Rapid identification of immobilized bacteria by aminopeptidase profiling
Abstract
At the present time a major goal of "improved" microbial identification techniques is to reduce the time required for identification. The turn-around time can be broken into two specific elements. The first is the growth period required to isolate and obtain enough sample to analyze, and the second is the time required to process the sample, including measurement and data reduction steps. Identification methods that utilize inherent enzyme reactions in the organism can provide a "measurement advantage" due to the chemical amplification that is present with such systems and should have the most success in reducing the "total" analysis time. One such technique is aminopeptidase profiling, which has been employed since the early 1970's to elucidate the metabolic characteristics of bacterial pathogens. The method is based upon microbial metabolism of non-fluorescent L-amino acid-$\beta$-napthylamide to produce the highly fluorescent tag, $\beta$-naphthylamine ($\beta$NA). Histograms that plot the normalized hydrolysis of these substrates provide a distinct aminopeptidase profile pattern which reveals various metabolic characteristics of that microbial species. This metabolic "fingerprint" can be utilized as a means for identification and differentiation of pathogen species. Conventional aminopeptidase methods require cell concentrations of approximately 10$\sp8$-10$\sp{10}$ cells/ml and an incubation period in the labeled substrates of 20 hours. In order to obtain the large number of cells required to perform this assay, a 36-48 hour growth period must precede the assay. An improved procedure is described which combines time-resolved fluorimetry and a non-destructive whole cell immobilization procedure. This method has reduced cell concentrations 80,0000 fold from the standard assay, has reduced the room temperature incubation period from 20 hours to 3 minutes, and has shortened the total turn-around time for the assay from 2.5 days to approximately 3-7 hours.
Degree
Ph.D.
Advisors
Lytle, Purdue University.
Subject Area
Analytical chemistry
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