Growth is limited by substrate transfer from the solid phase to the aqueous phase. Maximization of the surface area of the solid substrate is one strategy to maximize contact of the cells with substrate. Kiyohara et al. (59) dissolved phenan-threne in ether to a concentration of 10% (wt/ vol) and sprayed the liquid onto the surface of agar plates. The ether immediately vaporizes andleaves a fine layer of the solid substrate on the surface of the plate. If the plate was previously inoculated, the ether is toxic to the cells. Inoculation after formation of the solid substrate layer can be difficult, as the film frequently breaks if an inoculating loop is used. Problems are encountered when the inoculum is pipetted to the solid layer since the layer is hydrophobic. When successful, however, it is immediately apparent which colonies that develop are able to metabolize the hydrocarbon; a cleared zone is easily seen around the colony (Fig. 6.2). A similar approach was adopted by Bogardt and Hem-mingsen (15), but soil and water dilutions were incorporated into an agarose overlayer with fine particles of phenanthrene. The overlayer was poured onto an agar mineral salts underlayer, and phenanthrene-degrading colonies were also recognized by a halo of clearing.
To overcome the problems with the sprayplate method, Alley and Brown (3) described a technique in which the solid substrate was sublimed onto the surface of the agar plate after inoculation. This technique is more controlled than the spray-plate technique and allows a reproducible quantity of substrate to be delivered without the use of toxic solvents.
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