Fluorescent Stain Method for Viable Microorganism Counting
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Solution Overview
Problem
Current methods for determining the concentration of microorganisms in a sample are limited by their inability to differentiate between viable and non-viable cells, and require lengthy incubation periods or complex instrumentation.
Innovation Solution
A method involving live/dead staining with cell-permeable and cell-impermeable fluorescent dyes, followed by imaging and image analysis to determine the concentration of intact microorganisms, which serves as an indicator of viable cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If turbidity measurement is used to determine microorganism concentration, then the measurement is rapid and easy to implement, but it cannot distinguish between viable and non-viable cells and has low sensitivity
Solution Approach 1:
The patent uses fluorescent dyes that emit different colors/wavelengths when bound to DNA in intact versus disrupted cells. Intact cells emit fluorescence at a first wavelength while disrupted cells emit at a second wavelength, enabling optical differentiation of viable from non-viable cells while maintaining rapid measurement capability
Solution Approach 2:
The patent replaces mechanical counting methods (microscopy, plating) with optical detection using flow cytometry or plate reader instrumentation. This substitution enables automated, rapid analysis of fluorescent signals to determine the concentration of intact microorganisms without manual intervention
2Measurement precision
If plating on solid growth medium is used to estimate viable microorganisms, then quantitative measurement is achieved, but lengthy incubation is required
Solution Approach 1:
The patent applies fluorescent stains to microorganisms before any incubation or growth step. The stains immediately bind to DNA and provide fluorescent signals that can be detected right away, eliminating the need to wait for colony formation while still providing quantitative data on viable cell concentration
Solution Approach 2:
The patent replaces the biological incubation process with direct optical detection of fluorescently-stained cells. Flow cytometry or plate readers can count and characterize intact cells within seconds or minutes, substituting the days-long incubation requirement with rapid instrumental analysis
3Measurement precision
If flow cytometry with live/dead staining is used to differentiate viable cells, then accurate differentiation is achieved, but complex instrumentation and calibration are required
Solution Approach 1:
The patent develops fluorescent dye combinations that can be detected by multiple types of instrumentation (flow cytometers, plate readers, microplate spectrophotometers). This universality allows the same staining protocol to work across different devices, reducing the need for device-specific calibration and simplifying the overall system complexity
Solution Approach 2:
The patent optimizes the fluorescent dye staining protocol to work with standard, widely-available instrumentation rather than requiring specialized flow cytometers. By adjusting staining conditions and detection parameters, the method achieves accurate viable cell differentiation using simpler, more common laboratory equipment
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method allows for rapid and accurate determination of the concentration of viable microorganisms, reducing the need for lengthy incubation and complex instrumentation, and enabling more efficient preparation of microbial inocula and performance of antimicrobial susceptibility tests.
Implementation Method 1
a first stain which is cell-permeable and comprises a fluorescent marker having a first emission wavelength
Implementation Method 2
a second stain which is cell-impermeable and comprises a fluorescent marker having a second emission wavelength
Data Source
AI summary
The present invention relates to a method of determining the concentration of intact microorganisms in a sample comprising optionally diluting an aliquot of the sample to provide a diluted aliquot at a dilution value; contacting at least a portion of an aliquot or of a diluted aliquot of the sample with first and second stains capable of binding to DNA, wherein the first stain is a fluorescent stain, is cell-permeable, and has a first emission wavelength, and the second stain is cell-impermeable, and is capable of acting as an acceptor molecule in a FRET pair with the first stain acting as a donor molecule; imaging the aliquot-stain mixture at the first emission wavelength and determining an image analysis value for the number of objects corresponding to intact microorganisms in the imaged mixture; and comparing the image analysis value for said aliquot to a pre-determined calibration curve, as well as an apparatus, a consumable and a kit therefor.