Rapid Microbiological Methods for a New Generation

These are exciting times, as 19th-century microbiological methods make way for rapid detection, quantification and characterization technologies.

By Michael J. Miller, Ph.D., Senior Research Fellow, Eli Lilly and Co.

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    • the MIDI (Newark, Del.) Sherlock Microbial Identification System;


    • the Waters (Milford, Mass.) MicrobeLynx system utilizing MALDI time-of-flight mass spectrometry;


    • SELDI time-of-flight mass spectrometry using Ciphergen (Fremont, Calif.) ProteinChip Arrays;


    • a portable, hand-held assay platform for the detection of endotoxin and Gram-negative bacteria using the Charles River Laboratories (Wilmington, Mass.) Endosafe PTS system;


  • the Cambrex (East Rutherford, N.J.) PyroSense on-line endotoxin detection system, which is currently in development, and is intended to monitor purified water systems.

 

Nucleic acid-based technologies rely on PCR DNA amplification, 16S rRNA typing, gene sequencing and other novel applications. Many of these systems are used for the rapid and accurate detection of a specific target microorganism or for the identification of an unknown isolate. Although these systems can provide results much faster than the growth-based detection and identification technologies, they still require starting material from a microbial culture (e.g., an isolated colony on an agar surface), and therefore will add additional time in obtaining the final result. Commercially available technologies that utilize nucleic acid-based technologies include:

 

    • the DuPont Qualicon (Wilmington, Del.) RiboPrinter and BAX systems;


    • Applied Biosystems’ (Foster City, Calif.) MicroSeq;


    • Sequenom’s (San Diego, Calif.) MassARRAY platform;


    • Bacterial BarCodes’ (Houston) DiversiLab System;


  • the Ibis (Carlsbad, Calif.) PCR-mass spectrometry TIGER system.

 

On the cutting edge of miniaturization

Other technology platforms are much smaller than the technologies previously discussed, and should have a significant impact in the way microbiological assays will be performed in the future. Some of these platforms offer continuous and instantaneous microbial detection, and are excellent candidates for at-line, on-line or in-line microbiological PAT applications. The technologies include biochips, microarrays, biosensors, and an instantaneous detection system for airborne microorganisms.

Micro-Electro-Mechanical Systems (MEMS)

Imagine, for a moment, a machine so small that the human eye cannot see it and thousands of these machines are manufactured on a single piece of silicon. Imagine a future where gravity and inertia are no longer important, but atomic forces and surface sciences dominate. This is the world of MEMS, and the future is now.

MEMS integrate mechanical, electrical, fluidic and optical elements, sensors, and actuators on a common silicon substrate, using microfabrication technology. Use of MEMS is growing the fastest in drug discovery and delivery. However, many of the same technologies used in these applications already have, or soon will have, a place as RMMs. They include the following platforms:

Lab-On-A-Chip is based on an automated, micro- or nano-scale laboratory that enables sample preparation, fluid handling and analysis and detection steps to be carried out within the confines of a single microchip. The technology is based on microfluidics, and the most familiar consumer application is ink-jet printing. Microfluidics allows for the manipulation of minute amounts of liquid in miniaturized systems that are composed of a network of channels and wells that are etched onto glass or polymer chips. Pressure or voltage gradients move pico- or nanoliter volumes through the channels in a finely controlled manner that enables sample handling, mixing, dilution, electrophoresis and chromatographic separation, staining and detection. Currently available lab chips analyze protein, DNA, RNA and whole cells in fluid samples. Examples that are now available include:

 

    • Bacterial Barcodes’ DiversiLab Microbial Typing System, which uses a microfluidics chip (the DNA LabChip) to separate rep-PCR amplicons. The chip is processed in a bioanalyzer, where the amplicons pass through a laser, causing fluorescence of an intercalating dye. The resulting rep-PCR fingerprints are compared with a database, and a detection result, or microbial identification, is provided.


  • A micro-scale impedance-based detection system is currently being developed by BioVitesse (San Francisco, Calif.) and Purdue University. In impedance microbiology-based systems, bacterial growth is detected by monitoring the movement of ions between two electrodes (conductance) or the storage of charge at the electrode surface (capacitance). Conventional systems require 104 - 105 cells in milliliter-sized samples in order to elicit a positive response over time. However, if the number of cells can be concentrated into a small incubation chamber, the time to detect microbial growth should decrease. This is the basis for the BioVitesse system. On a single chip, sample channels and incubation chambers are etched onto a crystalline silicon substrate. Platinum microelectrodes, which are located in the incubation chambers, measure impedance changes. Because the sample volume in this system is less than 1 nanoliter, the time to detect microbial growth may be considerably less than what is currently available today.

 

Microarrays (Biochips or DNA Chips)

Microarrays are collections of miniaturized test sites, arranged on solid substrates, that permit many tests to be performed at the same time. They are composed of an orderly arrangement of protein or thousands of DNA or RNA fragments on glass, silicon or nylon substrates. This technology evolved from Southern Blotting technology, in which fragmented DNA is attached to a substrate and then probed with a known gene or DNA fragment, using fluorescent tags to allow visual detection.

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