# US 10,870,876 B2 · Method for detecting a presence or absence of at least one first zone of inhibition

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- Number: US 10,870,876 B2
- Document ID: US10870876B2
- Status: Granted
- Kind: B2
- Application number: 15/743,148
- Filed: 2016-07-06
- Published: 2018-07-26
- Granted: 2020-12-22
- Earliest claimed: 2015-07-09
- Assignees: Biomerieux
- Applicants: BioméRieux
- Inventors: Laurent Drazek; Agnés Dupont-Fillard; Frédéric Pinston; Hervé Rostaing
- Web page: https://patentgrep.com/patent/US10870876B2

## 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)}
- C12M 41/36: {of biomass, e.g. colony counters or by turbidity measurements (electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404)}
- C12Q 1/18: Testing for antimicrobial activity of a material
- C12Q 1/20: using multifield media
- G01N 33/521: {Single-layer analytical elements}
- 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
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

A method for detecting a presence or an absence of at least one zone of inhibition, the method including a step consisting in depositing a volume of the sample in liquid form along a deposition zone extending along an axis at the surface of the agar culture medium and a step consisting in depositing a determined amount of a chemical agent at the surface of the agar culture medium, the deposit defining a potential zone of inhibition, the axis of the zone of deposition of the sample intersecting the potential zone of inhibition.

## Claims

### Claim 1

A method for detecting a presence or an absence of at least one zone of inhibition, the method comprising:
providing a support impregnated with a determined amount of a chemical agent on a surface of an agar culture medium before or after sample deposition such that the chemical agent defines a potential inhibition zone;
depositing a volume of a sample in liquid form that contains or is liable to contain microorganisms on the surface of the agar culture medium within a deposition zone extending along an axis at the surface of the agar culture medium so that only a portion of the surface of the agar culture medium is covered with the volume of the sample;
incubating the agar culture medium with the chemical agent and the volume of the sample on the surface of the agar culture medium; and
determining the presence or absence of the zone of inhibition after incubation, wherein:
the deposition zone intersects the potential inhibition zone; and
the volume of the sample is deposited in a series of droplets in which each droplet of the series of droplets is from the same sample.

### Claim 2 (depends on 1)

The method of claim 1, further comprising obtaining the sample by pre-culturing a crude sample that includes isolating a microorganism strain and incubating the microorganism strain for less than 10 hours so as to increase microorganism biomass.

### Claim 3 (depends on 2)

The method of claim 2, wherein the sample contains a known type of microorganism prior to the volume of the sample being deposited on the surface of the agar culture medium.

### Claim 4 (depends on 1)

The method of claim 1, wherein the droplets are spaced apart by a predetermined interval.

### Claim 5 (depends on 1)

The method of claim 1, wherein the droplets are spaced apart by an interval in a millimeter range.

### Claim 6 (depends on 1)

The method of claim 1, wherein each droplet has a volume between 1 nl and 10 μl.

### Claim 7 (depends on 1)

The method of claim 1, wherein an amount of microorganisms contained in each droplet is known and is between 1 microorganism per droplet and 106 microorganisms per droplet.

### Claim 8 (depends on 1)

The method of claim 1, further comprising determining number of inhibited droplets and/or non-inhibited droplets.

### Claim 9 (depends on 1)

The method of claim 1, wherein the support is a disk that contains the determined amount of the chemical agent.

### Claim 10 (depends on 9)

The method of claim 9, further comprising measuring a distance between center of the disk and the zone of inhibition so as to estimate a sensitivity of the microorganisms contained in the sample to the chemical agent.

### Claim 11 (depends on 10)

The method of claim 10, further comprising classifying the microorganisms as Sensitive, Intermediate or Resistant to the chemical agent.

### Claim 12 (depends on 1)

The method of claim 1, wherein the support is a strip containing a concentration gradient of the chemical agent and the volume of the sample is deposited parallel and adjacent to a long edge of the strip.

### Claim 13 (depends on 12)

The method of claim 12, further comprising:
locating a boundary between the zone of inhibition and a zone of growth of the microorganisms; and
determining a minimum inhibitory concentration of the chemical agent from location of the boundary.

### Claim 14 (depends on 1)

The method of claim 1, wherein deposition of the volume of the sample is automated.

### Claim 15 (depends on 1)

The method of claim 1, wherein the volume of the sample is deposited so as to be spaced from a periphery of the agar culture medium.

### Claim 16 (depends on 1)

The method of claim 1, wherein the volume of the sample is deposited so as to not contact sides of a Petri dish containing the agar culture medium.

### Claim 17

A method for detecting a presence or an absence of at least one zone of inhibition, the method comprising:
obtaining a sample by pre-culturing a crude sample that includes isolating a microorganism strain and incubating the microorganism strain for less than 10 hours so as to increase microorganism biomass;
providing a determined amount of a chemical agent on a surface of an agar culture medium before or after sample deposition such that the chemical agent defines a potential inhibition zone;
depositing a volume of the sample in liquid form on the surface of the agar culture medium within a deposition zone extending along an axis at the surface of the agar culture medium so that only a portion of the surface of the agar culture medium is covered with the volume of the sample;
incubating the agar culture medium with the chemical agent and the volume of the sample on the surface of the agar culture medium; and
determining the presence or absence of the zone of inhibition after incubation, wherein:
the deposition zone intersects the potential inhibition zone; and
the volume of the sample is deposited in a continuous line.

### Claim 18 (depends on 17)

The method of claim 17, wherein the sample contains a known type of microorganism prior to the volume of the sample being deposited on the surface of the agar culture medium.

### Claim 19 (depends on 17)

The method of claim 17, wherein the sample has a concentration of between 0.0005 McFarland and 0.5 McFarland.

### Claim 20 (depends on 17)

The method of claim 17, wherein a support impregnated with the determined amount of the chemical agent is provided on the surface of the agar culture medium.

### Claim 21 (depends on 20)

The method of claim 20, wherein the support is a disk that contains the determined amount of the chemical agent.

### Claim 22 (depends on 21)

The method of claim 21, further comprising measuring a distance between center of the disk and the zone of inhibition so as to estimate a sensitivity of the microorganisms contained in the sample to the chemical agent.

### Claim 23 (depends on 22)

The method of claim 22, further comprising classifying the microorganisms as Sensitive, Intermediate or Resistant to the chemical agent.

### Claim 24 (depends on 20)

The method of claim 20, wherein the support is a strip containing a concentration gradient of the chemical agent and the volume of the sample is deposited parallel and adjacent to a long edge of the strip.

### Claim 25 (depends on 24)

The method of claim 24, further comprising:
locating a boundary between the zone of inhibition and a zone of growth of the microorganisms; and
determining a minimum inhibitory concentration of the chemical agent from location of the boundary.

### Claim 26 (depends on 17)

The method of claim 17, wherein deposition of the volume of the sample is automated.

### Claim 27 (depends on 17)

The method of claim 17, wherein the volume of the sample is deposited so as to be spaced from a periphery of the agar culture medium.

### Claim 28 (depends on 17)

The method of claim 17, wherein the volume of the sample is deposited so as to not contact sides of a Petri dish containing the agar culture medium.