# US 11,306,344 B2 · Spectral intensity ratio (SIR) analysis for rapid live microbial enumeration

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- Number: US 11,306,344 B2
- Document ID: US11306344B2
- Status: Granted
- Kind: B2
- Application number: 15/542,922
- Filed: 2016-01-09
- Published: 2018-01-11
- Granted: 2022-04-19
- Earliest claimed: 2015-01-12
- Assignees: Tacount Exact Ltd.
- Applicants: Tacount Exact Ltd.
- Inventors: Eran Zahavy
- Web page: https://patentgrep.com/patent/US11306344B2

## CPC

- C12Q 1/06: Quantitative determination
- 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)}
- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- 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
- 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

Single dye fluorescent staining (with a membrane-associated dye such as FM 1-43 or FM 4-64) and the combination of differences in both intensity and spectral emission discriminate live from inactivated/dead bacteria and provides for rapid and accurate detection of live bacteria in mixed populations.

## Claims

### Claim 1

A method of differentiating between live and inactivated bacteria in a treated sample comprising:
staining the sample with a N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide dye;
illuminating the sample with an incident light at an excitation wavelength of 488 nm, wherein an illuminated live bacteria stained with the N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide dye exhibits a first maximum emission peak, and wherein an illuminated inactivated bacteria stained with the N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide dye exhibits a second maximum emission peak that is shifted compared to the first maximum emission peak;
measuring, for each bacterial cell (i) the intensity I1 of emitted light at wavelength 1 of 530 nm and (ii) the intensity I2 of emitted light at wavelength 2 of 610 nm;
calculating a ratio I2/I1; and
determining that a bacterial cell is live when I2/I1<2.0 and determining that the bacterial cell is inactivated when I2/I1>2.0.

### Claim 2

A method of differentiating between live and inactivated bacteria in a treated sample comprising:
staining the sample with a N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide dye;
illuminating the sample with an incident light at excitation between 488 and 570 nm, wherein an illuminated live bacteria stained with the N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide dye exhibits a first maximum emission peak, and wherein an illuminated inactivated bacteria stained with the N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide dye exhibits a second maximum emission peak that is shifted compared to the first maximum emission peak;
measuring, for each bacterial cell (i) the intensity 11 of emitted light at wavelength 1 of 670 nm and (ii) the intensity I2 of emitted light at wavelength 2 of 780 nm;
calculating a ratio I2/I1; and
determining that a bacterial cell is live when I2/I1<2.0 and determining that the bacterial cell is inactivated when I2/I1>2.0.

### Claim 3 (depends on 1)

The method of claim 1, further comprising treating live bacteria cells contained in the sample in an effort to inactivate the bacteria cells prior to the staining step.

### Claim 4 (depends on 3)

The method of claim 3, further comprising the step of filtering the stained cells prior to the illuminating step and subsequent to the staining step.

### Claim 5 (depends on 3)

The method of claim 3, wherein the treating step comprises interacting the live bacteria with an antimicrobial drug.

### Claim 6 (depends on 3)

The method of claim 3, wherein the treating step comprises heating, adding an antibiotic drug, adding an antibacterial drug, adding ethanol or ultraviolet (UV) irradiation.

### Claim 7 (depends on 2)

The method of claim 2, further comprising treating live bacteria cells contained in the sample in an effort to inactivate the bacteria cells prior to the staining step.

### Claim 8 (depends on 7)

The method of claim 7, further comprising the step of filtering the stained cells prior to the illuminating step and subsequent to the staining step.

### Claim 9 (depends on 7)

The method of claim 7, wherein the treating step comprises interacting the live bacteria with an antimicrobial drug.

### Claim 10 (depends on 7)

The method of claim 7, wherein the treating step comprises heating, adding an antibiotic drug, adding an antibacterial drug, adding ethanol or ultraviolet (UV) irradiation.