carrageenan ratio

[0064] Medium 3 includes a 50:50 agar:K-carrageenan ratio

[0065] Medium 4 includes a 30:70 agar:K-carrageenan ratio

[0066] Medium 5 includes a ratio of agar:K-carrageenan 0:100

[0067] Figure 5(b) represents the viscosity of TSA media modified with different agar:K-carrageenan ratios. Medium 1 comprises 100% agar

[0068] Medium 2 includes a 70:30 agar:K-carrageenan ratio

[0069] Medium 3 includes a 50:50 agar:K-carrageenan ratio

[0070] Medium 4 includes a 30:70 agar:K-carrageenan ratio

[0071] Medium 5 includes a ratio of agar:K-carrageenan 0:100

[0072] Figure 6 represents the gelation temperature of CHAPMAN media modified with different agar:K-carrageenan ratios.

[0073] Medium 1 consists of 100% agar

[0074] Medium 2 includes an agar:K-carrageenan ratio of 80:20

[0075] Medium 3 includes a 70:30 agar:K-carrageenan ratio

[0076] Medium 4 includes a 60:40 agar:K-carrageenan ratio

[0077] Medium 5 includes a 50:50 ratio of agar:K-carrageenan

[0078] Medium 6 includes a ratio of agar:K-carrageenan 0:100

[0079] Figure 7(a) represents the gelation temperature of SDA media modified with different agar:K-carrageenan ratios

[0080] Medium 1 consists of 100% agar

[0081] Medium 2 includes an agar:K-carrageenan ratio of 80:20

[0082] Medium 3 includes a 70:30 agar:K-carrageenan ratio

[0083] Medium 4 includes a 60:40 agar:K-carrageenan ratio

[0084] Medium 5 includes a 50:50 ratio of agar:K-carrageenan

[0085] Medium 6 includes a 30:70 agar:K-carrageenan ratio

[0086] Medium 7 includes a ratio of agar:K-carrageenan 0:100

[0087] Figure 7(b) represents the elastic modulus of SDA media modified with different agar:K-carrageenan ratios

[0088] Medium 1 consists of 100% agar

[0089] Medium 2 includes an agar:K-carrageenan ratio of 80:20

[0090] Medium 3 includes a 70:30 agar:K-carrageenan ratio

[0091] Medium 4 includes a 60:40 agar:K-carrageenan ratio Medium 5 includes a 50:50 agar:K-carrageenan ratio

[0092] Medium 6 includes a 30:70 agar:K-carrageenan ratio

[0093] Medium 7 includes a ratio of agar:K-carrageenan 0:100

[0094] Figure 8 corresponds to photos after incubation of culture media inoculated with different microorganisms.

[0095] Line (a) corresponds to a TSA medium comprising 100% agar and line (b) corresponds to a modified TSA medium comprising an agar:K-carrageenan ratio of 70:30

[0096] Column 1 corresponds to a seeding of S. aureus

[0097] Column 2 corresponds to a seeding of B. subtilis

[0098] Column 3 corresponds to a seeding of P. aeruginosa

[0099] Column 4 corresponds to a seeding of C. albicans

[0100] Column 5 corresponds to a seeding of A. brasiliensis

[0101] Column 6 corresponds to a seeding of E.coli

[0102] Figure 9 corresponds to photographs of the modified CHAPMAN culture media after incubation inoculated with Staphylococcus aureus.

[0103] Medium 1 corresponds to a medium with 100% agar.

[0104] Medium 2 comprises an agar: K-carrageenan ratio of 80:20.

[0105] Medium 3 includes a 50:50 agar:K-carrageenan ratio.

[0106] DETAILED DESCRIPTION OF THE INVENTION

[0107] Certain terms and expressions used in the context of the invention are detailed below.

[0108] A first subject of the invention relates to a culture medium for the detection of microorganisms comprising

[0109] - T agar or T agarose

[0110] - K-carrageenan, the weight ratio [agar, K-carrageenan]: [other gelling agent] being greater than 70:30.

[0111] The culture medium according to the invention further comprises a peptone as a source of amino acids and nitrogen.

[0112] The term "culture medium" means a medium comprising all the elements necessary for the growth of microorganisms. Generally speaking, the agar culture medium comprises:

[0113] - a source of carbon generally provided by sugars

[0114] - a source of amino acids and nitrogen provided by peptones

[0115] - different salts and buffer

[0116] - demineralized water

[0117] - a gelling agent

[0118] Other ingredients may be added, such as selective agents. The medium may also include a colorant.

[0119] According to the present invention, the culture medium may be in the form of a ready-to-use gel, i.e. ready for inoculation in a Petri dish. This is also referred to as a gelled culture medium, or a medium in solid form. The culture medium may also be in powder form. In this case, it is then rehydrated, heated / sterilized and then poured into a Petri dish to form a ready-to-use gelled medium. Agar is the traditional gelling agent used in microbiology for the cultivation of microorganisms, but it is also possible to use other gelling agents such as gellan gum, pectin, guar gum, gelatin, as well as other natural or artificial gelling agents.

[0120] Thus, according to the present invention, the weight ratio [agar, K-carrageenan]: [other gelling agent] is greater than 70:30. Preferably the weight ratio [agar, K-carrageenan]: [other gelling agent] is greater than 80:20, even more preferably greater than 90:10, even more preferably greater than 95:5.

[0121] The ratio [agar, K-carrageenan]: [other gelling agent] can be 100:0.

[0122] Agar is an unbranched polysaccharide obtained from the cell walls of certain species of red algae, mainly tengusa (Gelidiaceae) and ogonori (Gracilaria). Agar provides a sufficiently stable network conducive to bacterial proliferation without serving as food for them, preserving the matrix structure of the gelling agent. A number of preparations are commercially available, such as Columbia agar, Trypcase-soy agar, Mac Conkey agar, Mueller Hinton agar or more generally those described in the Handbook of Microbiological Media. According to the present invention, the culture medium may comprise agarose which is a polysaccharide extracted from agar, composed of the repetition of a diholoside. Agarose is the gelling part of agar.

[0123] According to the present invention, the culture medium comprises K-carrageenan. Carrageenans are anionic polysaccharides extracted from red algae mainly from Gigarina, Chondrus and Eucheuma. They exist in several forms and the main commercial classes are:

[0124] - The K-carrageenan form, a rigid gel in the presence of monovalent ions such as potassium, sodium, rubidium, lithium, etc. or divalent ions such as calcium or magnesium. It comes mainly from the algae kappaphycus alvarezii

[0125] - The r-carrageenan form, soft gel in the presence of calcium ions. It comes mainly from the seaweed Eucheuma denticulatum.

[0126] - The X-carrageenan form which does not form a gel.

[0127] Surprisingly, it was found that a culture medium comprising a mixture of agar and K-carrageenan improved the shrinkage resistance of the medium. Indeed, culture media, in their routine use, will be incubated at temperatures between 25 and 45°C for periods of up to 28 days in some cases. For pharmaceutical environmental control applications, agar media are exposed for several hours to dehydrating laminar air flows. In all these cases of use, a strong dehydration of the culture media occurs and will lead to destabilization of the agar network and the appearance of tensions inside the gel. For culture media with agar, this phenomenon can lead to the appearance of shrinkage, cracks and / or detachments. These defects have a direct impact on the microbiological performance of the media.Technical solutions that limit their appearance despite dehydration represent a significant advantage in the quality of the results provided by these products. Thus, greater resistance to shrinkage of the agars allows for longer shelf lives.

[0128] Preferably, the concentration of the agar and K-carrageenan mixture is between 10g / l and 30g / l, preferably between 10 and 20g / l. Even more preferably between 13 and 17g / l. The agar and K-carrageenan mixture is the main gelling agent in the culture medium according to the invention. It constitutes the main gelling agent. Thus, the medium according to the invention comprises a mixture of agar and K-carrageenan at a weight ratio [agar, K-carrageenan]: [other gelling agent] greater than 70:30, even more preferably greater than 80:20, even more preferably greater than 90:10, even more preferably greater than 95:5. The weight ratio [agar, K-carrageenan]: [other gelling agent] may be 100:0.

[0129] Another gelling agent may be added in a minor proportion to the medium according to the invention if the shrinkage resistance of said medium remains greater than that of a medium not comprising any other gelling agent than agar at the same concentration of gelling agents. Examples include gelatin, pectin, guar gum. Thus, a medium according to the invention may comprise 15g / l of agar and K-carrageenan mixture supplemented with 2g / l of guar gum, this medium exhibiting improved shrinkage behavior compared to a medium containing 17g / l of agar alone.

[0130] Advantageously, the weight ratio of agar: K-carrageenan is between 80:20 and 30:70.

[0131] Shrinkage refers to a partial or complete detachment of the culture medium from the side wall of the Petri dish. This then leads to a reduction in surface area. Shrinkage resistance can be measured by the "Extreme Dehydration Test" which involves exposing the dishes under a vertical laminar air flow hood for 60 hours without a lid. The culture media will dehydrate during exposure. The surface area of ​​each dehydrated medium is then measured using a dish reading instrument such as the Scan® 4000. Culture medium with low shrinkage resistance will quickly detach from the side wall of the dish during the test and will continue to shrink until a fraction of the initial surface area is completely dry.On the other hand, a culture medium with good shrinkage resistance will only detach from the side wall of the dish later or never during the test and the surface area of ​​the dried medium will decrease slightly or not at all.

[0132] The agar:K-carrageenan ratio also has an effect on the gelation of the agar. These properties are measured by the gelation temperature, viscosity, and elastic modulus of the agar.

[0133] The gelation temperature is measured using a rheometer. Liquid culture media samples are placed on the rheometer's Peltier plate, preheated to a temperature higher than the sample's gelation temperature. The samples are then cooled to 20°C, with a cooling rate of 3°C / min, a rate similar to that in production. During cooling, an oscillating deformation is imposed on the sample, which allows the study of viscoelastic properties and changes from "liquid" to "solid" phases of the sample. The gelation temperature is defined by a rapid increase in the elastic modulus or viscosity, which represents the beginning of the formation of gelling network and solidification of the medium. The gelation temperature may also depend on the physicochemical conditions of the medium, such as the ion charge or the pH.Some media contain high concentrations of salts (Potassium, Sodium, Calcium), such as some selective media. However, the presence of ions increases the gelation temperature. A gelation temperature between 30 and 45°C is particularly interesting because it allows for easily industrialized production of the medium. Indeed, it will not be necessary to maintain the medium at a high temperature to prevent it from gelling before being poured into the dishes. In addition, in the case of preparation of the medium in the laboratory, a temperature below 60°C prevents burns. Furthermore, such a medium also allows the addition of heat-sensitive compounds such as blood or antibiotics, which are then not damaged by heat.

[0134] Viscosity is measured using a rheometer. Viscosity is the resistance of a fluid to changing shape. It determines the speed of movement of the fluid. The more viscous the liquid, the slower the movement. For the culture medium with an agar: K-carrageenan mix, a viscosity similar to a medium with 100% agar is particularly interesting because it allows the production of the medium without the need to modify the manufacturing process parameters, namely the stirring speed, or the pump pressure for distribution.

[0135] The elastic modulus is an intrinsic quantity of a material, defined by the ratio of a stress to the elastic deformation caused by this stress. For the culture medium, it represents the hardness of the medium. The softer the medium, the lower the elastic modulus. A culture medium with an elastic modulus greater than 5 kPa is required for the medium to be correctly inoculated manually or automatically. It is measured using a rheometer. Liquid culture media samples are placed on the rheometer's Peltier plate preheated to a temperature higher than the sample's gelation temperature. The samples are then cooled to 20°C, with a cooling rate of 3°C / min, a rate similar to that in production.During cooling, an oscillating deformation is imposed on the sample which allows to study the viscoelastic properties and the changes of the "liquid" to "solid" phases of the sample. During cooling, the elastic modulus increases with the gelation of the medium. It then reaches a stable value which signifies the completion of the gelation. This final value corresponds to the elastic modulus of the medium.

[0136] Thus, in a particular embodiment of the invention, the medium has a salt concentration of less than 5 g / l and a weight ratio of agar: K-carrageenan of between 80:20 and 60:40. Media having a salt concentration of less than 5 g / l are well known to those skilled in the art. Examples include Sabouraud Dextrose agar medium. Substituting the gelling agent of a medium with a low ionic charge with a mixture of agar: K-carrageenan having a ratio of between 80:20 and 60:40 makes it possible to obtain a medium having an appropriate elastic modulus, i.e. greater than 5 kPa, facilitating its seeding. It also makes it possible to obtain a gelling temperature of between 30°C and 45°C, and a viscosity similar to an unmodified medium with 100% agar, which facilitates its industrialization.

[0137] This ratio of between 80:20 and 60:40 is also particularly suitable for a medium with a high salt concentration above 20 g / l. Thus, in a particular embodiment of the invention, the medium has a salt concentration greater than 20 g / l and the weight ratio of agar:K-carrageenan is between 80:20 and 60:40. Media having a high salt concentration, i.e. greater than 20 g / l, are well known to those skilled in the art. For example, CHAPMAN medium can be cited. Substituting the gelling agent of a medium with a high salt concentration with a mixture of agar:K-carrageenan having a ratio of between 80:20 and 60:40 makes it possible to have an appropriate elastic modulus, i.e. greater than 5 kPa, facilitating its inoculation. It also has a gelation temperature between 30°C and 45°C, and a viscosity similar to an unmodified medium with 100% agar, facilitating its industrialization.

[0138] In another particular embodiment of the invention, the medium according to the invention has a salt concentration of between 5 g / l and 20 g / l, and a weight ratio of agar: K-carrageenan of between 80:20 and 30:70. Media having a conventional salt concentration, i.e. between 5-20 g / l, are well known to those skilled in the art. Examples include TSA medium. Substituting the gelling agent of a medium having a salt concentration of between 5-20 g / l with a mixture of agar: K-carrageenan having a ratio of 80:20 - 30:70 makes it possible to obtain a medium with an elastic modulus compatible with seeding, i.e. greater than 5 kPa. This ratio also allows to obtain a gelation temperature between 30°C and 45°C, a viscosity similar to an unmodified medium with 100% agar, which facilitates its industrialization. In a preferred embodiment, the culture medium further comprises a thermosensitive compound.The heat-sensitive compound can be an antibiotic and / or blood. Thus, a culture medium according to the invention with a gelling temperature between 30°C and 45°C allows the preservation of heat-sensitive compounds.

[0139] Another subject of the invention relates to a process for obtaining a medium characterized in that it essentially consists of:

[0140] - supercool the compounds in the culture medium in order to mix them

[0141] - cool to a first temperature above the gelation temperature of the culture medium

[0142] - Fill the Petri dishes with this culture medium in solution

[0143] - allow to cool to reach the gelling temperature and allow the formation of a gelled culture medium, the gelling temperature being between 30°C and 45°C. Generally speaking, the compounds in the medium are supercooled to a temperature above 80°C, or even above 100°C to allow their sterilization.

[0144] The compounds are then mixed. The supercooled culture medium is then cooled to a temperature that remains above the gelation temperature of the culture medium. Typically, the temperature is between 50 and 60°C.

[0145] If heat-sensitive compounds are required, they are then added.

[0146] The culture medium is then poured into the Petri dishes. It then reaches its gelation temperature, which is the temperature that characterizes the phase transition from “liquid” to “solid”.

[0147] Another subject of the invention relates to a culture medium comprising a mixture of agar: K-carrageenan having a weight ratio of between 80:20 and 30:70 and a gelling temperature of between 30°C and 45°C, the weight ratio [agar, K-carrageenan]: [other gelling agent] being greater than 70:30,

[0148] This medium then constitutes an intermediate product in the process of obtaining the medium when its temperature reaches the phase transition temperature from “liquid” to “solid”.

[0149] Another subject of the invention relates to a medium according to the invention gelled in a Petri dish. Another subject of the invention relates to a method of in vitro microbiological culture, in which microorganisms likely to be present in a sample are inoculated into or onto a culture medium according to the invention.

[0150] Another subject of the invention relates to a method for detecting a target microorganism in a sample likely to contain it, comprising the following steps:

[0151] - bringing said sample into contact with a gelled culture medium according to the invention

[0152] - incubate

[0153] - detect the presence of said microorganism.

[0154] The present invention is illustrated in a non-limiting manner from the following examples

[0155] Examples

[0156] Example 1: Preparation of culture media according to the invention and other culture media Different concentrations of gelling agents (agar, carrageenan) were used: from 0 to 15 g / l.

[0157] Different agar:carrageenan weight ratios were used to make the agar:carrageenan gelling mix:

[0158] - 0:100; 30:70; 40:60; 50:50; 70:30; 20:80; 100:0.

[0159] Different media, called modified media, were prepared from a base of TSA, CHAPMAN or Sabouraud Dextrose Agar medium (tables below) in which the agar was replaced by an agar:carrageenan mix whose ratios are mentioned in the examples. [Table 1] Formula of modified TSA medium

[0160] [Table 2] Formula of modified CHAPMAN medium

[0161] [Table 3] Formula of the modified Sabouraud Dextrose Agar medium

[0162] The preparation follows the following steps: 1) Agars, carrageenans and other raw materials are weighed according to the formula of each culture medium.

[0163] 2) All raw materials are diluted with demineralized water.

[0164] 3) The whole is stirred and heated to boiling in order to completely dissolve the raw materials. 4) The solution is autoclaved in a liquid cycle, with a plateau at 120°C for 16 min.

[0165] Steps 3) and 4) can also be carried out with an automated culture media preparation instrument such as Masterclave®.

[0166] 5) 20-30 ml of medium are poured hot, i.e. at a temperature higher than the gelling temperature of the medium, into 90 mm Petri dishes without lids. 6) the lids of the dishes are replaced.

[0167] 7) The culture media dishes are stored at 2-8°C. Example 2: Shrinkage resistance test of media according to the invention comprising agar from different suppliers

[0168] Different types of agar were tested for the evaluation of shrinkage resistance:

[0169] European Agar (Roko; ref 03904182, Lot 180100340)

[0170] European Agar (Setexam; ref 03904185, Lot M2019)

[0171] American Agar (Roko; ref 3904170, Lot 200201654)

[0172] Commercial agar (Sigma Aldrich, ref 05040 CAS: 9002-18-0, Lot BCCF9819)

[0173] Agar Gracilaria (Roko, ref Rokoagar RGM LAB, Lot 200201758)

[0174] Agarose (Sigma Aldrich, ref A4718 CAS: 9012-36-6, Lot SLCK4183)

[0175] The media tested included either agar alone or mixed with K-carrageenan from the supplier Setexam (Setexam, Reference Danish agar: 03904187, Lot C19152).

[0176] For media with agar alone, the agar concentration is 15g / l.

[0177] For media with agar: K-carrageenan mix, a 50:50 ratio was used, the total gelling agent concentration is 15g / l.

[0178] The TSA formula was used for this test. Culture media were prepared using a Masterclave®. 30 ml of the medium was added to each 90 mm Petri dish.

[0179] The Extreme Dehydration Test method was used to evaluate the shrinkage resistance of each medium in Petri dishes:

[0180] - Thirty boxes of each formula were exposed under a vertical laminar air flow hood (V=0.45m / s) for 60 hours without a lid.

[0181] - The culture medium is completely dehydrated after exposure with total water loss.

[0182] - The appearance of each culture medium was observed post-exposure.

[0183] - The surface area of ​​each dried culture medium was measured using a Scan® 4000 automatic dish reader instrument (ref 438000, supplier Interscience).

[0184] The results were presented as a percentage, calculated relative to the initial surface area of ​​the culture medium.

[0185] [Table 4] Results of the TSA Extreme Dehydration Test method with agar: K-carrageenan mix including different agars.

[0186] Conclusion: As can be seen in Figure 1, all media with 100% agar (media 1a, 2a, 3a, 4a, 5a, 6a) have very poor shrinkage resistance. All agars detach from the wall of the dish and shrink during the test. The use of an agar: K-carrageenan mix (50:50) significantly improves shrinkage resistance (media 1b, 2b, 3b, 4b, 5b, 6b).

[0187] On the other hand, it was observed that the number of intact culture media post-exposure increases. The results are presented in Figure 1. For example, for the European-Roko agar: K-carrageenan mix (medium 4b), 43% of the culture media do not show any shrinkage after the Extreme Dehydration Test. The surface area of ​​the dried culture medium is also increased from 46.5% to 91.9% as shown in Table 4.

[0188] Thus, the improvement in shrinkage resistance was observed on the agar: K-carrageenan mix with all types of agar and agarose tested in this study, as shown in Figure 1, Figure 2 and Table 4.

[0189] Example 3: Shrinkage resistance test of media comprising different carrageenans (kappa, lambda, iota) and culture media according to the invention

[0190] Different types of carrageenan were tested for shrinkage resistance assessment:

[0191] K-carrageenan (Setexam, Reference Danish agar: 03904187, Lot C19152) K-carrageenan (Roko, Reference Rokogel 4600: MV210120, Lot 210200659)

[0192] K-carrageenan (Sigma Aldrich, ref 22048, Lot BCCF0613)

[0193] - r-carrageenan (Sigma Aldrich ref Cl 138, Lot SLCG0678) k-carrageenan (Sigma Aldrich, ref 22049, Lot BCBP8978V) Each carrageenan was tested in mix with European agar (Setexam, ref. 03904185, Lot M2019). An agar:carrageenan ratio = 50:50 was used. The TSA formula was used for this test. The culture media were prepared using a Masterclave®. 30ml of the medium was added to each 90mm Petri dish.

[0194] The Extreme Dehydration Test method as described in Example 2 was used to evaluate the shrinkage resistance of each medium in Petri dishes. The results were presented as a percentage calculated relative to the initial surface area of ​​the culture medium.

[0195] [Table 5] Results of the TSA Extreme Dehydration Test method with agar:carrageenan mix including different carrageenans. As can be seen in Table 5 and Figure 3, the use of agar: K-carrageenan mix improves the shrinkage resistance (media 2, 3, 4). This improvement was observed for the three tested K-carrageenans from different suppliers. For example, for agar: K-carrageenan mix (Setexam), the surface area of ​​the post-exposure culture medium is approximately 91% of the initial size instead of 46% for the medium with agar. For agar: K-carrageenan mix (Sigma Aldrich) and agar: K-carrageenan mix (Roko), the surfaces of the post-exposure agars are also improved.

[0196] However, mixing with α-carrageenan (medium 5) or i-carrageenan (medium 6) does not improve the shrinkage resistance of the medium. Over time, all media shrink and become a small dried film with only 50% residual surface area compared to their initial surface area.

[0197] In conclusion, the improvement in shrinkage resistance was observed in the mixes with all K-carrageenans tested in this study. In contrast, X-carrageenan and i-carrageenan did not improve shrinkage performance.

[0198] Example 4: Shrinkage resistance test of culture media according to the invention with different agar: K-carrageenan ratios

[0199] A series of agar: K-carrageenan ratios were tested for shrinkage strength evaluation (100:0, 70:30, 60:40, 50:50, 30:70, 0:100) with a total gelling agent concentration equal to 15g / l. European agar (Setexam, ref 03904185, Lot M2019) and K-carrageenan (Setexam, ref 03904187, Lot C19152) were used. The TSA formula was used for this test. Culture media were prepared using a Masterclave®. 30ml of the medium was added to each 90mm Petri dish.

[0200] The Extreme Dehydration Test method as described in Example 2 was used to evaluate the shrinkage resistance of these media.

[0201] [Table 6] Results of the TSA Extreme Dehydration Test method with agar: K-carrageenan mix (ratio 100:0, 70:30, 60:40, 50:50, 30:70 and 0:100)

[0202] The medium with 100% agar has poor shrinkage resistance. All media detach from the plate wall and shrink during the test. The surface area of ​​the dried medium is only 46.5% of its initial surface area.

[0203] Using an agar:K-carrageenan mix significantly improves shrinkage resistance compared to agar medium with 100% agar.

[0204] With an agar:K-carrageenan ratio of 70:30, all media retracted during the test. However, the average surface area of ​​the dried medium was 73.9% of the initial surface area, instead of 46.5% for TSA with 100% agar as shown in Table 6.

[0205] With an agar:K-carrageenan ratio of 50:50 (medium 4), 43% of the media show no shrinkage after Extreme Dehydration Test, as shown in Figure 4. The average surface area of ​​the dried medium is 91.8% compared to the initial surface area as shown in Table 6.

[0206] With an agar: K-carrageenan ratio of 30:70 (medium 5), 70% of the media do not show any shrinkage after the Extreme Dehydration Test as shown in Figure 4. The average surface area of ​​the dried medium is increased to 97.4% compared to the initial surface area as shown in Table 6.

[0207] Conclusion: The use of the agar: K-carrageenan mix allows a significant improvement in the shrinkage resistance of the medium. This improvement was observed on different agar: K-carrageenan ratios: 70:30; 60:40; 50:50; 30:70. Example 5: Gelling test of culture media according to the invention with different agar: K-carrageenan ratios

[0208] A series of agar: K-carrageenan ratios were tested (100:0, 80:20, 70:30, 60:40, 50:50, 30:70, 0:100) with a total gelling agent concentration of 15g / l. European agar (Setexam) and K-carrageenan (Setexam or Roko) were used with TSA, CHAPMAN, and Sabouraud Dextrose Agar formulas. The solutions were autoclaved in a liquid cycle, with a plateau at 120°C for 16 min and then placed in a water bath preheated to 80°C. The gelling temperature of each medium was measured using a Discovery HR-2 rheometer (TA Instruments).

[0209] The results obtained with the modified TSA medium are shown in Figure 5 (a) and 5 (b).

[0210] For the TSA medium with agar: K-carrageenan mix, the formulas with ratio 70:30, 50:50 and 30:70 have a gelation temperature between 30°C and 45°C. If the K-carrageenan share is increased by more than 70% (medium 5), the gelation temperature exceeds 45°C as shown in Figure 5 (a). The viscosity of the medium also becomes very important, as shown in Figure 5 (b).

[0211] Increasing the proportion of K-carrageenan beyond 70% of the mix can make industrialization difficult. Changes to the production process are necessary. Furthermore, the production of media with heat-sensitive additives (antibiotics) or blood media (sheep or horse blood agar) also becomes impossible.

[0212] Conclusion: For modified TSA medium or culture media with salt concentrations between 5-20g / l, it is preferable to have a K-carrageenan proportion equal to or less than 70% in the agar:K-carrageenan mix.

[0213] The results obtained with the modified CHAPMAN medium are shown in Figure 6.

[0214] The gelation temperature for a CHAPMAN medium with 100% agar is approximately 40°C (medium 1). For the agar: K-carrageenan mix, media with a ratio of 80:20 to 60:40 (mediums 2, 3, 4) have a gelation temperature similar to that of the CHAPMAN medium.

[0215] Media with a 50:50 ratio or with a K-carrageenan content greater than 50% (media 5 and 6) have a very high gelling temperature (>60°C). This can make industrialization difficult. The production of media with heat-sensitive additives (antibiotics) or blood media (sheep or horse blood agar) also becomes impossible.

[0216] Conclusion: For CHAPMAN medium or other culture media with high ion loads including a salt concentration greater than 20g / l, the use of a ratio of 80:20 to 60:40 is preferred. The presence of ions accelerates the gelation of K-carrageenan and increases the gelation temperature.

[0217] The results obtained on Sabouraud Dextrose Agar (SDA) medium are shown in Figure 7.

[0218] The gelation temperature (Figure 7a) for an SDA medium with 100% agar is approximately 34°C (medium 1). For the agar: K-carrageenan mix, media with a ratio of 80:20 to 50:50 (medium 2 to medium 5) have a gelation temperature between 30°C and 45°C. Media with a ratio of 30:70 or with a K-carrageenan content greater than 70% (mediums 6 and 7) have a very low gelation temperature (<30°C) as shown in Figure 7(a).

[0219] The elastic modulus of the medium (Figure 7b) is also measured by the rheometer. For the agar: K-carrageenan mix, media with a ratio of 80:20 to 60:40 (media 2, 3, 4) have an acceptable elastic modulus (> 5 kPa). Media with a ratio of 50:50 or with a K-carrageenan content greater than 50 (media 5, 6, 7) are very soft. The media are not solidified enough to be handled or inoculated.

[0220] Conclusion: For SDA medium or other culture media with low ion loads having a salt concentration lower than 5g / l, the use of a ratio of 80:20 to 60:40 is preferred.

[0221] Example 6: Detection of microorganisms using culture media according to the invention

[0222] To assess the microbiological performance of the media, microorganism growth tests are performed using the strains listed in the table below. The strains used are from the BioBall® MultiShot 550.

[0223] Two plates of each medium were inoculated for each microorganism and incubated as specified in the table below. After incubation, the plates were counted using a Scan® 4000 automatic plate reading instrument (ref 438000, supplier Interscience). The recovery rate (RR) was calculated. It corresponds to the ratio of the number of colonies on tested plates to the number of colonies on control plates with unmodified culture medium.

[0224] The desired recovery rate is 50 to 200% as recommended by the Pharmacopoeias.

[0225] The TSA formula and the CHAPMAN formula were used for this test. [Table 7] Microorganism tested for TSA medium: [Table 8] Microorganism tested for CHAPMAN medium:

[0226] The results are shown in the table below.

[0227] [Table 9] TSA recovery rate with agar: K-carrageenan mix (ratio 70:30, 50:50, and 30:70) compared to control plates with unmodified TSA medium.

[0228] [Table 10] Recovery rate of CHAPMAN with agar: K-carrageenan mix (ratio 80:20, 50:50, 60:40) compared to unmodified CHAPMAN medium. Conclusion: The media with agar: K-carrageenan mix show good detection and good growth of microorganisms. The recovery rates are similar to the unmodified medium, and well in accordance with the Pharmacopoeia request, i.e. between 50%-200% for all the microorganisms tested. The sizes and morphologies of the colonies of plates 1b to 6b (figure 8) inoculated respectively by the microorganisms mentioned in table 9 on a TSA medium with an agar: K-carrageenan ratio of 70:30, are also very similar with those of the control plates (plates 1a to 6a) comprising a TSA medium with 100% agar. The same applies to Staphylococcus aureus colonies (figure 9) inoculated on a CHAPMAN medium with an agar mix: K-carrageenan 80:20 (box 2) and 50:50 (box 3) versus a CHAPMAN medium with 100% agar (box 1). BIBLIOGRAPHICAL REFERENCES

[0229] Lines, “value of the K+ salt of carrageenan as an agar substitute in Routine bacteriological media”, Applied and Environmental Microbiology, Dec 1977, p 637-639

Claims

CLAIMS 1- Culture medium for the detection of microorganisms including - agar or agarose - K-carrageenan the weight ratio [agar, K-carrageenan]: [other gelling agent] being greater than 70:

30. 2- Culture medium according to claim 1 further comprising peptones. 3- Culture medium according to claim 1 or 2 characterized in that the concentration of the agar and K-carrageenan mixture is between 10g / l and 30g / l, preferably between 10g / l and 20g / l. 4- Culture medium according to any one of the preceding claims, characterized in that the weight ratio of agar: K-carrageenan is between 80:20 and 30:

70. 5- Culture medium according to any one of the preceding claims, characterized in that the concentration of salts present in the medium is less than 5 g / l and the weight ratio of agar: K-carrageenan is between 80:20 and 60:

40. 6- Culture medium according to any one of the preceding claims, characterized in that the concentration of salts present in the medium is between 5g / l and 20g / l and the weight ratio of agar: K-carrageenan is between 80:20 and 30:

70. 7- Culture medium according to any one of the preceding claims, characterized in that the concentration of salts present in the medium is greater than 20 g / l and the weight ratio of agar: K-carrageenan is between 80:20 and 60:

40. 8- Culture medium according to any one of the preceding claims, characterized in that said culture medium further comprises a heat-sensitive compound. Tl 9- Culture medium according to claim 8 characterized in that the heat-sensitive compound is an antibiotic. 10- Culture medium according to any one of claims 8 or 9 characterized in that the heat-sensitive compound is blood. 11- Culture medium according to any one of claims 1 to 10 characterized in that it is gelled in a Petri dish. 12- Method for obtaining a culture medium according to any one of claims 1 to 11, characterized in that it essentially consists of: - supercool the compounds in the culture medium in order to mix them - cool to a first temperature above the gelation temperature of the culture medium - fill the Petri dishes with this culture medium in solution - allow to cool to reach the gelling temperature and allow the formation of a gelled culture medium, the gelling temperature being between 30°C and 45°C. 13- Culture medium comprising a mixture of agar: K-carrageenan having a weight ratio of between 80:20 and 30:70 and a gelling temperature of between 30°C and 45°C, the weight ratio [agar, K-carrageenan]: [other gelling agent] being greater than 70:

30. 14- Method of in vitro microbiological culture, in which microorganisms likely to be present in a sample are inoculated into or onto a culture medium in a Petri dish as described in any one of claims 1 to - Method for detecting a target microorganism in a sample likely to contain it comprising the following steps: - bringing said sample into contact with a gelled culture medium according to any one of claims 1 to 11 - incubating - detect the presence of said microorganism.

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