The accuracy of an identification scheme heavily depends on the ability to reliably detect whether a bacterial isolate has utilized the substrates composing the identification battery. The sensitivity and strength of the detection signal can also contribute to how rapidly results are available. No matter how quickly an organism may metabolize a particular substrate, if the end products are slowly or weakly detected, the ultimate production of results will still be "slow."
Detection strategies for determining the end products of different metabolic pathways use one of the following: colorimetry, fluorescence, and turbidity.
Several identification systems measure color change to detect the presence of metabolic end products. Most frequently the color change is produced by pH indicators in the media. Depending on the byproducts to be measured and the testing method, additional reagents may need to be added to the reaction before lesult interpretation. An alternative to the use of pH indicators is the oxidation-reduction potential indicator tetrazolium violet. Organisms are inoculated into wells that contain a single, utiHzable carbon source. Metabolism of that substrate generates electrons that reduce the tetrazolium violet, resulting in production of a purple color (positive reaction) that can be spectro-photometrically detected. In a third approach, the substrates themselves may be chromogenic so that when they are "broken down" by the organism the altered substrate produces a color.
Some commercial systems use a miniaturized modification of conventional biochemical batteries, with the color change being detectable with the unaided eye. Alternatively, in certain automated systems, a photoelectric cell measures the change in the wavelength of light transmitted through miniaturized growth cuvettes or wells, thus eliminating the need for direct visual interpretation by laboratory personnel. Additionally, a complex combination of dyes and filters may be used to enhance and broaden the scope of substrates and color changes that can be used in such systems. These combinations hasten identification and increase the variety of organisms that can be reliably identified.
There are two basic strategies for using fluorescence to measure metabolic activity, to one approach, substrate-
fluorophore complexes are used. If a bacterial isolate processes the substrate, the fluorophore is released and assumes a fluorescent configuration. Alternatively, pH changes resulting from metabolic activity can be measured by changes in fluorescence of certain fluorophore markers. In these pH-driven, fluorometric reactions, pH changes result in either the fluorophore becoming fluorescent or, in other instances, fluorescence being quenched or lost To detect fluorescence, ultraviolet light of appropriate wavelength is focused on the reaction mixture and a special kind of photometer, a fluorometer, measures fluorescence.
Turbidity measurements are not commonly used for bacterial identifications but do have widespread application for determining growth in the presence of specific growth inhibitors, induding antimicrobial agents, and for detecting bacteria present in certain clinical specimens.
TUrbidity is the ability of partides in suspension to refrart and deflect light rays passing through the suspension such that the light is reflected back into the eyes of the observer. The optical density (OD), a measurement of turbidity, is determined in a spectrophotometer. This instrument compares the amount of light that passes through the suspension (the percent transmittance) with the amount of light that passes through a control suspension without partides. A photoelectric sensor, or photometer, converts the light that impinges on its surface to an electrical impulse, which can be quantified. A second type of turbidity measurement is obtained by nephelometry, or light scatter. In this case, the photometers are placed at angles to the suspension, and the scattered light, generated by a laser or incandescent bulb, is measured. The amount of light scattered depends on the number and size of the partides in suspension.
Was this article helpful?