Gram-stain differentiation with nonlinear light scattering

Patent metadata

Field	Value
Publication number	US-12578271-B2
Application number	US-202118252724-A
Country	US
Kind code	B2
Filing date	Nov 12, 2021
Priority date	Nov 12, 2020
Publication date	Mar 17, 2026
Grant date	Mar 17, 2026

Abstract

The present invention generally relates to improved methods for characterizing the cell wall complex composition of a bacterial specimen via differential transport kinetics of a small molecule. The present invention also relates to improved and automated methods for Gram differentiation of a bacterial specimen using nonlinear optical techniques.

First claim

What is claimed is:

1 . A method of determining bacterial cell wall composition comprising the steps of:
a. measuring a first second-harmonic scattering (SHS) signal of a crystal violet (CV) solution to generate a baseline; b. adding a bacterial specimen to said solution to generate a suspension; c. measuring a second SHS signal of said suspension to generate a response; d. determining whether said response is above or below said baseline; and e. identifying the bacterial cell wall of the bacterial specimen as that of a Gram-positive bacterium if the response is below said baseline or identifying the bacterial cell wall of the bacterial specimen as that of a Gram-negative bacterium if the response is above said baseline by a Gram-stain biosensor using nonlinear optical scattering.

2 . The method of claim 1 , wherein said measuring of said first or said second SHS signal comprises:
a. exposing said solution or said suspension to a fundamental light source; and b. detecting the second-harmonic wavelength of said light.

3 . The method of claim 2 , wherein said fundamental light has a wavelength of about 800 nm.

4 . The method of claim 3 , wherein the source of said about 800 nm fundamental light is a titanium-sapphire laser.

5 . The method of claim 2 , wherein said second-harmonic wavelength is about 400 nm.

6 . The method of claim 5 , wherein said about 400 nm second-harmonic wavelength is passed through a band-pass filter and monochromator, detected by a photomultiplier, amplified, and processed through a correlated photon counting system.

7 . The method of claim 6 , wherein said band-pass filter and monochromator excludes light of a wavelength of said fundamental light.

8 . The method of claim 1 , wherein said small molecule is taken up by said bacterial specimen.

9 . The method of claim 1 , wherein said CV is present at a concentration of at least 35 μM.

10 . The method of claim 9 , wherein said CV is present at a concentration of about 50 μM.

11 . The method of claim 1 , wherein said baseline is measured for between 1-100 seconds.

12 . The method of claim 1 , wherein said response is measured for at least 100 seconds.

Assignees

• Temple University—Of the Commonwealth System of Higher Education
• Univ Temple

Inventors

• Dai Hai-Lung
• Sharifian Mohammad
• Wilhelm Michael

Classifications

• G01N21/636 Primary

  using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties; (non-linear optics per se G02F1/35) · CPC title

• G01N15/1433

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• G01N15/01

  specially adapted for biological cells, e.g. blood cells (investigating sedimentation of particle suspensions in blood G01N15/05) · CPC title

• G01N15/1434

  Optical arrangements · CPC title

• G01N2015/0038

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