Direct Epifluorescent Filtration Technique DEFT

This technique has also been extensively used in the food industry. It utilizes the observation that viable cells, when exposed to acridine orange, can be visualized under a fluorescent microscope, appearing bright orange, while nonviable cells appear green. By sample filtration, the viable cells can be stained in situ and immediately counted, giving a result within one hour or less. The most obvious drawback is that the technique is limited to filterable samples and its sensitivity, to some extent, determined by how much of a sample can be filtered. Although some workers have detected down to 10 to 20 organisms per ml by filtering large volumes (litre quantities), generally the detection limit is of the order of 102-104 organism/ml. Other issues are the occasional nonselectivity of acridine orange, which can stain certain nonviable cells, and has a tendency to also stain other noncellular material in a sample.

However, because of the rapidity of the method, considerable development of the fundamental technique has taken place. The commercial equipment manufacturers Chemunex (an international company with its head offices in Paris, France) have developed equipment that overcomes at least two of the major drawbacks of the acridine orange methodology.

The first is the utilization of a more specific "stain" based on fluorescein. Samples are filtered, and then treated with the stain. Once taken into the cell, the stain is cleaved by an esterase releasing a fluorochrome that can be detected by laser scanning. Additionally, only cells with an intact cell membrane (i.e., viable cells) can retain sufficient amounts of the stain to be detected. Following laser scanning, the associated software logs every fluorochrome "hit," enabling true enumeration to be achieved since it has been "taught" to ignore fluorescing debris below a predetermined threshold. At worst this would probably lead to falsely high counts. The use of the laser and software also removes the labour-intensive microscopic examination required by the acridine orange method.

One area where the Chemscan RDI system has been used is in the monitoring of pharmaceutical water systems, where the provision of real-time results can potentially prevent the use of out-of-specification (OOS) water being used in the compounding of an expensive active. An interesting corollary is that the system appears to result in higher counts than traditional methods. This may be due to normal culture media offering less than optimal recovery for all organisms, or possibly enumeration of so-called viable nonculturable organisms by the Chemscan system. This poses a regulatory dilemma, in that although the water quality is no worse than it has ever been, it is possible that it will fail the pharmacopoeial standards. This dilemma has yet to be resolved in all countries but the MCA (now MHRA) at least recognizes the issue and accepts that counts may be higher without "failing" pharmacopoeial standards.

A further development of the fluorochrome labelling system is its combination with flow cytometry. The latter technique has been around since the early 1960s and basically consists of passing microorganisms, diluted in an electrolyte, through a very small aperture across which an electric current is applied. As the microorganisms pass through the aperture the electrical resistance changes can be measured. The disadvantages of this technique are that many other substances can alter the resistance, and the aperture can easily become blocked. The so-called D-Count apparatus uses the fluorochrome to label viable cells, which are then flowed past a laser beam that excites the fluorochrome, subsequently measuring it using a photomultiplier. As yet the sensitivity at reportedly 50 to 100 cfu/ml is still an issue, although recently sensitivity claims down to 1 cfu/ml have been made.

As might be expected from such a sophisticated computer-based system, the initial capital outlay for such systems is an issue. In today's climate, the more sophisticated the systems, particularly when involving computer software and hardware, the more sophisticated and time-consuming the equipment qualification will be, such as conformance to Good Automated Manufacturing Practice (GAMP) and CFR 211 Part 11 regulatory compliance. To our knowledge this technique has now gained regulatory approval for process water testing, and release testing for nonsterile products in Europe.

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