Rapid Microbiological Methods and the PAT Initiative - Numerous new RMM systems are available to replace traditional testing methods - BioPharm International

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Rapid Microbiological Methods and the PAT Initiative
Numerous new RMM systems are available to replace traditional testing methods


BioPharm International
Volume 18, Issue 12

Direct Epifluorescent Filter Technique (DEFT)

Type of Technology: Viability-based.

Premise of Technology: In the direct epifluorescent filter technique (DEFT), samples are filtered and stained using a fluorescent viability indicator. Acridine orange was used originally, but more recent applications use 4',6-diamidino-2-phenylindole. Epifluorescence microscopy detects fluorescing microorganisms. The sensitivity of the technique depends on the volume filtered and the number of fields viewed under the microscope. Micro-colony formation can further enhance the accuracy of the technique and the detection of cell viability. Automated and semi-automated systems have been developed to speed up the process and increase the accuracy of the technique.

Commercial Systems Available: Microscopes with epifluorescence capabilities.

Other: The ability to differentiate between fluorescing organisms and auto-fluorescing particles can be a concern. The robustness of the test can be affected by the distribution of the microorganisms on the membrane. This methodology is best suited for low viscosity fluids, although it may be possible to use pre-filtration to allow testing of other solutions.7

Endospore Detection

Type of Technology: Cell-component-based.

Premise of Technology: A major component of the spore case is calcium dipicolinate (Ca[dpa]). Dipicolinate anions are present only in bacterial endospores. Ca (dpa) and dipicolinate anions (dpa2- ), when dissolved, are not photoluminescent. It has been shown that terbium (Tb3+ ) is able to complex with dpa2- , forming a photoluminescence complex. The procedure for detecting spores (US patent issued to the US Army Research Laboratory) involves the following steps:11

  • Mixing: Terbium chloride (TbCl3) is added to the aqueous-based test solution (potentially including endospores). It may be desirable also to add aluminum chloride (AlCl3), as it has been shown to bind to phosphates that may interfere with the results obtained. Buffers can compete with the dipicolinate for the Tb3+ , thereby reducing the expected photoluminescence. Tris buffer was found to be a good choice for the solution.
  • Filtration: A 0.22-m filter is used to remove excess residual bacterial particles from the terbium-treated solution as the solution is passed from the mixing container to a cuvette containing [Tb(dpa)]+ .
  • Analysis: The sample is exposed to an ultraviolet (UV) source and excited. The corresponding emission is perceived by a detector and is used to generate an emission spectrum (UV-visible spectrophotometer used with wavelength showing range of 250-300 nm).

Commercial Systems Available: The equipment needed to perform this procedure is commercially available as individual instruments.

Other: Insoluble items may be photoluminescent, and there may be light loss due to the presence of insoluble particulate matter.

Enzyme-Linked Immunosorbent Assay (ELISA)

Type of Technology: Cell-component-based.

Premise of Technology: In the ELISA technique, an antigen-antibody reaction detects unique microorganisms or cellular components.

Commercial Systems Available: VIDAS and Mini-VIDAS (bioMerieux), Tecra Salmonella ELISA (International Bioproducts), and Salmonella Tek ELISA (Organon Teknika).

Other: This technology has been used for many years in clinical labs.


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