日本Rapid Detection based on Non-destructive, Fluorescent Staining
Rapid detection of microbial contamination is challenging and demands highly sensitive and reliable technologies.
The Milliflex Quantum system enables the rapid detection of microorganisms in filterable samples. Detection is based on a universal enzymatic fluorescent staining of viable microorganisms. The staining is non-destructive enabling specific identification following a positive result using standard ID technology.
Principle
The fluorescence detection is based on an enzymatic reaction. The fluorogenic substrate used is a non–fluorescent viability marker that is cleaved by non-specific intracellular enzymes resulting in a fluorescent product. Accumulation of fluorescence inside cells is an indicator of microbial metabolism activity and membrane integrity. The dye is diluted in a staining buffer allowing cell membrane permeabilization and the introduction of dye into the cells.The procedure is easy-to-use and requires minimal training. Sample contaminants are trapped by membrane filtration using Millipore’s Milliflex system. After a short incubation period, the media cassettes are removed and each Milliflex membrane is transferred onto a cellulose pad pre-wetted with 2 mL of staining solution and re-incubated for 30 minutes at 32.5°C ± 2.5 °C. The fluorescent microcolonies are then visually counted through the window of the Milliflex Quantum reader (specific LED system) or from a PC screen using the camera and software (see figure 1). After detection, membranes can be reapplied onto the media for traditional plate count (viability assay), enabling identification of microorganisms, if needed, using any existing ID technology. The typical time from sample preparation to final result is typically ~24-48 h.
Figure 1: Typical Milliflex Quantum results displayed on a computer screen or through the window of the Milliflex Quantum reader.
| Bacillus cereus | Staphylococcus epidermidis | Propionibacterium acnes |
Results in 2 to 3 easy steps
Two reagent kits are available with the Milliflex Quantum system. The Universal Milliflex Quantum kit is used for the detection of microorganisms in all sample matrices. The Water Control kit is designed specifically for the microbiological testing of your water system.Figure 2: Workflow of the Milliflex Quantum protocols
Since the methodology is non-destructive, the membrane can be re-incubated for later identification using any ID technology, if contamination is detected
Applications
The Milliflex Quantum system can be successfully used in all applications where the sample is filterable and the target is an enumeration of the microorganisms, e.g., TAMC and TYMC. The following matrices have been tested or are already in use in the industry:- Raw material:
- Media
- Buffers
- Pharmaceutical ingredients
- Water
- In-process samples:
- Bioburden prior to sterilization
- CIP/SIP samples
- Cell culture/fermentation samples
- Media for fermentation
- Buffers for manufacturing
- Intermediate process samples
- Final Product
- Environmental samples
The system also allows the rapid detection of contaminants present in mammalian cells cultures.
Using a specific lysis buffer, mammalian cells are removed. Microorganisms that grow on the membrane are not affected by this treatment and are easily detected by the system.
Validation Strategy
The Milliflex Quantum method was validated in accordance with US and European pharmacopeia (USP ≤1223≥ and EP 6.3 chapter 5.1.6). The performances of the Milliflex Quantum method were compared to the compendial Milliflex filtration method. The Milliflex Quantum system falls under the definition of a quantitative test, since the test not only detects but enables users to manually enumerate contaminating microorganisms. For this kind of test, the pharmacopeia recommends validating the following parameters:- Robustness
- Ruggedness
- Accuracy
- Precision
- Linearity
- Range
- Limit of Quantification
- Limit of Detection
- Specificity
All parameters have been shown to comply with the recommended specifications from the compendia.
Millipore provides a complete service package around this product, including IQ/OQ protocols, onsite validation services and method development. Additionally, repair and maintenance contracts and services are available to support the implementation at a customer site.
| Products | Services |
| Milliflex Quantum Detection System | On site IQ/OQ Services |
 | PQ Consultancy Services |
| Maintenance contracts |
| Hardware Rental Services |
| Application Development Services |
