Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/045—Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/06—Quantitative determination
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/06—Quantitative determination
  • C12Q1/08—Quantitative determination using multifield media

Definitions

• TITLE OF THE INVENTION Culture medium for detecting microorganisms comprising a mixture of agar and kappa-carrageenan as a gelling agent.
• the present invention relates to the field of microbiology and more particularly that of industrial and clinical microbiology. More specifically, the present invention relates to a new culture medium for detecting microorganisms comprising a mixture of agar and K-carrageenan as a gelling agent.
• microorganisms are very important in clinical and industrial fields.
• 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 food.
• media are used mainly to carry out environmental controls (air, surface, operator, etc.) and to verify that there is no bacterial contamination in the products in progress Manufacturing.
• the checks 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 homogeneous and reproducible results. Media requirements can be specific to both the sample and the strains to be tested for.
• They can be in the form of liquid, solid or semi-solid medium supplied in boxes, vials, tubes or other containers.
• a major compound of solid or semi-solid culture media is the gelling agent and in particular agar.
• the latter is extracted from red algae from the Rhodophytes family.
• the station has the disadvantage of coming from a limited natural resource.
• 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 culture medium whose agar has been substituted with K-carrageenan is that it begins to solidify at high temperatures. While agar is dispensed at an approximate temperature of 45-55°C, a culture medium based on K-carrageenans requires, to be dispensed in boxes, to be at a temperature above 60°C.
• the gelling agent is also the key compound for improving the lifespan of culture media.
• agar-based culture media although widely used, have disadvantages such as syneresis (leakage of water from the gel network) and retraction which it is desirable to avoid.
• document WO 2004050675 describes the use of a mixture of agar and iota-carrageenan in an agar culture medium. This mixture limits the syneresis of the gel, thus improving its stability and lifespan.
• An objective of the present invention is to provide a culture medium resistant to dehydration through its capacity not to shrink, or very weakly.
• Another objective of the invention is to provide a culture medium having a gelling temperature compatible with industrialization or the use of thermosensitive compounds.
• the weight ratio of agar: K-carrageenan in the culture medium is between 80:20 and 30:70.
• the weight ratio of agar: K-carrageenan is between 80:20 and 60:40.
• the concentration of salts present in the medium is between 5 g/1 and 20 g/1
• the weight ratio of agar: K-carrageenan is between 80: 20 and 30 : 70.
• the concentration of salt present in the medium is greater than 20 g/1 and the weight ratio of agar: K-carrageenan is between 80:20 and 60:40.
• the culture medium further comprises a heat-sensitive compound, such as an antibiotic and/or blood.
• a heat-sensitive compound such as an antibiotic and/or blood.
• the culture medium is gelled in a Petri dish.
• the gelling temperature being between 30°C and 45°C.
• 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 gelation temperature of between 30°C and 45°C.
• Another object of the invention relates to an in vitro microbiological culture method, in which microorganisms likely to be present in a sample are seeded in or on a culture medium according to the invention.
• Another object of the invention relates to a method for detecting a target microorganism in a sample likely to contain it comprising the following steps:
• Figure 1 is a diagram representing the percentage of intact (non-retracted) culture medium after the Extreme Dehydration Test.
• Series “a” media include 100% agar (or agarose for medium 1).
• the “b” series media include an agar:K-carrageenan ratio of 50:50 (or an agarose:K-carrageenan ratio of 50:50 for medium 1).
• Middle la and middle lb Agarose from Sigma Aldrich
• Medium 2a and medium 2b Sigma Aldrich agar
• Medium 3a and medium 3b American Roko agar
• Medium 4a and medium 4b European agar from Roko
• Medium 5a and medium 5b European agar from Setexam
• Figure 2 shows photos of the culture media after the Extreme Dehydration Test.
• Series “a” media include 100% agar (or agarose for medium 1).
• the “b” series media include an agar:K-carrageenan ratio of 50:50 (or an agarose:K-carrageenan ratio of 50:50 for medium 1). Origin of T agar (or agarose for medium 1):
• Middle la and middle lb Agarose from Sigma Aldrich
• Medium 2a and medium 2b Sigma Aldrich agar
• Medium 3a and medium 3b American Roko agar
• Medium 4a and medium 4b European agar from Roko Medium 5a and medium 5b: European agar from Setexam
• Figure 3 shows photos of the agar mix culture media with different carrageenans after the Extreme Dehydration Test.
• Medium 1 includes 100% European agar (Setexam)
• Medium 2 includes an agar: K-carrageenan (sigma Aldrich) ratio 50:50
• Medium 3 includes an agar: K-carrageenan (Setexam) ratio 50:50
• Medium 4 includes an agar: K-carrageenan (Roko) ratio 50:50
• Medium 5 includes an agar: k-carrageenan ratio (Sigma Aldrich) 50:50
• Medium 6 includes a ratio of agar: i-carrageenan (Sigma Aldrich) 50:50.
• Figure 4 represents the percentage of non-retracted culture media for different agar: K-carrageenan ratios after the extreme dehydration test.
• Medium 1 includes 100% European agar
• Medium 2 includes an agar: K-carrageenan ratio 70:30
• Medium 3 includes an agar: K-carrageenan ratio 60:40
• Medium 4 includes an agar:K-carrageenan ratio 50:50
• Medium 5 includes an agar: K-carrageenan ratio 30:70
• Medium 6 includes an agar: K-carrageenan ratio 0:100
• Figure 5(a) shows the gelation temperature of TSA media modified with different agar:K-carrageenan ratios.
• Medium 1 includes 100% agar
• Medium 2 includes an agar: K-carrageenan ratio 70:30
• Medium 3 includes an agar: K-carrageenan ratio 50:50
• Medium 4 includes an agar: K-carrageenan ratio 30:70
• Medium 5 includes an agar: K-carrageenan ratio 0:100
• Figure 5(b) shows the viscosity of TSA media modified with different agar:K-carrageenan ratios.
• Medium 1 includes 100% agar
• Medium 2 includes an agar: K-carrageenan ratio 70:30
• Medium 3 includes an agar: K-carrageenan ratio 50:50
• Medium 4 includes an agar: K-carrageenan ratio 30:70
• Medium 5 includes an agar: K-carrageenan ratio 0:100
• Figure 6 represents the gelation temperature of CHAPMAN media modified with different agar: K-carrageenan ratios.
• Medium 1 includes 100% agar
• Medium 2 includes an agar: K-carrageenan ratio 80:20
• Medium 3 includes an agar: K-carrageenan ratio 70:30
• Medium 4 includes an agar: K-carrageenan ratio 60:40
• Medium 5 includes an agar:K-carrageenan ratio 50:50
• Medium 6 includes an agar: K-carrageenan ratio 0:100
• Figure 7(a) represents the gelation temperature of modified SDA media with different agar:K-carrageenan ratios.
• Medium 1 includes 100% agar
• Medium 2 includes an agar: K-carrageenan ratio 80:20
• Medium 3 includes an agar: K-carrageenan ratio 70:30
• Medium 4 includes an agar: K-carrageenan ratio 60:40
• Medium 5 includes an agar:K-carrageenan ratio 50:50
• Medium 6 includes an agar: K-carrageenan ratio 30:70
• Medium 7 includes an agar: K-carrageenan ratio 0:100
• Figure 7 (b) represents the elastic modulus of modified SDA media with different agar:K-carrageenan ratios.
• Medium 1 includes 100% agar
• Medium 2 includes an agar: K-carrageenan ratio 80:20
• Medium 3 includes an agar: K-carrageenan ratio 70:30
• Medium 4 includes an agar: K-carrageenan ratio 60:40
• Medium 5 includes an agar:K-carrageenan ratio 50:50
• Medium 6 includes an agar: K-carrageenan ratio 30:70
• Medium 7 includes an agar: K-carrageenan ratio 0:100
• Figure 8 corresponds to photos after incubation of culture media inoculated with different microorganisms.
• 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 70:30
• Figure 9 corresponds to photos of CHAPMAN culture media modified after incubation inoculated with Staphylococcus aureus.
• Medium 1 corresponds to a medium with 100% agar.
• Medium 2 comprises an agar: K-carrageenan ratio of 80:20.
• Medium 3 includes a ratio of agar: K-carrageenan 50:50.
• a first object of the invention relates to a culture medium for the detection of microorganisms comprising
• the culture medium according to the invention further comprises a peptone as a source of amino acids and nitrogen.
• culture medium we mean a medium comprising all the elements necessary for the growth of microorganisms. Generally speaking, the agar culture medium understand:
• the medium may also include a dye.
• the culture medium can be in the form of a ready-to-use gel, that is to say ready for seeding in a Petri dish.
• a ready-to-use gel that is to say ready for seeding in a Petri dish.
• the culture medium can also be in powder form. In this case, it is then rehydrated, heated/sterilized then poured into a Petri dish to form a gelled medium ready for use.
• 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.
• the weight ratio [agar, K-carrageenan]: [other gelling agent] is greater than 70: 30.
• 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.
• the ratio [agar, K-carrageenan]: [other gelling agent] can be 100: 0.
• Agar is an unbranched polysaccharide obtained from the cell walls of certain species of red algae, mainly tengusa (Gelidiaceae) and ogonori (Gracilaria).
• Agar makes it possible to have a network that is sufficiently stable and conducive to bacterial proliferation without serving as food for them, preserving the matrix structure of the gelling agent.
• a certain 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.
• the culture medium may comprise agarose which is a polysaccharide extracted from agar, composed of the repetition of a disaccharide. Agarose is the gelling part of agar.
• the culture medium comprises K-carrageenan.
• Carrageenans are anionic polysaccharides extracted from red algae, mainly Gigarina, Chondrus and Eucheuma. They exist in several forms and the main commercial classes are:
• the K-carrageenan form rigid gel in the presence of monovalent ions such as potassium, sodium, rubidium, lithium, etc. or divalent ions like calcium or magnesium. It comes mainly from the algae kappaphycus alvarezii
• the r-carrageenan form soft gel in the presence of calcium ions. It mainly comes from Eucheuma denticulatum algae.
• a culture medium comprising a mixture of agar and K-carrageenan made it possible to improve the resistance to retraction of the medium.
• the culture media in their routine use, will be incubated at temperatures between 25 and 45°C for periods which can in some cases go up to 28 days.
• 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 a 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, cracking and/or detachment.
• 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 17 g/1.
• the agar and K-carrageenan mixture is the majority gelling agent in the culture medium according to the invention. It constitutes the main gelling agent.
• 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] can be 100:0.
• Another gelling agent can be added in a minority proportion to the medium according to the invention if the resistance to retraction of said medium remains greater than that of a medium comprising no other gelling agent than agar at the same concentration of gelling agents.
• examples include gelatin, pectin and guar gum.
• a medium according to the invention can comprise 15g/l of a mixture of agar and K-carrageenan supplemented with 2 g/l of guar gum, this medium exhibiting improved behavior with respect to retraction compared to a medium containing 17g/l of agar alone.
• the weight ratio of agar: K-carrageenan is between 80:20 and 30:70.
• retraction we mean a partial or total separation 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 consists of exposing the boxes under a vertical laminar air flow hood for 60 hours without a cover. The culture media will dehydrate during exposure. The surface area of each dehydrated medium is then measured using a box reading instrument such as the Scan® 4000. A culture medium with low resistance to shrinkage will quickly detach from the side wall of the box during the test and will continue to shrink until a fraction of the initial surface is completely dry. On the other hand, a culture medium with good resistance to retraction will only come off the side wall of the box later or never during the test and the surface area of the dried medium will decrease slightly or not at all.
• the agar:K-carrageenan ratio also has an effect on agar gelation. These properties are measured by the gelation temperature, viscosity and elastic modulus of the agar.
• the gelation temperature is measured using a rheometer.
• the liquid culture media samples are placed on the Peltier plate of the rheometer preheated to a temperature higher than the gelling temperature of the sample.
• the samples are then cooled to 20°C, with a cooling rate of 3°C/min, a speed similar to that in production.
• an oscillating deformation is imposed on the sample which allows the viscoelastic properties and changes from “liquid” to “solid” phases of the sample to be studied.
• the gelling temperature is defined by a rapid increase in elastic modulus or viscosity which represents the beginning of the formation of gelling agent networks and solidification of the medium.
• the gelation temperature can also depend on the physicochemical conditions of the medium such as the ion load or the pH.
• Certain media include high concentrations of salts (Potassium, Sodium, Calcium) such as certain selective media.
• salts Potassium, Sodium, Calcium
• a gelling temperature of between 30 and 45°C is particularly interesting because it allows production of the medium which can be easily industrialized. In fact, it will not be necessary to maintain the medium at a high temperature to prevent it from gelling before being poured into the boxes.
• a temperature below 60°C helps avoid burns.
• such a medium also allows the addition of heat-sensitive compounds such as blood or antibiotics which are then not damaged by heat.
• 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.
• a viscosity similar to a medium with 100% agar is particularly interesting because it allows production of the medium without the need to modify the parameters of the manufacturing process, namely the stirring speed, or even the pump pressure for distribution.
• 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 properly seeded manually or automatically. It is measured using a rheometer. The liquid culture media samples are placed on the Peltier plate of the rheometer preheated to a temperature higher than the gelling temperature of the sample. The samples are then cooled to 20°C, with a cooling rate of 3°C/min, a speed similar to that in production.
• the medium has a salt concentration of less than 5 g/1 and a weight ratio of agar: K-carrageenan of between 80:20 and 60:40.
• Media having a salt concentration of salt less than 5g/l are well known to those skilled in the art.
• Substituting the gelling agent of a medium with a low ionic charge by a mixture of agar: K-carrageenan having a ratio 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 between 30°C and 45°C, and a viscosity similar to an unmodified medium with 100% agar, which facilitates its industrialization.
• the medium has a salt concentration greater than 20 g/1 and the weight ratio of agar: K-carrageenan is between 80:20 and 60:40.
• the media having a high salt concentration, that is to say greater than 20 g/1, are well known to those skilled in the art. We can cite for example the CHAPMAN environment. Substituting the gelling agent of a medium with a high salt concentration with a mixture of agar: K-carrageenan having a ratio of 80:20 and 60:40 makes it possible to have an appropriate elastic modulus, greater than 5 kPa, facilitating its seeding. It also has a gelling temperature between 30°C and 45°C, and a viscosity similar to an unmodified medium with 100% agar, facilitating its industrialization.
• the medium according to the invention has a salt concentration of between 5 g/l and 20 g/1, and a weight ratio of agar: K-carrageenan of between 80: 20 and 30. : 70.
• Media having a conventional salt concentration, that is to say between 5-20 g/1, are well known to those skilled in the art. We can cite for example the TSA environment. Substituting the gelling agent of a medium having a salt concentration of between 5-20 g/1 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.
• the culture medium further comprises a heat-sensitive compound.
• the heat-sensitive compound may be an antibiotic and/or blood.
• a culture medium according to the invention with a gelling temperature of between 30°C and 45°C allows the conservation of heat-sensitive compounds.
• the compounds in the medium are supercooled at a temperature above 80°C, or even above 100°C to allow their sterilization.
• the compounds are then mixed.
• the supercooled culture medium is then cooled to a temperature which remains above the gelation temperature of the culture medium.
• the temperature is between 50 and 60°C.
• the culture medium is then poured into the Petri dishes. It then reaches its gelling temperature which is the temperature which characterizes the phase transition from “liquid” to “solid”.
• 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,
• 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”.
• Another object of the invention relates to a medium according to the invention gelled in a Petri dish.
• Another subject of the invention relates to an in vitro microbiological culture method, in which microorganisms likely to be present in a sample are seeded in or on a culture medium according to the invention.
• Another object of the invention relates to a method for detecting a target microorganism in a sample likely to contain it comprising the following steps:
• 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/1.
• gelling agents agar, carrageenan
• modified media 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.
• modified TSA medium formula
• the preparation follows the following steps: 1) Agars, carrageenans and other raw materials are weighed according to the formula for each culture medium.
• Steps 3) and 4) can also be carried out with an automated instrument for preparing culture media such as Masterclave®.
• Agar Gracilaria (Roko, ref Rokoagar RGM LAB, Lot 200201758)
• the media tested included either agar alone or mixed with K-carrageenan from the supplier Setexam (Setexam, Reference Danish agar: 03904187, Lot C19152).
• the agar concentration is 15g/l.
• 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.
• the Extreme Dehydration Test method was used to evaluate the shrinkage resistance of each Petri dish medium:
• the culture medium is completely dehydrated after exposure with total water loss.
• Example 3 Test of resistance to retraction of media comprising different carrageenans (kappa, lambda, iota) and culture media according to the invention
• K-carrageenan (Setexam, Reference Danish agar: 03904187, Lot C19152) K-carrageenan (Roko, Reference Rokogel 4600: MV210120, Lot 210200659)
• K-carrageenan (Sigma Aldrich, ref 22048, Lot BCCF0613)
• Example 2 The Extreme Dehydration Test method as described in Example 2 was used to evaluate the resistance to retraction of each medium in a Petri dish. The results were presented as a percentage calculated relative to the initial surface area of the culture medium.
• Example 4 Test of resistance to retraction of culture media according to the invention with different agar: K-carrageenan ratios
• a series of agar:K-carrageenan ratio was tested for evaluation of shrinkage resistance (100:0, 70:30, 60:40, 50:50, 30:70, 0:100) with concentration total gelling agent equal to 15g/l.
• European agar Setexam, ref 03904185, Lot M2019
• K-carrageenan Setexam, ref 03904187, Lot C19152
• 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.
• Example 2 The Extreme Dehydration Test method as described in Example 2 was used to evaluate the resistance to retraction of these media.
• the medium with 100% agar has poor resistance to retraction. All media will peel away from the side of the box and shrink during testing.
• the surface area of the dried medium is only 46.5% compared to its initial surface area.
• K-carrageenan mix significantly improves the resistance to retraction compared to the agar medium with 100% agar.
• K-carrageenan ratio was tested (100: 0, 80: 20, 70: 30, 60: 40, 50: 50, 30: 70, 0: 100) with a total concentration of the gelling agent equal to 15g /L.
• European agar (Setexam) and K-carrageenan (Setexam or Roko) were used with the TSA, CHAPMAN, and Sabouraud Dextrose Agar formulas.
• the solutions were autoclaved in a liquid cycle, with a tray at 120°C for 16 min then placed in a water bath preheated to 80°C.
• the gelation temperature of each medium was measured using a Discovery HR-2 rheometer (TA Instruments).
• the formulas with ratio 70:30, 50:50 and 30:70 have a gelling temperature between 30°C and 45°C. If we increase the share of K-carrageenan 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).
• K-carrageenan share equal to or less than 70% in the agar mix: K- carrageenan.
• the gelling temperature for CHAPMAN medium with 100% agar is around 40°C (medium 1).
• the media with a ratio of 80:20 to 60:40 (media 2, 3, 4) have a gelling temperature similar to that of the CHAPMAN medium.
• Media with a 50:50 ratio or with a K-carrageenan content greater than 50% have a very high gelation temperature (>60°C). This can make difficult industrialization.
• the manufacture of media with heat-sensitive additives (antibiotics) or blood media (sheep or horse blood agar) also becomes impossible.
• the gelation temperature (figure 7a) for an SDA medium with 100% agar is around 34°C (medium 1).
• the media with a ratio of 80:20 to 50:50 (medium 2 to medium 5) have a gelling temperature between 30°C - 45°C.
• Media with a 30:70 ratio or with a K-carrageenan content greater than 70% (medium 6 and 7) have a very low gelling temperature ( ⁇ 30°C) as indicated in Figure 7(a).
• the elastic modulus of the medium ( Figure 7b) is also measured by the rheometer.
• the media with a ratio of 80:20 to 60:40 (media 2, 3, 4) have an acceptable elastic modulus (> 5 kPa).
• Media with a 50:50 ratio 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.
• Example 6 Detection of microorganisms using culture media according to the invention
• microorganism growth tests are carried out with the strains listed in the table below.
• the strains used come from the BioBall® MultiShot 550.
• TR recovery rate
• the desired recovery rate is 50 to 200% as recommended by the Pharmacopoeias.

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  • 2022-11-10 EP EP22206665.6A patent/EP4368723A1/de not_active Withdrawn
• 2023
  • 2023-11-07 JP JP2025526267A patent/JP2025540916A/ja active Pending
  • 2023-11-07 AU AU2023376570A patent/AU2023376570A1/en active Pending
  • 2023-11-07 KR KR1020257019009A patent/KR20250105447A/ko active Pending
  • 2023-11-07 CN CN202380077820.8A patent/CN120187862A/zh active Pending
  • 2023-11-07 WO PCT/EP2023/081050 patent/WO2024100070A1/fr not_active Ceased
  • 2023-11-07 EP EP23828972.2A patent/EP4615992A1/de active Pending

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