AQU@Sense MB

	Heterotrophic Plate Count	AQU@Sense MB
Technology	Growth based, dependent on culture medium	Fluorescence analysis after DNA-specific staining
Sampling	Manual	Automatic
Measuring unit	Colony Forming Unit (CFU)	Intact Cell Count (ICC)
Time to result	3-7 Days	20 Minutes (30 Minutes measurement interval)
LOD	1 Colony	1 ICC
Range	1-300 colonies per sample	1-1 000 000 ICC per sample
Identification	Colonies Limited morphological determination	Individual Cells Dot-plot / fingerprint of a population Interpretation of the results according to different events (trend monitoring)
Enumeration	Visual counting by humans	Automatic
Sample size	100 or 200ml	90µl

The AQU@Sense MB employs two specific staining agents: SYBR Green I and Propidium Iodide. SYBR Green I penetrates intact cell membranes and selectively binds to the cell’s DNA. In contrast, Propidium Iodide cannot penetrate intact membranes and binds exclusively to the DNA of compromised cells.

To determine whether only SYBR Green I or both SYBR Green I and Propidium Iodide are bound to a cell, the cells are passed through a very thin capillary and illuminated with a laser, stray light and fluorescence light from is generated and analyzed with a specific detection system. As SYBR Green I and Propidium Iodide emit fluorescence at distinct wavelengths, the system can differentiate between intact or damaged cells.

• Primary-validated technology by independent scientists, ensuring high credibility and reliability
• Clear and unambiguous results, with differentiation between intact and damaged cells/debris
• Reproducible results
• High autonomy with a user-friendly plug-and-play setup, minimizing manual intervention
• Robust/industrial design
• No direct user contact with chemicals or waste
• Minimal maintenance requirements
• Advanced detection capabilities, identifying individual cells, even within conglomerates or biofilm fragments, enabling comprehensive trend monitoring.

Intact Cell Counts (ICC) and Colony Forming Units (CFU) do not exhibit a direct correlation. Primarily due to the presence of Viable but Non-Culturable Cells (VBNC), which are not detected on HPC but easily detected in flow cytometer. Secondly, due to difference in time and environmental factors between two methods. Flow cytometry provides an instantaneous snapshot of cell presence. Therefore, cells that may not thrive under HPC conditions might still be detected by flow cytometry.

Consequently, when the AQU@Sense MB is employed as a tool in Quality Control (QC), a performance qualification (PQ) is necessary at the customer's site to establish parameters, including baseline, warning, and action levels.

For microorganisms, entering the VBNC (viable but non-culturable) stage is a survival strategy in response to stress conditions like unfavorable environments, nutrient depletion, and temperature fluctuations, which are main features of PW/WFI water systems.
These VBNC bacteria remain alive and metabolically active but are unable to grow in standard laboratory conditions. Recent studies have shown that the VBNC state is reversible, meaning these microorganisms can return to a multiplying state, posing a potential bioburden, and in turn, health risk. Flow cytometry helps detect VBNC microorganisms in pharmaceutical water because it uses non-growth-based methods.

No, the AQU@Sense MB does not identify the strain. However, since flow cytometry is applied, a fingerprint of the distribution of the cells is given which can give more information about the state of the microbial population.

There are two possible fields of application for daily routine analysis. In-Process Control (IPC) and Quality Control (QC).
For IPC it can be can be integrated into the water system as an online control system for monitoring bioburden. When operating at its maximum capacity, the AQU@Sense MB can perform batchwise continuously up to 48 measurements per day (minimum measurement interval: 30 minutes). The measurement interval is adjustable according to the needs and standard operation of water system. The benefits of frequent measurements are noticeable - for rapid detection of even minor changes, in contrast to the conventional plate count method. This system can be utilized not only to continuously monitor bioburden levels in the plant but also e.g., to release the water system after sanitation or maintenance for technical water release.

Yes, it is possible to perform a QC release using the AQU@Sense MB. However, to incorporate it into current QC release process, customers must conduct a performance qualification (PQ) to confirm its suitability for the intended purpose. According to regulatory guidelines, the AQU@Sense MB or any other Rapid Microbiological Method (RMM) can be validated as a QC release method. Since the AQU@Sense MB is already primary validated the performance qualification procedure is much simpler.

An equivalence testing must be conducted using appropriate parameters, followed by statistical analysis, since it indicates a new measuring unit (Intact Cell Counts, ICC) instead of Colony Forming Units (CFU). The results of the testing must demonstrate that the results of the AQU@Sense MB allow for an unequivocal decision regarding compliance with the standards outlined in the monographs, even when compared to the conventional HPC.

The AQU@Sense MB, along with other RMM devices (Status November 2023), is not specifically authorized by name by any regulatory body. However, it can be validated and qualified as a QC release method, since the EU GMP Annex I strongly recommend to incorporate RMM’s.

At BWT, we are working on obtaining authorization for the applied method (flow cytometry).