Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N2021/7769—Measurement method of reaction-produced change in sensor
  • G01N2021/7783—Transmission, loss

Definitions

• the technical field of the invention is the determination of the presence of bacteria in an agar or on the surface of an agar, by optical measurements.
• Observation of bacterial colonies by imaging is a technique that has been known for a long time in the field of microbiology, for monitoring the development of microorganisms or cells.
• a Petri dish it is possible to follow the development of colonies, for example of bacterial colonies, and to count them.
• the shape of the colonies gives information on the type of microorganism.
• by combining the use of different culture media, allowing or not allowing the development of colonies it is possible to identify the type of microorganism forming colonies.
• Agarose-based structured optical fiber describes the use of an optical fiber to characterize a liquid in which the fiber is immersed. The fiber response depends on the refractive index of the liquid.
• This publication also describes the use of an optical fiber in agarose gel, comprising cells. Optical fiber is used as waveguide, for guiding excitation light and fluorescence light from the cells. More precisely, the fiber makes it possible to evaluate the spectral response of the cells as a function of the environment.
• Patent application US20200327306 describes a method allowing early detection of the development of bacteria in an agar, in particular an opaque agar. This is a method conferring a wide field of observation, and which can be implemented from a simple and inexpensive device.
• the inventors propose another approach, suitable for agars having a certain level of transparency, and also making it possible to carry out a rapid determination of the presence of microorganisms in a sample.
• a first object of the invention is a method for determining the presence of microorganisms in an agar component, the agar component being formed from an agar, the agar comprising nutrients favorable to the development of microorganisms, the method comprising:
• agar component extending between a first end and a second end, the agar component being at least partially transparent to an illumination wavelength
• agar component obtained during a), in contact with at least one ambient medium, the ambient medium having a refractive index lower than the refractive index of the agar, such that the agar component is adapted to confine a light, at the illumination wavelength, between the first end and the second end;
• step c) repeating step c) at different times;
• the transmittance of the agar component is greater than 0.1 (10%) between the first end and the second end.
• step e) can comprise a measurement of a decrease in a quantity of light detected at different instants.
• the agar contains a chromogenic agent, capable of inducing a change in color of the agar under the effect of the development of microorganisms;
• step e) comprises detection of a variation of a wavelength of the detected light.
• the ambient medium includes an analysis medium (2), which may include microorganisms;
• the method comprises, following step e), a determination of the presence of microorganisms in the analysis medium.
• the determination of the presence of microorganisms in the medium can in particular be carried out according to the evolution of the light detected at the different instants.
• the agar component is formed from an agar potentially comprising microorganisms
• the method comprises, following step e), a determination of the presence of microorganisms in the agar.
• the determination of the presence of microorganisms in the agar can in particular be carried out according to the evolution of the light detected at the different instants.
• the first end and the second end are two opposite ends of the agar component, the agar component forming a waveguide between the first end and the second end. According to such an embodiment,
• the agar component extends along a direction of extension between the first end and the second end;
• the agar component extends along a diameter or a greater diagonal less than one tenth of the length.
• the surrounding medium contains water
• the agar component is placed on an auxiliary agar
• a layer of water is interposed between the auxiliary agar and the agar component.
• the helper agar may include nutrients such that nutrients may migrate to the agar component through the water layer.
• the waveguide has several second ends
• the waveguide comprises several branches, each branch extending between the same section of the agar component, and each respective second end;
• the section extends between the first end and each branch
• the method comprises, following step e), a determination of the presence of microorganisms in the medium, the first medium and/or in the second medium.
• the agar component extends between a base plane and at least one opposite face
• the agar component is configured to reflect incident light, emitted by the light source, and reaching the base plane along an axis of incidence, forming, after propagation of the incident light through the agar component, a reflected light , the reflected light emanating from the base plane along a reflection axis.
• the method comprises, following step e), a determination of the presence of microorganisms in the analysis medium.
• the opposite face can be a hemispherical face.
• the opposite face can comprise three elementary faces, each elementary face extending from the same vertex, the three elementary faces forming a trirectangle trihedron. The axis of reflection is then parallel to the axis of incidence.
• a second object of the invention is a device for determining the presence of microorganisms in an agar component, the device comprising:
• agar component extending between a first end and a second end, the agar component being at least partially transparent to an illumination wavelength
• the agar component being configured to confine light, at the illumination wavelength, between the first end and the second end;
• - a light source configured to illuminate the first end
• a photodetector configured to detect light emanating from the second end after having propagated between the first end and the second end;
• step e) of a method according to the first object of the invention so as to determine the presence of a microorganism in the agar component.
• the agar component may have the characteristics as described in connection with the first object of the invention.
• Figure IA shows an agar component according to a first embodiment.
• Figure IB shows a top view of a device using the agar component shown in Figure IA.
• Figure IC represents a possible implementation of the device described in connection with Figure IB.
• FIG. 1D represents another possible implementation of the device described in connection with FIG. IC.
• Figure 2A shows an example of agar component according to the first embodiment.
• Figure 2B is a photograph of an agar component as described in connection with Figure 2A.
• Figure 3 shows a variation of the intensity transmitted by a rectilinear agar component, respectively in the absence and in the presence of bacteria in the agar component.
• FIG. 4A shows an agar component according to a second embodiment.
• FIG. 4B represents a possible implementation of the agar component described in connection with FIG. 4A.
• Figure 4C shows a variant of the second embodiment.
• the invention exploits a variation in the optical properties of an agar component, under the effect of the development of microorganisms on the surface of the latter or in a volume delimited by the latter.
• the agar component comprises an agar, comprising nutrients conducive to the growth of microorganisms.
• microorganisms is meant, without limitation, bacteria, viruses, fungi, yeasts or microalgae.
• the agar can in particular be formed from a hydrogel.
• the agar can for example comprise agar-agar, according to a mass fraction of between 0.5% and 10% or 0.5% and 20%.
• the agar is formed by adding a gelling agent, for example a cold gelling agent, to an aqueous solution.
• the cold gelling agent is water-soluble. It is configured to gel when it is brought into contact with the aqueous solution, at room temperature, thus forming a hydrogel.
• the cold gelling agent can be chosen from: water-soluble polysaccharide, carboxymethylcellulose, guar gum, gum arabic, gellan gum, xanthan gum, or a gelling agent obtained from animal bones, for example pork, beef, chicken.
• the agar component is at least partially transparent, or considered so, to at least one illumination wavelength.
• transmittance denotes a ratio between a light intensity transmitted by the agar component to a light intensity incident on the agar component, at the illumination wavelength.
• Agar has a refractive index, the latter being generally greater than 1.33 (refractive index of water).
• One aspect of the invention is to place the agar component in contact with an ambient medium, the refractive index of which is lower than the refractive index of the agar. Due to the variation in refractive index at the interface between the agar component and the ambient medium, the agar component allows confinement of a light propagating in the latter.
• the agar component 10 extends between a first end 11 and a second end 12.
• One aspect of the invention is to determine an evolution of the transmittance of the agar component, between the first end 11 and the second end 12, under the effect of growth of microorganisms in the agar component or at the interface between the agar component and the surrounding environment. In a complementary or alternative way, another aspect of the invention is to determine a temporal evolution of the wavelength of a light emanating from the second end.
• FIGS. 1A to 1D A first embodiment of the invention has been shown in FIGS. 1A to 1D, in which the agar component 10 extends along a rectilinear direction of extension D.
• the direction of extension D is parallel to a longitudinal axis Y, between the first end 11 and the second end 12.
• the agar component extends along the direction of extension, between the first end and the second end .
• the length of the agar component depending on the direction of extension, can be between 1 cm and a few tens of cm, for example 5 cm.
• the diameter or the longest diagonal of the component 10 is preferably less than one tenth of the length.
• the agar component extends in an elongated shape between the first end and the second end.
• the agar component extends, parallel to the X axis, and parallel to the Z axis, along a dimension comprised between 5 ⁇ m and 100 ⁇ m.
• the agar component 10 is placed in an ambient medium.
• the ambient medium is formed of air 14 extending around the agar component 10, with the exception of a peripheral part of the component 10, at the level of which the ambient medium is a layer of water 15.
• the ambient medium surrounding the agar component has a refractive index lower than the refractive index of the agar forming the agar component.
• the agar component can then form a light guide, between the first end 11 and the second end 12.
• the agar component 10 is placed in indirect contact with an auxiliary agar 17, the water layer 15 being interposed between the auxiliary agar 17 and the agar component 10.
• the contact is said to be indirect because of the presence of water at the interface between agar component 10 and auxiliary agar 17.
• Auxiliary agar 17 may be the same as or different from the agar from which agar component 10 was formed.
• the agar and the auxiliary agar are hydrogels, the water layer 15 can spontaneously form between the two hydrogels.
• the agar component 10 and the agar auxiliary 17 are prepared separately and then assembled together. For example, agar component 10 is cast onto helper agar 17 after helper agar 17 has solidified.
• agar component 10 and auxiliary agar 17 are solidified separately and then joined together.
• the agar component 10 and the auxiliary agar are not mechanically attached to each other.
• the layer of water 15 is formed at the interface, by capillarity, the water diffusing from each of the hydrogels.
• the auxiliary agar 17 constitutes a reserve of nutrients, the nutrients being able to diffuse, from the latter, towards the agar component 10, through the water layer 15.
• the water layer 15 thus forms an interface between the agar component and the auxiliary agar 17. It is noted that the refractive index of the water layer 15 is lower than that of the agar component 10.
• the presence of a layer of water 15 at all or part of the periphery of the agar component 10 also makes it possible to prevent the agar component from drying out during its use.
• FIG. 1B shows, in top view, a device 1 enabling the invention to be implemented.
• the device comprises a light source 21, placed opposite the first end 11.
• the light source 21 is configured to emit light so as to illuminate the first end 11 in a wavelength illumination at which the agar component is considered to be at least partially transparent.
• the light source 21 can be a laser light source, or a white light source, or a light emitting diode.
• the light source 21 can be associated with an optical fiber, the optical fiber extending between the light source and the first end 11.
• the light source emits light in an illumination spectral band.
• a filter making it possible to adjust the spectral band of illumination, can be arranged between the light source 21 and the first end 11.
• the spectral band of illumination according to which the first end 11 is illuminated, has a bandwidth preferably less than 100 nm. According to one possibility, several spectral illumination bands can be used successively or simultaneously.
• the device 1 comprises a photodetector 22, configured to detect light emanating from the second end 12.
• the photodetector 22 can be a photodiode or a pixelated photodetector, for example a CMOS or CCD type image sensor.
• an incident light beam of incident intensity hn
• the light is confined inside the agar component, the latter forming a waveguide between the first end 11 and the second end 12.
• a light beam transmitted by the agar component, of intensity l or t propagates between the second end 12 and the photodetector 22.
• the photodetector 22 detects the intensity l out of the light transmitted by the agar component 10.
• the device 1 comprises a processing unit 30, connected to the photodetector 22, so as to perform the processing operations described below, in particular a monitoring of the intensity transmitted or a monitoring of a wavelength of the light beam transmitted.
• the processing unit 30 may in particular comprise a microprocessor.
• the agar component is illuminated at different instants, preferably according to the same incident intensity hn.
• the photodetector 22 measures the intensity l out of the beam transmitted by the agar component.
• the transmitted intensity I out decreases, under the effect of diffusion or absorption of light by the microorganisms.
• the evolution, over time, of the transmitted intensity l or t makes it possible to determine the presence of microorganisms developing in the agar component, or at the interface between the agar component and the ambient medium.
• microorganisms can also generate a morphological variation of the agar component 10, for example discontinuities at the level of the interface with the ambient medium. Such a modification of the surface condition can also lead to a gradual decrease in the transmitted intensity l out .
• the agar component perpendicular to the direction of extension D, the better the sensitivity of the measurement. For example, when the height (along Z) and the width (along X) are between 5 and 12 ⁇ m, the agar component behaves like a monomode waveguide. The inventors consider that this configuration is particularly sensitive because the microorganisms develop on the surface and this configuration is more sensitive to the surface state.
• Figure IC illustrates a first mode of implementation, according to which the agar, forming the agar component 10, constitutes an analyzed medium.
• the temporal evolution of the intensity the output detected by the photodetector 22 is representative of a quantity of microorganisms initially present in the agar.
• initially present is meant present upon formation of the agar component 10 from the agar.
• the agar forming the agar component 10 may have been seeded with microorganisms.
• the agar can comprise a known concentration of an agent which inhibits the development of microorganisms. It may be an agent specific to a given species of microorganisms.
• the evolution of the intensity of the light beam transmitted can make it possible to determine a sensitivity of the microorganisms with regard to the inhibiting agent.
• the temporal evolution of the intensity l out detected by the photodetector 22 is then representative of the ability of the microorganisms to develop in the presence of the inhibiting agent.
• FIG. 1D illustrates a second mode of implementation, according to which the agar component 10 is brought into contact with an analyzed medium 2, the latter forming part of the ambient medium, in which the agar component 10 is placed.
• the analyzed medium 2 extends around all or part of the agar component. When the analyzed medium contains microorganisms, the latter can develop at the level of the interface between the analyzed medium 2 and the agar component 10. This results in a diffusion or an absorption of the light propagating through the agar component 10. This results in a gradual decrease in the transmitted intensity l out .
• the analyzed medium 2 can be gaseous or liquid.
• the refractive index of the analyzed medium 2 is lower than the refractive index of the agar forming the agar component.
• the temporal evolution of the intensity I out detected by the photodetector 22 is then representative of a quantity of microorganisms present in the analyzed medium.
• the agar, forming component 10 may comprise a known concentration of an agent which inhibits the development of microorganisms. It may be an agent specific to a given species of microorganisms.
• FIG. 2A represents a variant of the embodiment described above.
• the agar component comprises several branches 13i, 132, each branch extending from the same initial section 13, respectively towards several second ends 12i, 122.
• the agar component comprises two branches, but it can include more.
• the initial section 13 extends, from the first end 11, towards each branch.
• the branches converge towards the same point of the central section.
• the optical paths between the first end 11 and respectively each second end 12i, 122, along each branch are identical.
• component 10 extends along a non-rectilinear extension direction D between first end 11 and each second end 12i, 122.
• the first branch 13i is brought into contact with a first analyzed medium 2i and the second branch 13 2 is brought into contact with a second analyzed medium 2 2 .
• the intensities l out ,i, Lut, 2 respectively from the branches 13i and 132 can be used to compare the developments, on the agar component, of microorganisms present respectively from the first medium 2i and from the second medium 22.
• FIG. 2B represents a photograph of a waveguide forming two branches, as schematized in FIG. 2A.
• a light source 21 fibered, injecting an intensity light at the first end of the agar component.
• This figure illustrates the ability of an agar component to guide light.
• the agar component was TSA agar (Trypticase Soy Agar: trypticase soy medium).
• the section of the waveguide was 1*1 mm 2 .
• Figures 2A and 2B illustrate configurations in which the agar component does not extend straight between the first end 11 and the second end 12.
• the direction of extension may be curved, or have straight portions and curved portions. . It can be in the form of a spiral, or a zig-zag, this type of geometry making it possible to obtain great lengths between the ends 11 and 12.
• FIG 3 shows an experimental run, performed using a straight agar component, as depicted in Figures 1A and 1B.
• the agar component was produced using TSA agar, with a section of 1 mm 2 , and a length of 2 cm.
• the agar component was obtained by molding, in an aluminum mould, a liquid solution to which a gelling agent (agar-agar - Sigma Aldrich 11296) -concentration 15 g/liter - was added.
• a gelling agent agar-agar - Sigma Aldrich 11296) -concentration 15 g/liter - was added.
• the agar contained no bacteria.
• the agar contained bacteria.
• the bacterial species was Escherichia coli, initially present in suspension at 2.10 8 cfu/mL, cfu meaning colony forming unit, in physiological serum.
• the agar of the agar component was inoculated en masse at 40° C. at 1/100 th from suspension, which corresponded to an initial microbial load of 2.10 th cfu/mL in the agar component.
• the agar component was illuminated with a laser type fiber light source (wavelength 546 nm).
• the intensity detected by a photodetector, optically coupled to the second end, was measured.
• the photodetector was a image sensor, arranged in contact with the second end.
• FIG. 3 shows the time course of the intensity detected respectively using agar without bacteria (curve a) and agar containing bacteria (curve b).
• the ordinate axis corresponds to light intensity (gray levels), while the abscissa axis corresponds to time (unit: minute).
• a clear and rapid decrease in light intensity is observed in the presence of bacteria in the agar.
• This figure illustrates an important aspect of the invention: the use of an agar both to allow bacteria to grow, but also to guide light between two ends of the agar component, for analysis purposes.
• Figure 4A shows another embodiment, in which the agar component 10 forms a half-ball.
• the half ball extends between a base plane P and an opposite face F.
• the opposite face F is hemispherical.
• the first end 11 and the second end 12 are coplanar and formed on the base plane P.
• a light source 21 emits an incident light beam of intensity l in towards the first end 11.
• a photodetector 22 detects a transmitted light beam coming from the second end 12.
• the transmitted light beam is a reflected light beam.
• the light beam is reflected by successive internal reflections, on the hemispherical face F.
• the intensity l out reflected by the agar component is attenuated, under the effect of diffusion or absorption light propagating through the agar component.
• FIG. 4B an example of implementation has been shown in which the hemispherical face is placed in an analyzed medium 2, the latter possibly being gaseous or liquid.
• the refractive index of the analyzed medium 2 is lower than the refractive index of the agar forming the agar component.
• the analyzed medium 2 can be water or gas.
• FIG. 4C a variant of this embodiment has been shown, in which the agar component extends between a base plane P and an opposite face F, the latter forming an isosceles trirectangle trihedron.
• An isosceles trirectangle trihedron shape is known in the field of optics.
• the opposite face F has three elementary faces Fi, F 2 , F 3 that are orthogonal, planar, secant at the same point, the latter forming a vertex S.
• Each face forms a right-angled triangle, preferably isosceles, the right angle of which is located at the level of the vertex S.
• the base plane P is perpendicular to a height of the trihedron passing through the vertex S.
• the base plane P forms a base of the trihedron, opposite to the vertex S.
• base of the trihedron we mean a plane s 'extending perpendicularly to a height from the vertex S of the trihedron.
• the agar component behaves like a reflector: when an incident light beam propagates towards the plane of base P, parallel to an axis of incidence, the agar component forms a reflected wave which propagates, from the base plane P, along an axis of reflection parallel to the axis of incidence.
• the transmitted light beam is a reflected light beam.
• the light beam is reflected by successive internal reflections, on the faces Fi, F 2 and F 3 .
• the intensity I out reflected by the agar component is attenuated, under the effect of the diffusion or absorption of the light propagating in the component.
• the reflector represented in FIG. 4C can be used according to the embodiment described in connection with FIG. 4B.
• the agar component can comprise a chromogenic substrate, configured to induce a variation in color of the agar in the presence of microorganisms.
• the substrate can for example lead to a variation in color under the effect of the metabolism of the microorganisms.
• the photodetector 22 is configured to detect a color variation of the light beam emanating from the agar component 10. The greater the quantity of bacteria present in the agar component, or on the surface of the agar component, the more the color change of the light is marked.
• the detection of a development of microorganisms in the component, or on the surface of the latter can be carried out by measuring a change in the wavelength of the light emanating from the agar component.

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• 2020
  • 2020-12-27 FR FR2014164A patent/FR3118465A1/fr active Pending
• 2021
  • 2021-12-24 EP EP21840064.6A patent/EP4267943A1/de active Pending
  • 2021-12-24 WO PCT/EP2021/087643 patent/WO2022136701A1/fr active Application Filing

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