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


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

BioPharm International
Volume 18, Issue 12

Lab-on-a-Chip (LOC), Arrays, Microarrays, and Microchips

Type of Technology: Combination.

Premise of Technology: An array is defined as an orderly arrangement of data. For example, a microtiter well plate can be considered an array of rows and columns of data. Arrays of data have been used in many microbiology applications to perform a single test or series of tests. Microchips are manufactured using microminiaturization technologies, such as photolithography, hot embossing, reactive ion etching, and microinjection molding. Each microchip is much like a miniature laboratory, and some scientists refer to these as "lab on a chip" devices. The chips have become miniature analyzers, i.e., small versions of analytical instrumentation. Reagents used with these chips have been manufactured utilizing micro-fabrication technologies, e.g., micro-dispensing, ink-jet printing. Typical microbiological reagents include oligonucleotides, proteins, and DNA. One application of this technology is the antibody dot or microspot assay. A small amount of antibody, typically 10-100 mL, is placed on the bottom surface of a plastic well. This antibody dot is used as the capture antibody in a microimmunoassay.8 This technology can be used for a variety of applications, e.g., microbial virulence factors, antimicrobial resistance and identification, and bacterial discrimination.14-16

Commercial Systems Available: Various sources are available for these technologies. Others choose to "build" a chip to meet specific laboratory requirements.

Other: Many of these technologies are very expensive. As more commercial systems become available, the costs may be reduced.

Limulus Amebocyte Lysate Endotoxin (LAL) Testing

Type of Technology: Cell-component-based.

Premise of Technology: Amebocyte lysate recovered from horseshoe crabs (Limulus) has similarities with blood coagulation in humans. This similarity allows this reagent to be used to detect the presence of bacterial endotoxins. Quantitation of the endotoxin present requires the use of standards that are appropriately certified or licensed. Three different methods are available: gel clot, kinetic turbidometric, and chromagenic. The gel clot method is an endpoint determination of the amount of endotoxin present. Several different dilutions are evaluated as part of the test to determine the lowest concentration of endotoxin at which a clot forms. This type of test can require a large test sample to perform the various dilutions. The kinetic-turbidometric test allows for faster handling and smaller sample sizes. The chromogenic test is another variation of this method.

Commercial Systems Available: Pyrogent Gel Clot (BioWhittaker), Pyrotell (Associates of Cape Cod), BioTek, and handheld unit (Charles Rivers Endosafe).

Other: This technology has been available for many years as a replacement for the rabbit pyrogen test. Many systems are available, and they have gained widespread regulatory acceptance. Several characteristics, such as pH composition and ions present, can affect results.

Mass Spectrometry (Matrix-Assisted Laser Desorption-Time Of Flight, MALDI-TOF)

Type of Technology: Cell-component-based.

Premise of Technology: When microbial isolates are heated in a vacuum, the gaseous breakdown products can be analyzed using mass spectrometry. A spectrum can be generated and compared to a database of known organisms for identification. When subjected to intense ionization (MALDI-TOF), intact cells will release charged particles in distinct patterns. These patterns can be compared to a database of known microorganisms.

Commercial Systems Available: MALDI-TOF (Kratos Analytical Systems) and Voyager (Perspective Biosystem).

Other: This technology has been used for microbial identifications. The size of the database is important in evaluating the effectiveness of the system.

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