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

Gram Stains

Type of Technology: Cell-component-based.

Premise of Technology: This technology uses a single solution, without fixatives or washes. Results are obtained in a few minutes. Syto-9 stain and red-fluorescent hexidium iodide nucleic-acid stain are used. The method can be used with mixed cultures. Using the LIVE Bac Light Bacterial Gram Stain Kit, Gram-positive organisms stain a reddish-orange, and Gram-negative organisms stain green. The fluorescent stains can be viewed and assessed using a fluorescent microscope (with a standard fluorecein long-pass optical filter set) or using flow cytometry. The reagents have been designed to show low background (intrinsic) stain. Dead cells do not show a predicted staining pattern. There are also procedures specified for use with DEFT. A second staining kit — ViaGram Red+ Bacterial Gram Stain and Viability Kit — is similar to the kit described above, but it uses two stains and three colors, so that viable and non-viable cells can be readily detected in addition to knowing the Gram reaction. Plasma membrane integrity is used as the distinguishing factor of live bacterial cells. Intact membranes are detected with a blue stain, while damaged membranes stain green. The red stain identifies Gram-positive bacteria.13

Commercial Systems Available: LIVE Bac Light Bacterial Gram Stain Kit and ViaGram Red+ Bacterial Gram Stain.

Immunological Methods

Type of Technology: Cell-component-based.

Premise of Technology: One can use an antigen-antibody reaction to detect unique microorganisms or cellular components.

Commercial Systems Available: Pathogen detection kits are available for various types of pathogen. ELISA is available.

Other: Immunological methods are useful for pathogen detection, and they may also be used for identification. In some cases, the systems may not distinguish whether the detected cells are viable.7

Impedance (Electrochemical) Methods

Type of Technology: Growth-based.

Premise of Technology: Growing microorganisms metabolize large complex constituents, such as proteins and carbohydrates, and convert them to smaller charged by-products such as amino acids, carbon dioxide, and acids. These smaller by-products of metabolism build up and eventually change the electrical conducting properties of the supporting growth medium. When an alternating current is applied across electrodes to this growth media, a change in impedance can be observed. Impedance is the resistance to the flow of an alternating current through a conducting material. Microbial detection systems based on impedance technology are classified in two types of systems: direct and indirect impedance. Direct impedance systems work by detecting changes in electrical conductivity of growth media when an a.c. current is passed across two electrodes. Indirect impedance systems detect carbon dioxide produced by metabolizing organisms, via the use of chemical sinks such as potassium hydroxide. As the carbon dioxide is ionized, changes in impedance result. There is no direct contact between the electrodes and the microorganisms under investigation. When microorganisms multiply, a detection threshold is reached, above which an electrical signal is detected by both types of systems. Generally, this detection limit is approximately 106 cfu/mL for many microbial species. The lower the initial population, the longer the time taken to reach the detection threshold.

Commercial Systems Available: Bactometer (bioMerieux), BacTrac (Sy-Lab), RABIT (Don Whitley Scientific Ltd), and the Malthus Microbial Detection System (Malthus Diagnostics, Inc.).

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