WO1990010712A1

WO1990010712A1 - Method for detection and/or identification of living micro-organisms by vibrometry

Info

Publication number: WO1990010712A1
Application number: PCT/FR1990/000171
Authority: WO
Inventors: Nicolas De Sainte-Agathe
Original assignee: Sainte Agathe Nicolas De
Priority date: 1989-03-15
Filing date: 1990-03-15
Publication date: 1990-09-20
Also published as: JPH04504055A; EP0463042A1; FR2644476A1

Abstract

The invention relates to a method for detecting and/or identifying living micro-organisms contained in a substrate by vibrometry,which consists in sensing the acoustics signals of very low amplitudes emited by micro-organisms within the substrate by means of a ultra-sensitive sensor and eventually comparing them with reference signals. Applications : control of sterilization and fermentation, antibiograms.

Description

Method for detecting and / or identifying living microorganisms by vibrometry.

The present invention relates to a method for detecting and / or identifying living microorganisms by vibrometry.

It relates more precisely to a rapid and automatic method for the remote detection and / or identification of living microorganisms contained in a given substrate by vibrometry. In the present description, 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.

Currently, this type of analysis requires, after sampling, either the cultivation of the 20 microorganisms contained in it and then the identification of said microorganisms, or the carrying out of biochemical assays. The processes commonly used do not allow an immediate result to be obtained, in fact these types of processes include several stages of incubation, washing, etc. These methods of analysis therefore require a great deal of time; moreover they require experienced personnel.

It has also been proposed to detect or identify microorganisms by studying their movements in a given medium by direct optical observation of these movements, for example by optical spectrometry and dopplerometry. To this end, mention may in particular be made of patent EP 179,270 and patents DE 888,593 and

3,039,825.

We have now found a method for rapid detection and / or identification of living microorganisms contained in a substrate by vibrometry, which consists in collecting the signals very low amplitude acoustics emitted by these microorganisms within said substrate, using at least one ultrasensitive sensor and optionally comparing them with reference signals. According to the invention, the term "emission of acoustic signals" means all piezo-mechanical phenomena generated by the microorganisms and transmitted within the substrate which propagates these phenomena.

It will be noted that according to the method of the invention, the acoustic signals are collected not in contact with the microorganisms but within the substrate containing them, for example on the surface thereof.

For the purposes of the invention, all the sensors and transducers which can detect pressures can be used.

_5 weak acoustics, preferably less than about 2 x 10

Pa, such as, for example, piezoelectric sensors, electrodynamic sensors, electrostatic sensors, capacitor sensors, tunnel effect sensors and interferometric sensors. Advantageously, the laser type interferometric sensor will be used according to the invention.

The invention will now be described in detail with reference to this type of sensor without however limiting it to this single embodiment. In this case, 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.

Advantageously, the substrate whose albedo is at least 0.02% will be used. When 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.

Preferably, a gold leaf having a thickness of between 1 and 5 μm, preferably 2 μm, will be used.

By "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.

By way of examples of microorganisms, mention may especially be made of bacteria, actiπo 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. In addition, the acoustic signals emitted by the microorganisms are characteristic of the species considered, of their physiological and physiopathological state, and of the physicochemical conditions which prevail in the environment in which these microorganisms are found. 0 By "sample" is meant according to the invention any substrate capable of containing microorganisms. As examples of samples, mention may be made in particular of all 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:

- samples of solid viscous consistency;

- samples of solid to liquid consistency; - 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 homodyπe 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.

In a heterodyne interferometer, a small frequency difference A is entered using two acousto-optic modulators A T enters the optical frequencies S and -Λ_p of the two interfering beams. Given this difference in frequency, 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.

Thus 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:

1) narrow beam adapted to the microscopic scale, 2) sufficient albedo of the vibrating part,

-11

3) detection range of at least 10 meters,

4) extended spectral width 100 Hz - 300 MHz,

5) extended linearity along the spectrum,

6) sensitivity little disturbed by low frequencies, 7) caloric power adapted to biology.

Heterodyne interferometers have been described in particular by:

- B. CRETIN et al. Optic Communications vol. 65 no.3, 1988, p. 157-162; - J.F. ILLEMIN et al. J. Acoustic. Soc. Am. 83 (2),

1988, p 787-795,

- Sietse M. VAN NETTEN J. Acoustic. Soc. Am. 83 (4), 1988, p. 1667-1674;

- M.A. N0KES, Rev. Sci. Instrum. 49 (6) 1978, p. 723-728. In these articles it is indicated that the heterodyne interferometers are suitable for measuring the vibrational movements of the order of the πaπometer 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.

However, it has never been proposed to use a heterodyne interferometer for the detection of acoustic signals emitted by microorganisms; such use provides considerable advantages over conventional analysis techniques, in particular the speed and the elimination of all the conventional steps.

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.

The invention will now be described in more detail with reference to the appended figures, in which: - 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). Note that the sample to be tested (7), when it is solid, can be placed directly on the sample holder (9). When the albedo of the substrate is not sufficient, a sheet of a reflective material is interposed between the laser beam of frequency _ ^" _. And 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. It will be noted that 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.

If 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.

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.

The invention will now be described by the following nonlimiting illustrative examples: EXAMPLE 1: Leaf blade of the celery root APIUM GRAVELENS

Two fragments of this blade, with a diameter of around 3 mm, taken from a fresh plant, were placed and held in place by a metal clip at the bottom of a 1 ml tube. The tube was placed on the sample holder and then filled with water. On the surface of the liquid, a fragment of gold leaf with a diameter of 2 mm and a thickness of 2 μm was deposited. This fragment of gold was maintained on the surface of the liquid by simple capillary effect.

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.

The next step consisted in analyzing and the spectra of the signal transmitted by the photo-detector of the interferometer to see Figure 2 on which appear the spectra emitted by this sample: (curves 2 to 4) placed under different conditions as well as the background noise of the empty sample carrier interferometer assembly of biological elements (curve 1) ~~ \ the spectra represented by curves 2 and 3 were obtained under the following conditions: Curve 2: spectrum emitted by the sample according to the operating mode defined above, the sample being in the dark.

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

60 seconds.

It is observed by observing curves 2 and 3 that the acoustic emission spectra of the sample placed under different conditions are also different. Various tests have shown that the spectra of curves (2) and (3) were always obtained when the sample was placed either in the dark or when it was photostimulated. EXAMPLE 2.

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.

The introduction into the tube containing the above sample of 3 drops of "F0NGIBACTYL" modifies the recorded spectrum.

In fact, as soon as the measurement is resumed, a gradual extinction of the emission and a return to the background noise of the system is slowly observed (approximately 200 seconds). We conclude that FONGIBACTYL has spermicidal properties.

This example shows that the method of the invention makes it possible to test the action of a substance with respect to a given sample, for example to determine the doses toxic for the microorganism contained in the sample to be tested. EXAMPLE 3

The end of the aerial stem of a young GLYCENE HISPIDA common soybean plant obtained after 10 days of germination was placed in the optical focus directly on the sample holder.

The surface of this plant being sufficiently smooth and reflective the measurement was carried out directly on the sample without the interposition of a gold leaf.

Analysis of the detected signal gave curve 4 (see Figure 2).

Claims

1. Method for rapid detection and / or identification of living microorganisms contained in a substrate by vibrometry, characterized in that it consists in collecting the acoustic signals of very low amplitudes emitted by these microorganisms within said substrate using at least one ultra-sensitive sensor.

2. Method according to claim 1, characterized in that the ultrasensitive sensor is a piezoelectric sensor, an electrodynamic sensor, an electrostatic sensor, a capacitor sensor, a tunnel effect sensor or an interferometric sensor.

3. Method according to one of claims 1 or 2, characterized_en that the sensor _. Ultrasensitive is a laser-type interferometric sensor.

4. Method according to claim 3, characterized in that it consists: a) of 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; c) possibly to compare this with a reference spectrum.

5. Method according to claim 4, characterized in that the albedo of the substrate is at least 0.02%.

6. Method according to claim 4, characterized in that there is interposed between the test sample and the laser beam a sheet of a thin reflective material.

7. Method according to claim 6, characterized in that the sheet of reflective material is a gold leaf.

8. Method according to any one of claims l to 7, characterized in that the microorganisms are subjected to a disturbance during the measurement.

9. Apparatus for implementing the method according to any one of claims 4 to 8, characterized in that it 12

includes a heterodyne laser interferometer, a sample holder consisting of a support that can be moved along the three axes, an analyzer of the beam reflected by the substrate of the sample to be tested, and possibly a sheet of reflective material of small thickness acoustically coupled to microorganisms via the substrate.