Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
• • 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
  • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles
  • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction

Definitions

• the present invention relates to the isolation of microorganisms.
• biofilms Many microorganisms develop by synthesizing a biofilm. In addition to bacteria, fungi, algae and protozoa are also organized in biofilms.
• Biofilms are therefore ubiquitous in many areas for which they pose health risks and sometimes cause relatively significant damage.
• Conditioning of the surface The organic or mineral molecules present in the liquid phase will adsorb on the surface, to form a "conditioning film”.
• Adhesion or reversible adhesion The microorganisms present approach the surfaces by gravimetry, brownian movements or by chemotaxis if they possess flagella. During this first fixing step, involving only purely physical phenomena and weak physicochemical interactions, the microorganisms can still be easily unhooked.
• - Adhesion This slower stage involves interactions of higher energy as well as microbial metabolism and cellular appendages of the microorganism (flagella, pilis, .7) • Adhesion is an active and specific phenomenon.
• the first colonizers will attach irreversibly to the surface thanks to the synthesis of exopolysaccharides (EPS). This process is relatively slow and depends on environmental factors and microorganisms.
• EPS exopolysaccharides
• the maturation of the biofilm (development and colonization of the surface): After adhering to a surface, the bacteria multiply and regroup to form microcolonies surrounded by polymers. This matrix of polymers (or glycocalyx) will act as a "cement” and strengthen the association of bacteria with each other and with the surface to finally form a biofilm and reach a state of equilibrium.
• the biofilm generally develops into a three-dimensional structure that constitutes a place of confinement. This microenvironment will be the site of many physiological and molecular changes with respect to the planktonic growth pattern.
• the biofilm thus formed will occupy all the surface that is offered to him if the conditions allow it.
• the maturation of the biofilm is correlated with the production of EPS even if certain species of microorganisms that do not synthesize or few polymers can also adhere and form biofilms on surfaces.
• Biofilms are structures in perpetual dynamic equilibrium that evolve according to the support, the microorganisms and the environment. This evolution can be reflected by cell or aggregate detachments.
• the adhesion and fixation property of the microorganisms is used to allow the isolation thereof.
• the principle of the invention is to add in a crude sample, more or less liquid, a medium which we want to study the contamination by a microorganism, particles, particularly beads, magnetic or magnetizable, to leave the beads in contact with the medium for a time sufficient for the adhesion of microorganisms to the surface of the particles, to isolate said particles from the medium by any appropriate means, particularly with the aid of a magnet and to spread said particles on a solid culture medium suitable for culturing said microorganisms entrapped with the particles.
• the subject of the invention is a process for isolating at least one microorganism from a medium containing them, comprising the following steps:
• the method may comprise a preliminary step of pre-culturing the sampling of said medium containing the microorganism that is to be isolated.
• the sample is brought to a temperature compatible with the viability of the microorganisms.
• living microorganisms exist under extreme conditions in terms of temperature, partial pressure of gas (oxygen, nitrogen, carbon dioxide, etc.), salinity, pH (acid, basic) under oxido-reduction conditions and / or under aerobic or anaerobic conditions. From then on, the skilled person will adapt the culture temperature to the requirements of the organism that he cultivates.
• culturing temperatures may be from 20 to 50 degrees Celsius, preferably from 30 to 40 degrees Celsius.
• This preculture step which aims to enrich the culture medium with microorganisms can be carried out for a very variable time depending on the microorganisms, which can be between 20 minutes and 7 to 10 days, preferably between 1 and 48 hours, possibly with stirring.
• the method may comprise an additional step interposed between steps c) and d) of the process according to the invention, consisting of the optional immersion of the beads obtained in step c ) in an advantageously aqueous rinsing solution for removing the previous medium and for removing non-adherent microorganisms (present in the ball imbibition liquid).
• This step makes it possible to select the most adherent microorganisms whose adhesion is the most irreversible.
• This step can also be the occasion of rapid or prolonged treatments for testing the adhesion properties of microorganisms (preventive or curative treatments in the rinsing solution).
• the skilled person can easily determine the amount of beads that he introduced into the environment.
• step b) the incubation of step b) continues for a time that can be between a few seconds and a few hours, preferably between 15 seconds and 45 minutes depending on the microorganisms.
• the microorganisms could adhere to the particles (beads).
• the separation of the particles and the medium in step c) can be carried out according to any method known to those skilled in the art.
• these particles could be removed by centrifugation and elimination of the culture medium, or alternatively, and preferably according to the invention, by using a system generating a magnetic or electrical field capable of attracting particles, particularly a magnet.
• the particles are removed by means of a magnet, which is advantageously immersed in the sample.
• the system generating a magnetic or electrical field capable of attracting particles, particularly a magnet may be protected by any system, particularly a removable cover or cover, made of any material, for example plastic , which does not interfere with magnetic or electric waves. Even more advantageously, said hood is disposable after use. We could then reuse this magnet.
• the method may comprise an additional step of washing the system generating a magnetic or electric field in order to eliminate the non-adherent microorganisms present in the wetting liquid, or the weakly adherent microorganisms.
• said system is immersed in a wash solution which may, for example, be sterile culture medium.
• a wash solution which may, for example, be sterile culture medium.
• the spreading of said particles on a support compatible with the development of the microorganisms can be carried out by depositing said particles on the surface of a microorganism culture device, for example a Petri dish containing a suitable culture medium. to the development of microorganisms.
• a microorganism culture device for example a Petri dish containing a suitable culture medium.
• the deposit can be made by removing the magnet from the plastic cover while bringing the plastic cover closer to the surface of the microorganism culture device.
• the beads can be deposited using another magnet placed under the surface of the culture device.
• any system known to those skilled in the art such as for example a manual spreader, may be used.
• a rotating magnet placed under the surface of the microorganism culture device is used.
• the dispersion of the beads can also be obtained by a liquid vortex generated by the rotation of the microorganism culture device.
• the culture device is placed in an incubator for a time sufficient for the development of microorganisms on the surface of the device.
• a person skilled in the art will adapt the incubation time and the temperature to the microorganism that he wishes to isolate. This time can be between a few hours and several days, preferably between 4 hours and 48 hours.
• the incubation temperature may be between 30 and 40 degrees Celsius.
• Figure 1 represents a comparison of traditional methods of sampling microorganisms (A) and the method according to the invention (B);
• Figure 2 shows the steps of sampling and rinsing particles
• Figure 3 shows the deposition of particles on the surface of a Petri dish
• Figure 4 shows the dispersion of the particles on the surface of the petri dish using a rotating magnet.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Analytical Chemistry (AREA)
• Organic Chemistry (AREA)
• Physics & Mathematics (AREA)
• Pathology (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Medicinal Chemistry (AREA)
• Cell Biology (AREA)
• Food Science & Technology (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Biophysics (AREA)
• Virology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Genetics & Genomics (AREA)
• General Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
• Coating Of Shaped Articles Made Of Macromolecular Substances (AREA)

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Publications (1)

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• 2005
  • 2005-03-15 FR FR0502553A patent/FR2883296B1/fr not_active Expired - Fee Related
• 2006
  • 2006-03-15 MX MX2007011380A patent/MX2007011380A/es active IP Right Grant
  • 2006-03-15 AU AU2006224472A patent/AU2006224472A1/en not_active Abandoned
  • 2006-03-15 RU RU2007137792/13A patent/RU2007137792A/ru not_active Application Discontinuation
  • 2006-03-15 EP EP06743578A patent/EP1859281A1/fr not_active Withdrawn
  • 2006-03-15 WO PCT/FR2006/000578 patent/WO2006097631A1/fr active Application Filing
  • 2006-03-15 US US11/886,082 patent/US20080213856A1/en not_active Abandoned
  • 2006-03-15 CN CNA2006800084091A patent/CN101156070A/zh active Pending
  • 2006-03-15 CA CA002601366A patent/CA2601366A1/fr not_active Abandoned
  • 2006-03-15 BR BRPI0609830-4A patent/BRPI0609830A2/pt not_active Application Discontinuation
• 2007
  • 2007-10-10 NO NO20075157A patent/NO20075157L/no not_active Application Discontinuation

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