# US 2021/0277440 A1 · Method and device for detecting at least one microorganism according to the staining kinetics thereof, and detection support

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- Number: US 2021/0277440 A1
- Document ID: US20210277440A1
- Status: Published application
- Kind: A1
- Application number: 17/279,162
- Filed: 2019-10-04
- Published: 2021-09-09
- Earliest claimed: 2018-10-05
- Assignees: Redberry
- Applicants: Redberry
- Inventors: Joseph PIERQUIN
- Web page: https://patentgrep.com/patent/US20210277440A1

## CPC

- C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)}
- G06T 7/0014: {using an image reference approach}
- H04N 23/56: provided with illuminating means
- G06T 2207/10056: Microscopic image
- G06T 2207/10064: Fluorescence image
- G06T 2207/30024: Cell structures in vitro; Tissue sections in vitro
- G06T 2207/30204: Marker
- G06T 2207/30242: Counting objects in image
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- H04N: PICTORIAL COMMUNICATION, e.g. TELEVISION

## Abstract

A method for detecting, on a detection support, at least one microorganism present in a sample to be analysed and revealed by means of at least one cell marker includes a first taking of an image (I0, I′0) before a step of controlled release of said markerand bringing this marker into contact with said microorganism, and at least one taking of an image (Ii) following this contact step, so as to detect said microorganism according to the change in the staining kinetics thereof, by means of a comparative analysis between the different images that have been taken. An automated detection device is capable of implementing the method and a detection support is capable of being used in the method and which is part of the device.

## Claims

### Claim 1

A method for detecting, on a solid detection support, at least one microorganism, of the bacteria, yeast or mould type, present in a sample to be analysed and revealed by means of at least one cell marker, said method being comprising at least the following steps, in this order:
a) Depositing said sample to be analysed on said solid detection support;
b) Illuminating said detection support at least once with light radiation capable of revealing said at least one cell marker;
c) Acquiring at least one image (I0) in a first phase P0 of said sample on at least one portion of said detection support by means of an optical imaging device targeting a field of view that is at least 10 mm long by at least 10 mm wide;
d) Carrying out a controlled release of said at least one cell marker, through the detection support, to bring said marker into contact with said microorganism;
e) Acquiring, by means of said optical device, an image (Ii) (i≥1) of each portion of the detection support of which an image was acquired at P0 in at least one subsequent phase Pi after the step of the controlled release of said marker so as to detect said at least one microorganism according to the change in the staining kinetics of said microorganism; and
f) Carrying out a comparative analysis, per portion of the detection support, between said image (I0) acquired at P0 and each image (Ii) acquired in at least one subsequent phase Pi and drawing a conclusion as regards the presence or absence of at least one microorganism in said sample.

### Claim 2 (depends on 1)

The detection method according to claim 1, wherein said detection support is illuminated locally, with light radiation every time an image is acquired at P0, Pi.

### Claim 3 (depends on 1)

The detection method according to claim 1, further comprising at least the following steps, in this order:
a1) Depositing said sample to be analysed on said solid detection support;
b1) Illuminating locally said detection support by light radiation capable of revealing said at least one cell marker;
c1) Simultaneously with the illumination step b1), acquiring at least one image (I0, I′0) in a first phase P0 of said sample on at least one portion of said detection support by means of an optical imaging device targeting a field of view that is at least 10 mm long by at least 10 mm wide;
d1) Carrying out a controlled release of said at least one cell marker, through the detection support, to bring said marker into contact with said microorganism;
e1) Within a lapse of time of less than 5 seconds after step d1) of the controlled release of said cell marker, illuminating locally said detection support with said light radiation capable of revealing said at least one cell marker;
f1) Simultaneously with the illumination step e1), acquiring an image (I1, I′1), by means of said optical device, of each portion of the detection support of which an image was acquired at P0 in at least one subsequent phase P1 after the step of the controlled release of said markerl;
g1) Re-illuminating locally, at least once, said detection support with said light radiation;
h1) Simultaneously with the illumination step g1), acquiring an image (I2, I′2), by means of said optical device, of each portion of the detection support of which an image was acquired at P0 and at P1 in at least one second subsequent phase P2 in order to detect said at least one microorganism according to the change in the staining kinetics of said microorganism; and
i1) Carrying out a comparative analysis, per portion of the detection support, between said image (I0, I′0) acquired at P0 and said images (I1, I′1; I2, I′2) acquired in at least two subsequent phases P1 and P2 and drawing a conclusion as regards the presence or absence of at least one microorganism in said sample.

### Claim 4 (depends on 1)

The detection method according to claim 1, wherein the detection support is subjected to n displacements along distinct paths with a view to spatially dividing said detection support into n+1 portions, images whereof are acquired in at least two phases P0 and Pi, where n is an integer less than or equal to 20.

### Claim 5 (depends on 1)

The detection method according to claim 1, wherein an optical imaging device having a resolution at least equal to 20 megapixels, preferably more than 100 megapixels, is used in the image acquisition steps c) and e) or cl), f1) and h1).

### Claim 6 (depends on 1)

The detection method according to claim 1, wherein if it is concluded that at least one microorganism is present in said sample in step f) or i1), an enumeration of the microorganisms present in said sample is carried out.

### Claim 7 (depends on 1)

The detection method according to claim 1, wherein said cell marker is a fluorescent marker.

### Claim 8 (depends on 1)

The detection method according to claim 1, wherein said cell marker released in a controlled manner in step d) or d1) is encapsulated in an encapsulation means.

### Claim 9 (depends on 8)

The detection method according to claim 8, wherein said cell marker is encapsulated in an encapsulation means in solid form, for example in the form of a powder, and wherein said detection support comprises a solvent for dissolving said marker.

### Claim 10 (depends on 8)

The detection method according to claim 8, wherein said cell marker is encapsulated in liquid form.

### Claim 11 (depends on 8)

The detection method according to claim 8, wherein said encapsulation means is heat-sensitive and, in step d) or d1), a controlled release of the cell marker encapsulated in said heat-sensitive encapsulation means is carried out by subjecting the latter to a temperature rise capable of melting said heat-sensitive encapsulation means.

### Claim 12 (depends on 11)

The detection method according to claim 11, wherein the temperature is raised from ambient temperature to a maximum temperature of 45° C.

### Claim 13 (depends on 1)

The detection method according to claim 1, wherein the cell marker in a liquid solution is conveyed, in a controlled manner, to said solid detection support containing the sample to be analysed.

### Claim 14 (depends on 1)

The detection method according to claim 1, wherein a volume comprised between 10 and 2,000 μL of said sample to be analysed is deposited directly on said detection support.

### Claim 15 (depends on 1)

The detection method according to claim 1, wherein said sample to be analysed is filtered through a filtering membrane having a cut-off threshold comprised between 0.2 and 0.45 μm, preferably a cut-off threshold equal to 0.3 μm, said filtering membrane being positioned directly in said detection support.

### Claim 16 (depends on 1)

The detection method according to claim 1, wherein in order to distinguish microorganisms from dust on the detection supports having received the samples to be analysed, the following steps are implemented:
measuring, after each image acquisition, the brightness of the luminescent pixels or clusters of pixels detected;
comparing the brightness of each luminescent pixel or cluster of pixels detected in the different images;
identifying the pixels or clusters of pixels having a brightness that does not substantially change in at least two consecutive images during the staining kinetics; and
analysing and counting the pixels or clusters of pixels having a brightness that changes during the staining kinetics.

### Claim 17 (depends on 1)

A device for detecting at least one microorganism, of the bacteria, yeast or mould type, present in a sample to be analysed, and capable of implementing the method of claim 1, said device comprising at least:
a solid detection support intended to receive said sample to be analysed which is liable to contain at least one microorganism;
a means of storing at least one cell marker of said microorganism;
a means for the controlled release of said cell marker for bringing said marker into contact with said sample to be analysed;
a means of illuminating said detection support;
an optical device for acquiring images of at least one portion of said detection support, said optical device having a field of view that is at least 10 mm long by at least 10 mm wide; and
means for storing in memory, for comparing and for analysing the images acquired by the optical device so as to detect said at least one microorganism potentially present in said sample according to the change in the staining kinetics of said at least one microorganism.

### Claim 18 (depends on 17)

The detection device according to claim 17, further comprising means for displacing said detection support beneath said optical imaging device.

### Claim 19 (depends on 17)

The detection device according to claim 17, wherein said solid detection support includes at least one filtering membrane resting on a glass fibre support disc.

### Claim 20 (depends on 17)

The detection device according to claim 17, wherein said means for storing the cell marker is included in the detection support and consists of a layer of microcapsules formed by said cell marker encapsulated in an encapsulation means, said layer of microcapsules being disposed between said glass fibre support disc and said filtering membrane.

### Claim 21 (depends on 20)

The detection device according to claim 20, wherein said means for the controlled release of the cell marker includes heating means and temperature control and regulation means capable of producing a temperature rise inside the device and of melting said encapsulation means encapsulating the cell marker.

### Claim 22 (depends on 17)

The detection device according to claim 17, wherein said cell marker storage means consists of a reserve of liquid cell marker solution separate from the detection support and in that said detection device comprises means for conveying said cell marker from said storage means to said detection support.

### Claim 23 (depends on 22)

The detection device according to claim 22, wherein said means for the controlled release of the cell marker includes at least one controlled-flow pump.

### Claim 24 (depends on 17)

The detection device according to claim 17, wherein said optical imaging device has a depth of focus in the order of or equal to 0.5 mm.

### Claim 25 (depends on 17)

The detection device according to claim 17, wherein said optical imaging device is based on a CMOS sensor camera.

### Claim 26 (depends on 17)

The detection device according to claim 17, wherein said optical imaging device has a field of view of 25 mm*25 mm.

### Claim 27 (depends on 17)

The detection device according to claim 17, wherein said optical imaging device has a resolution at least equal to 20 megapixels and preferably greater than 100 megapixels.

### Claim 28 (depends on 1)

A detection support capable of being implemented in the detection method according to claim 1, wherein it consists, at least, of a rigid base through which passes at least one liquid feed line and at least one liquid aspiration and discharge line, said feed line being extended by a vent projecting from said base and passing through, on the one hand, a first orifice made in a rigid and permeable support means above said base and, on the other hand, a second orifice made in a flexible support disc made of glass fibres, said disc being positioned on said rigid and permeable support means and supporting a filtering membrane having a cut-off threshold comprised between 0.2 and 0.45 μm, capable of retaining microorganisms, said detection support further comprising a clamping ring fixed such that it is integral with said base and holding said base, said rigid and permeable support means, said support disc and said filtering membrane together.