Comparison of Different Fluorescence and Non-Fluorescence Staining Techniques for Rapid Detection of Airborne Micro-Organisms Collected on Room Temperature Vulcanizing (RTV) Silicones from Generated Aerosols and from Ambient Air

Abstract

For rapid detection, airborne micro-organisms can be collected by impaction on silicone sealants immediately followed by fluorescence or non-fluorescence staining and microscopy. At present, it is not known which combination of silicone, dye, and additional treatment provides best staining results. In laboratory experiments, selected groups of micro-organisms were prepared, aerosolized in a test tubing system, and collected by impaction on microscope slides that were coated with a silicone sealant before sampling. After sampling, different staining procedures were performed and the stained particles were counted under a fluorescence microscope. Two different sealants, 16 different dyes and staining procedures, and 21 additional reagents (buffers, solvents, and acids/bases) that may improve staining results were tested. In order to estimate the possible loss rate of particles caused by staining, washing, and additional treatment, the particles adhered to the silicones were counted under the microscope both before and after the procedures. The average losses of adhered micro-organisms and latex particles depended on the type of the silicone, the time between impaction of the particles and the application of the dye, the particle size, and the kind of treatment. Particle losses were less than 1% in most cases when staining solutions were applied 5 min after sampling. Most of the tested dyes showed good or even excellent staining properties on both types of silicone sealants and allowed to distinguish collected airborne bacteria from background debris. Best performance was achieved by “Grimm Silicone 1228” and the dye SYBR® Safe using a portable fluorescence microscope. The method was also tested successfully for the rapid detection of airborne micro-organisms in animal housings.

Copyright 2012 American Association for Aerosol Research

Acknowledgments

This research project was funded by the Federal Ministry of Economics and Technology based on a decision of the German Federal Parliament.