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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
  • G01N29/22—Details, e.g. general constructional or apparatus details
  • G01N29/24—Probes
  • G01N29/2418—Probes using optoacoustic interaction with the material, e.g. laser radiation, photoacoustics

Definitions

• the present invention relates to a method for detecting and / or identifying living microorganisms by vibrometry.
• vibrometry is understood to mean the study of pressure waves.
• the detection and / or identification of microorganisms living in an environment makes it possible to know its physiological state and its pathophysiological state.
• This analysis is " commonly used in particular in the medical field for the detection of pathogenic states and in the food field for the determination of possible contamination.
• the term "emission of acoustic signals” means all piezo-mechanical phenomena generated by the microorganisms and transmitted within the substrate which propagates these phenomena.
• the acoustic signals are collected not in contact with the microorganisms but within the substrate containing them, for example on the surface thereof.
• Pa such as, for example, piezoelectric sensors, electrodynamic sensors, electrostatic sensors, capacitor sensors, tunnel effect sensors and interferometric sensors.
• the laser type interferometric sensor will be used according to the invention.
• the method of the invention consists of: a) directing a laser beam onto the substrate of the sample to be tested; b) analyzing the spectrum of the light beam reflected by the substrate at a point located at a distance from the microorganisms, advantageously several micrometers. c) possibly to compare this with a reference spectrum.
• the substrate whose albedo is at least 0.02% will be used.
• the substrate albedo is not sufficient, that is to say is less than 0.02%, there is advantageously interposed between the test sample and the laser beam a device acoustically coupled with the microorganisms via the substrate.
• This device is advantageously a sheet of reflective material having a thickness sufficiently small to be able to transmit the vibrations of the substrate.
• a gold leaf having a thickness of between 1 and 5 ⁇ m, preferably 2 ⁇ m, will be used.
• living micro-organisms is meant in the present description and the claims all biological entities of small size, metabolically active, animal or vegetable, simple or complex capable of being born, of developing and normally of reproducing.
• microorganisms By way of examples of microorganisms, mention may especially be made of bacteria, acti ⁇ o ycetes, fungi, yeasts, viruses, phages, mycoplasmas, rickettsiae, eukaryotic cells, prokaryotic cells, fragments of 'DNA and RNA, neonodes and protozoa as well as simple or complex cell groups. 0 It has indeed been surprisingly found that living micro-organisms behave like micro-emitters of vibrations. It seems that everything happens as if the biochemical reactions which take place in microorganisms are at the origin of thermoacoustic phenomena which shake up the cellular structures.
• sample is meant according to the invention any substrate capable of containing microorganisms.
• biological media such as for example blood, urine, sperm and biological fragments or biopsies and food media, such as milk, juices fruit, preserving juices, food, etc.
• the process can be implemented with:
• powder samples In the case of powder samples, they should be placed in an environment conducive to the best acoustic collection, for example in water.
• the laser beam directed onto the test sample is emitted by a conventional heterodyne laser interferometer.
• Optical interferometers work by mixing two beams of light, one of the beams is reflected by the object under examination and the other serves as a phase reference. When the two beams have the same optical frequency, the interferometer is a homody ⁇ e interferometer. On the other hand, when the frequency of the reference beam is different from that of the object, we speak of a heterodyne interferometer.
• a small frequency difference A is entered using two acousto-optic modulators A T enters the optical frequencies S and - ⁇ _p of the two interfering beams.
• the interference between the reference beam ' and the beam reflected by the target produces a modulation of light intensity at the frequency of is detected by a photodetector.
• the length of the optical path and consequently the phase of the object beam is modified by the displacements of the object and directly converted into a phase change in the beat frequency.
• the linearity of a heterodyne interferometer is not limited to small variations in vibration, since variations in optical phases are not converted into variations in intensity as is the case in homodyne interferometers but in variations in signal phase at the beat frequency.
• heterodyne interferometers allow the detection of very small variations.
• the laser interferometers which are suitable for the purposes of the invention advantageously have the following characteristics:
• Heterodyne interferometers have been described in particular by:
• heterodyne interferometers are suitable for measuring the vibrational movements of the order of the ⁇ a ⁇ ometer of microscopic objects, such as the vibrations of the fish eye and of biological systems, such as the eyelashes of the organ of Corti or the giant axon of the dryer.
• the apparatus for implementing the method according to the invention consists of a laser interferometer, of a sample holder consisting of a support which can be moved according to the three . axes and a beam analyzer ⁇ reflected by the sample to be tested.
• FIG. 1 is a schematic representation of the apparatus for implementing the method of the invention
• - Fig. 2 represents the recordings of the spectra emitted by the sample to be tested according to examples 1 and 3 below:
• the interferometer 1 is made up of a He-Ne laser source (l), acousto-optical modulators (3) and (4) to shift the reference beam (RB) of frequency and the beam directed towards the sample to be tested (0B) from a DET photodetector (5) for detecting the frequency beat signal -J. —SI * ), which is analyzed in the analyzer (6).
• the laser beam of frequency- ⁇ is directed onto the substrate, of the sample to be tested (7) in which there is a colony of microorganisms (7a), contained in a tank (8), which is placed on a door -sample (9).
• the sample to be tested (7) when it is solid, can be placed directly on the sample holder (9).
• a sheet of a reflective material is interposed between the laser beam of frequency _ " _.
• the sample for example a gold sheet (10), which remains on the It will be indicated that the placement of the reflective material can be facilitated by using an acoustic coupling material, for example a gel.
• This apparatus may further include a manual or automatic control system (11) which, in the absence of a useful signal corresponding to usable information, controls the movement of the support of the sample along three axes up to obtaining a useful signal showing vibrations.
• a manual or automatic control system (11) which, in the absence of a useful signal corresponding to usable information, controls the movement of the support of the sample along three axes up to obtaining a useful signal showing vibrations.
• the laser beam can be conducted by an optical fiber and that the substrate can be opaque to photons.
• the sample or sheet of reflective material is placed in the path of the laser beam from the interferometer.
• the collimation is carried out by moving the sample support along three axes and the focusing is carried out by moving the objective (12) so that the focal point is located at the interface t.
• the signal detected by the photodetector (5) is analyzed and can be automatically compared with reference signals (for example time-frequency spectra), this is observed on suitable supports, such as paper supports, magnetic supports, supports optics, etc.
• the detected signal differs from that reflected by a stationary mirror located in place of the sample, we are in the presence of living microorganisms.
• the method of the invention therefore makes it possible to detect the presence of a living microorganism in a given sample. It also makes it possible to determine the sensitivity of a living micro-organism to a given substance; for example it will thus be possible to determine the compounds which inhibit or on the contrary promote the growth of microorganisms. Indeed, it has been found that under the effect of a disturbance (for example photostimulation, magnetic or electric stimulation, complex stimulation by electric spark, by IR, X or UV radiation; thermal, chemical or acoustic disturbance, action of a bactericide) the spectrum of the detected signal is different from that emitted by the micro-organism alone.
• a disturbance for example photostimulation, magnetic or electric stimulation, complex stimulation by electric spark, by IR, X or UV radiation; thermal, chemical or acoustic disturbance, action of a bactericide
• the method of the invention is also suitable for studying substances by analyzing the acoustic behavior of microorganisms or mixtures thereof which have been brought into contact with these substances. For example, it is suitable for carrying out antibiograms or studying the fungicidal, herbicidal, insecticidal, mutagenic or toxic properties, etc. of given substances.
• the method of the invention thus makes it possible, by multiplying the sensors at different points of the substrate and by comparing the detected signals, to locate the microorganisms and to follow in their trajectory in real time thanks to the acoustic signals they emit.
• This process can be used for the control of fermentations, sterilizations, the production of biosensors usable in analysis laboratories and for all the studies and the control of phenomena related to the environment of microorganisms.
• EXAMPLE 1 Leaf blade of the celery root APIUM GRAVELENS
• the gold fragment constitutes the reflective interface necessary for the measurement which is thus carried out at a distance of approximately 13 mm from the plant fragment.
• the sample holder made it possible by micro-displacements along three axes to move the tube, sample, medium, reflective interface and objective assembly.
• the focusing and collimation of the optical system were obtained by progressive adjustment of this set with respect to the interferometer.
• Curve 3 spectrum emitted by the sample according to the operating mode defined above, the sample being photostimulated by bringing a 100 W lamp 15 cm from the transparent tube for
• Example 1 The procedure of Example 1 was repeated, using a volume-to-volume mixture of sperm and "Glucose Elbiol 5%" as a sample.
• the emission spectrum of the sperm was recorded by analysis of the signal collected by the photodetector.
• FONGIBACTYL has spermicidal properties.

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• Chemical & Material Sciences (AREA)
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• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Physics & Mathematics (AREA)
• Biochemistry (AREA)
• Analytical Chemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
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• Wood Science & Technology (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
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• Biophysics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Toxicology (AREA)
• Genetics & Genomics (AREA)
• Optics & Photonics (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)
• Investigating Or Analyzing Materials By The Use Of Ultrasonic Waves (AREA)

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• 1989
  • 1989-03-15 FR FR8903403A patent/FR2644476A1/fr not_active Withdrawn
• 1990
  • 1990-03-15 EP EP19900904858 patent/EP0463042A1/fr not_active Withdrawn
  • 1990-03-15 WO PCT/FR1990/000171 patent/WO1990010712A1/fr not_active Application Discontinuation
  • 1990-03-15 JP JP50495490A patent/JPH04504055A/ja active Pending

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