Lyse and Wash Buffers for Rapid Microbial Isolation
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Solution Overview
Problem
Current methods for isolating viable microorganisms from positive blood culture samples, such as those containing Streptococcus pneumoniae, are inefficient and often result in compromised or non-viable cells due to interference from blood components, leading to delays in diagnosis and inappropriate treatment.
Innovation Solution
The development of 'lyse and wash' buffers containing salts, peptones, nutrients, and non-ionic detergents like Triton X-100, which lyse blood cells while maintaining microbial viability, allowing for rapid isolation and concentration of microorganisms suitable for MALDI-TOF/MS, phenotypic identification, and antimicrobial susceptibility testing without the need for sub-culturing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If sub-culturing is used to isolate microorganisms from PBC samples, then microbial viability is maintained, but the process takes up to 72 hours causing treatment delays
Solution Approach 1:
The invention extracts and removes blood components (red blood cells, white blood cells, platelets, plasma proteins) from the PBC sample through a series of washing steps using buffers. This extraction eliminates interfering substances while preserving the microorganisms, enabling rapid downstream analysis without the need for time-consuming sub-culturing procedures.
Solution Approach 2:
The invention performs preliminary preparation of the PBC sample by lysing blood cells and washing away debris before downstream analysis. This preliminary action removes interfering substances in advance, allowing direct use of the prepared sample for MALDI-TOF/MS, phenotypic identification, and AST testing, thereby eliminating the need for subsequent sub-culturing and saving up to 72 hours.
2Object-generated harmful factors
If liquid separation methods with lysis buffers containing detergents are used, then blood cells are lysed and microorganisms are retained, but the microorganisms become compromised or non-viable
Solution Approach 1:
The invention changes the parameters of the lysis buffer by using non-ionic detergents (such as Tween 20, Tween 80, Triton X-100) at optimized concentrations (0.01-2.0%, preferably 0.1-0.5%) instead of harsh ionic detergents. This parameter change allows effective lysis of blood cells while maintaining the integrity and viability of microorganisms, enabling their use in subsequent growth-based assays.
Solution Approach 2:
The invention uses composite buffer solutions containing multiple components: non-ionic detergents for lysis, salts (NaCl, KCl) for osmotic balance, peptones for nutrient support, and sometimes additional agents like saponin or EDTA. This composite formulation achieves selective lysis of host cells while preserving microbial viability through the synergistic effect of its components.
3Productivity
If PBC samples are used directly for downstream testing, then processing time is reduced, but blood components interfere with identification and testing accuracy
Solution Approach 1:
The invention extracts and removes interfering blood components (hemoglobin, plasma proteins, cellular debris) through washing steps with non-ionic detergent buffers. This extraction eliminates substances that would interfere with MALDI-TOF/MS spectral analysis, phenotypic identification reagents, and AST testing, thereby improving measurement precision while maintaining rapid processing.
Solution Approach 2:
The non-ionic detergent buffer acts as an intermediary substance that facilitates the separation of microorganisms from blood components. The buffer mediates the lysis of host cells and the aggregation/washing away of debris, leaving purified microorganisms ready for downstream analysis. This intermediary step enables both speed and accuracy by preparing the sample appropriately without requiring extensive processing.
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
These buffers enable rapid and effective isolation of viable microorganisms from a single blood sample, suitable for multiple downstream analyses, including MALDI-TOF/MS and AST testing, reducing interference from blood components and preserving microbial viability, especially for challenging organisms like S. pneumoniae.
Implementation Method 1
lyse and wash buffers containing salts, peptones, nutrients, and non-ionic detergents like Triton X-100, which lyse blood cells
Implementation Method 2
The mixture is centrifuged to form a pellet containing viable microorganism(s) and a supernatant containing blood cell debris
Data Source
AI summary
Various embodiments disclosed herein provide for reagents and methods for rapidly isolating viable microbial cells, including S. pneumoniae, from positive blood culture samples. The resulting microbial pellet can be used for both identification and growth-based methods such as antimicrobial susceptibility testing. The buffers described herein may contain a base solution, non-ionic detergents, thiols, and optionally, ammonium chloride. The disclosed methods provide a process for rapidly isolating and concentrating viable microorganism(s) from PBC samples using only one sample preparation tube and centrifugation while removing cellular debris from the mammalian blood cells that may interfere with identification methods.