Single Spore Viability Detection via Fluorescent Solid Medium
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Solution Overview
Problem
Current methods for determining the survival condition of spores, such as the α-glucosidase test method, have limited sensitivity and are not suitable for testing individual spores, restricting their application and accuracy.
Innovation Solution
A method involving sterilization treatment of spores, preparation of a spore suspension, and application of 4-MUG onto a solid medium, followed by observation under an excitation light to determine viable spores through fluorescent spots, allowing for rapid assessment of spore viability and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the α-glucosidase test method is used to determine spore survival condition, then the activity of spores can be determined through fluorescence intensity, but the sensitivity is relatively low and single spore testing cannot be performed
Solution Approach 1:
The patent divides the spore suspension into individual spores and distributes them onto the solid medium, allowing each spore to be tested independently. This segmentation enables single spore viability detection, transforming the traditional bulk testing method into a single-cell analysis approach, thereby significantly improving measurement precision and expanding application versatility.
Solution Approach 2:
The patent replaces the traditional liquid-phase α-glucosidase test with a solid medium-based fluorescence detection system. By substituting the liquid mechanical mixing and fluorescence measurement with a solid medium approach, the method achieves higher sensitivity and enables single spore detection while maintaining the biochemical principle of α-glucosidase activity measurement.
2Measurement precision
If 4-MUG is added in sample liquid for α-glucosidase test, then fluorescence intensity can indicate spore activity, but the method is limited to testing spores in liquid sample only
Solution Approach 1:
The patent changes the physical state parameter of the testing medium from liquid to solid. By incorporating 4-MUG into a solid medium matrix, the method maintains the fluorescence-based detection capability while expanding applicability to surface contamination testing and single spore analysis, thereby improving both measurement precision and ease of operation across different application scenarios.
3Reliability
If conventional sterilization verification methods are used, then sterilization efficacy can be tested, but the process is time-consuming and cannot rapidly determine spore survival condition
Solution Approach 1:
The patent incorporates the substrate 4-MUG into the solid medium in advance, so that when spores are plated and incubated, the hydrolysis reaction can immediately occur without requiring additional steps to add the substrate. This preliminary preparation enables rapid detection, significantly improving productivity while maintaining the reliability of sterilization verification through accurate spore viability assessment.
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 enables rapid determination of spore viability and activity, with the ability to identify individual spores, improving sensitivity and application scope compared to existing methods.
Implementation Method 1
4-methylumbelliferone (4-MU for short), a product after being hydrolyzed by the α-glucosidase, has a fluorescence characteristic, and an emission spectrum of the 4-MU has a significant peak at 440 nm to 465 nm under an appropriate excitation light (an excitation wavelength is about 365 nm)
Data Source
AI summary
The present disclosure discloses a method for rapidly determining survival condition of spores. The method includes the following steps: S100: performing sterilization treatment on the spore; S200: preparing a spore suspension with the spore subjected to the sterilization treatment; S300: dripping an appropriate amount of the above prepared spore suspension onto a solid medium, wherein 4-MUG is contained in the solid medium; S400: observing the solid medium under an excitation light source after the spore suspension fully infiltrates into the solid medium; and S500: determining the number of viable spores according to the number of the observed fluorescent spots on the solid medium. The method is based on an α-glucosidase test method and can rapidly determine activity of the spore and judge activity of a single spore.