Sunday, 2 May 2021

Rapid microbiology: Testing pharmaceutical grade water with Milliflex Quantum

Gram-negative bacteria


Control of pharmaceutical water systems represents an essential part of Good Manufacturing Practice and embedded within this is the quality control testing of water systems for viable microorganisms, and subjecting the data obtained to trend analysis. This has traditionally been achieved by membrane filtration and the use of a culture medium (latterly, Reasoner’s 2A agar or broth, with subculture onto R3A agar). While such test methodologies can recovery a level of the bioburden present, the incubation times are lengthy. To address the problem of time-to-result, rapid microbiological methods offer an alternative approach. One such example is the Milliflex Quantum.


The Milliflex Quantum rapid detection system utilizes the Milliflex membrane filtration system which was originally developed for conventional microbial culture work. With the Quantum system, using fluorescent based staining, the technology allows for earlier detection and enumeration of microorganisms before they are visible to the naked eye. This occurs by a fluorescent viability marker which is taken up by organisms and the microbial cell metabolism causes an enzymatic cleavage of a non-fluorescent substrate. This reaction causes the liberation of a free fluorochrome into the cytoplasm of the cells, allowing them to be counted.  This paper outlines studies undertaken to demonstrate how the Milliflex Quantum system can significantly decrease incubation time required for detection of colony forming units from water samples.


Tim Sandle has published a new paper:


Sandle, T. (2021) A Study Into the Use of Milliflex Quantum as Rapid Microbial Detection Method for Microorganisms in Pharmaceutical Grade Water, Journal of Validation Technology, at:

With the Milliflex Quantum technology, the detection method for microorganisms is based on a universal enzymatic fluorescent staining of viable microorganisms. The fluorescent viability marker is taken up by organisms, where bacterial metabolism causes an enzymatic cleavage of the non-fluorescent substrate. This reaction causes the liberation of a free fluorochrome into the cytoplasm of the cells. As such, after incubation for 30 minutes, when the membrane is viewed under a UV lamp, viable colonies fluoresce. The advantage of the fluorescence is that colonies not visible to the human eye can be detected earlier on in the incubation process, shortening incubation time. 


To illustrate the reduced incubation times that can be achieved using the technology, this paper looks at the incubation times for purified water.  Analysis of data using One-Way ANOVA demonstrated a minimum of 2 days and a maximum of 6 days Milliflex Quantum Method Incubation Time for purified water samples. Overall, the process comprises of 2 - 6 days incubation at 30-35°C post filtration (as determined by this study) followed by Milliflex Quantum dye incubation step with a 30-minute incubation and plate read using the Milliflex Quantum reader. Hence, it was determined that the Quantum system was able to detect colonies several days before they became visible to the naked eye. Therefore, allowing for shorter incubation times and faster release of water data.


Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (

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