The objective of the project was to develop a method employing fluorescence microscopy for the detection of legionella in industrial samples from a range of working areas. The method was intended for use in the BGMG hazardous substances measurement system as a time-saving alternative to existing detection methods. For this purpose, a number of fluorescence-based methods, such as the use of genetic probes and a range of dyes, were to be tested on laboratory and industrial samples. This was to be followed by standardization, quantification and validation of the methods.
In the first phase of the project, a fluorescence workplace equipped with the necessary microscopy apparatus was set up at the BGIA - Institute for Occupational Safety and Health. Fluorescence in-situ hybridization (FISH) was subsequently tested in conjunction with a range of oligonucleotide probes for the detection of legionella in environmental samples. This work was based upon the results of a previous project conducted in conjunction with the Jülich research centre and sponsored by the German Social Accident Insurance (DGUV). The hybridization technique developed in the "Development of a quick test for determination of legionella pneumophila by molecular biological methods" project No. FFFF0202 was to be optimized, and the time required for processing of the samples reduced as a result. In addition, a commercially available rapid test for the detection of legionella was to be examined for its suitability for use with industrial samples.
Based upon the studies conducted, the hybridization technique was developed further, the time required for processing of the samples reduced, and the quality of the fluorescence microscopy specimens improved. It was not possible at this stage to formulate a standard method for the detection of legionella from industrial samples which described direct detection of the legionella on a filter and computer-aided quantification of the cells. The reasons for this are, on the one hand, the sensitivity of the method to interference from chemical substances in these samples, and on the other, the associated microbiological flora, which is generally present here in high concentrations and which presents an obstacle to the automated microscopic detection of legionella. Computerized automated image analysis, however, first requires a reproducible analysis of the individual microscope images. Further work is required in this area in order for the hybridization and staining protocol to be optimized for different samples.
Like the conventional DIN detection method, the rapid test which was examined requires that single colonies be cultivated beforehand. Fluorescence microscopy is employed for analysis. As a detection method, it does not represent an improvement over the established method (cultivation on selective media and detection by means of a latex agglutination test), since the combination of commercial test system and fluorescence microscope entails relatively complex laboratory apparatus and expensive test kits.
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Messverfahren, Biologische ArbeitsstoffeDescription, key words:
legionella, fluorescence microscopy, molecular biology, gene probes, detection methods, fluorescence in-situ hybridization (FISH)