A method was developed to deposit a visible layer of water-insoluble compounds via sublimation onto the surface of solid media. The compound is sublimed from a heated aluminum dish containing the compound onto the surface of an inverted, ice-cooled, inoculated agar petri dish. The method results in the deposition of a thin, even layer on the agar surface without the use of solvent. After incubation, clearing zones around colonies indicate the presence of compound-degrading microorganisms.In 1982, Kiyohara et al. described a method for the screening of hydrocarbon-degrading bacteria (9). This method involved spraying polycyclic aromatic hydrocarbons (PAHs) with a thinlayer chromatography sprayer onto mineral salts agar plates. The spray-plate method was developed because water-insoluble compounds cannot be incorporated into agar in the usual way and must be dissolved in organic solvents. Colonies showing clearing zones around them after incubation were picked as degrading species. Because many of these compounds are carcinogenic, spraying had to be carefully performed in a hood with the appropriate personal safety equipment.The spray-plate method was reviewed in Manual of Environmental Microbiology (13), and many publications cite this method as the technique used for initial isolation of PAHdegrading bacteria (3-6, 8, 10). However, several problems exist with the spray-plate method. The compound must be solubilized with acetone or ether, which may have toxic effects on the bacteria or may be used as an alternate carbon source. If the spray-plate method is implemented before streaking, the compound layer may be disrupted. When plates are sprayed after inoculation, colonies may spread or be disrupted by impacting solvent droplets. The quantity of PAH sprayed onto the agar cannot be easily controlled, compound may be lost by overspraying, and overspraying may result in contamination of large areas of the hood. Because it is difficult to spray plates in an aseptic manner, this method cannot confidently be used for final isolation or enumeration of PAH-degrading bacteria. Other methods, such as spread-plating or the agar-overlay technique (1), involve either direct contact of solvent with the agar or incorporation of solvent directly into the agar. A sublimation method was developed to avoid the problems described above. This method safely deposits an even, thin, visible layer of compound onto the agar surface without the use of solvents.(This article was presented as poster Q-201 at the 99th General Meeting of the American Society for Microbiology in Chicago, Ill., on 1 June 1999.)The sublimation system consists of two aluminum dishes, an inoculated mineral salts agar (MSA) standard plastic petri plate (100 by 15 mm), a thermometer, and a heated Pyrex petri dish bottom (diameter, 140 mm) filled to a depth of ϳ16 mm with sand (Fig. 1). A thermostatically controlled, aluminumtop hot plate (type 2200; Thermolyne) with a sand bath was placed in a fume hood. The sand was packed and smoothed to a flat surface. An empty al...
