Dimeric Nucleic Acid Dye Staining for Rapid Bacterial Counting
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Solution Overview
Problem
Current methods for detecting bacteria in raw milk are either slow, use toxic reagents, are cumbersome, or expensive, making them unsuitable for high-throughput screenings and large production processes.
Innovation Solution
A method involving a staining composition of a dimeric nucleic acid dye and a buffering agent, with optional sonication and incubation at temperatures from 45°C to 95°C for less than 10 minutes, allowing for rapid and reliable counting of bacterial cells using flow cytometry without the need for ion-chelating agents, detergents, or centrifugation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard plate count method is used to detect and enumerate bacteria in raw milk, then reliability of bacterial counting is improved, but productivity is worsened due to approximately 48 hours processing time
Solution Approach 1:
The patent replaces the mechanical/cultural method (standard plate count requiring bacterial growth on agar plates) with a fluorescent staining method using flow cytometry. The dimeric nucleic acid dye stains bacterial DNA directly, allowing rapid detection and enumeration without requiring bacterial cultivation, thus reducing processing time from 48 hours to under 10 minutes while maintaining reliability through flow cytometric analysis
Solution Approach 2:
The patent changes the detection parameter from colony formation (macroscopic growth) to fluorescent signal intensity (molecular level detection). By using a dimeric nucleic acid dye that binds to bacterial DNA and emits fluorescence, the method enables direct detection of bacterial cells through flow cytometry, fundamentally changing the detection parameter to achieve both speed and reliability
2Measurement precision
If ion-chelating agents, detergents and toxic fluorochromes like ethidium bromide are used for bacterial detection, then measurement precision is improved, but object-affected harmful factors are worsened due to toxic waste requiring cumbersome handling
Solution Approach 1:
The patent extracts and removes the toxic components (ion-chelating agents, detergents, and toxic fluorochromes like ethidium bromide) from the detection system. Instead, it uses a dimeric nucleic acid dye that stains bacterial DNA effectively without requiring these harmful agents, thereby maintaining measurement precision while eliminating toxic waste and simplifying handling procedures
Solution Approach 2:
The patent employs a safer, less persistent staining approach using dimeric nucleic acid dyes that do not require toxic auxiliaries. The method uses disposable microplates and standard buffer solutions rather than requiring complex waste handling procedures associated with toxic reagents, effectively replacing hazardous materials with safer alternatives that maintain detection accuracy
3Measurement precision
If multiple agents including ion-chelating agents and detergents are used for bacterial lysis and staining, then measurement precision is improved, but device complexity is worsened due to cumbersome procedure
Solution Approach 1:
The patent merges multiple separate steps (bacterial lysis, protein degradation, and staining) into a single integrated procedure. The dimeric nucleic acid dye directly stains bacterial DNA in intact cells without requiring prior lysis or the use of ion-chelating agents and detergents. This consolidation simplifies the procedure to basic steps: sample preparation, fluorescent staining, and flow cytometric analysis, reducing device complexity while maintaining measurement precision
4Measurement precision
If centrifugation and viability-dependent dyes are used to determine percentage of viable cells, then measurement precision is improved for viable cell detection, but productivity is worsened due to time-consuming procedure and inability to detect all cells
Solution Approach 1:
The patent creates a universal detection method that stains all bacterial cells regardless of viability status. The dimeric nucleic acid dye binds to bacterial DNA in both live and dead cells, enabling comprehensive enumeration of total bacterial load. This multi-functional approach allows simultaneous detection of all cells in the sample without requiring viability assessment, thereby improving productivity by eliminating centrifugation and selective staining steps while maintaining accurate counting capability
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 provides a fast, safe, and cost-effective means to count bacteria in raw milk, reducing processing time to under 10 minutes and eliminating the use of toxic compounds, while maintaining high accuracy and reliability.
Implementation Method 1
a) mixing a staining composition comprising a dimeric nucleic acid dye and a buffering agent with said sample; e) counting the cells that are stained with said dye
Implementation Method 2
b) optionally sonicating the mixture of step a)
Implementation Method 3
c) incubating the mixture at a temperature from about 45° C. to about 95° C. for less than 10 minutes
Data Source
AI summary
The invention relates to a method for counting cells, such as bacteria and/or somatic cells in liquid samples, such as in dairy products, preferably raw milk. Disclosed is a method comprising a combination of steps that apply dimeric nucleic acid dyes that normally do not penetrate cells (=cell-impermeant dyes), which are rendered cell-permeant by using the right combination of pH, buffer and temperature.