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In biofilms, the matrix of extracellular polymeric substances (EPSs) retains water in the interstitial region of the EPS. This interstitial water is the ambient environment for microorganisms in the biofilms. The nutrient condition in the interstitial water may affect microbial activity in the biofilms. In the present study, we measured the concentrations of dissolved organic nutrients, i.e., saccharides and proteins, contained in the interstitial water of biofilms formed on the stones. We also analyzed the molecular weight distribution, chemical species, and availability to bacteria of some saccharides in the interstitial water. Colorimetric assays showed that the concentrations of saccharides and proteins in the biofilm interstitial water were significantly higher (ca. 750 times) than those in the surrounding lake waters (p < 0.05). Chromatographic analyses demonstrated that the saccharides in the interstitial waters were mainly of low molecular-weight saccharides such as glucose and maltose, while proteins in the interstitial water were high molecular-weight proteins (over 7000 Da). Bacterial growth and production of EPS occurred simultaneously with the decrease in the low molecular-weight saccharide concentrations when a small portion of biofilm suspension was inoculated to the collected interstitial water, suggesting that the dissolved saccharides in the interstitial water support bacterial growth and formation of biofilms.

We have developed a simple, sensitive and reliable assay procedure for cyclosporin A (CyA), a modified fluorescence polarization immunoasay method incorporating fat emulsion (FE-FPIA), to determine the CyA content in rat skin. The conventional fluorescence polarization immunoassay (FPIA) method for CyA using a commercially available FPIA kit, TDX[○!R] cyclosporine monoclonal whole blood, was modified. A fat emulsion for intravenous infusion, Intralips[○!R], was incorporated for dissolving the CyA extracted from the skin tissue, and a mixture of MeOH/purified water was used as the sample pretreatment medium instead of the precipitation reagent in the conventional FPIA kit intended for whole blood samples. These modifications enabled us to produce a reliable and the sensitive assay of CyA in skin tissue. The reproducibility (coefficient of variation), detection limit, and assay time for FE-FPIA were below 2%, 25 ng/ml, and about 24 min/24 samples, respectively, and were comparable with those for the whole blood samples determined by the conventional FPIA. Pre-purification of samples required by the HPLC assay is not needed in the FE-FPIA method. The usefulness of the FE-FPIA method in evaluating the topical pharmacokinetics of CyA in skin is discussed.