Dissolution of Natural Mineral and Man-Made Vitreous Fibers in Karnovsky's and Formalin Fixatives

Abstract

Karnovsky's fixative and formalin are commonly used for fixing and storing lung tissue from animal inhalation studies. The dissolution rates of three natural mineral fibers (wollastonite, chrysotile, and crocidolite asbestos) along with eight man-made vitreous fibers (MMVF; representing a broad range of commercial products, including aluminosilicates, mineral wools, borosilicates, and lime silicates) were determined in Karnovsky's fixative and formalin buffer. Twenty-five milligrams of fiber was mixed with 25 ml of fixative or buffer in plastic bottles and maintained at room temperature with gentle agitation for up to 30 days. At specified times, samples were filtered through 0.45– or 0.22–γm millipore filters, the residues were saved for SEM or TEM analyses, and the filtrates were analyzed for dissolved silica using inductively coupled plasma spectroscopy. Characteristic silica solubility curves were determined for each fiber type, and average silica solubility rates for 30 days were calculated. In Karnovsky's fixative, solubility rates (%SiO₂/day) ranged from ∼0.04 for chrysotile or crocidolite to 7.7 for wollastonite. The dissolution rate of silica from the MMVFs in Karnovsky's fixative varied among the fiber types tested from 0.004 to 1.4. Formalin buffer was more aggressive than Karnovsky's fixative to the fibers. The average silica solubility rate in formalin buffer varied from 0.06 for chrysotile or crocidolite to 3.7 for wollastonite. The dissolution rate of silica from the MMVFs in formalin buffer varied from 0.02 to 1.6. SEM photomicrographs comparing fiber surface morphologies before and after leaching for 30 days in Karnovsky's fixative showed that for some of the fibers studied, surface changes were observed. These findings demonstrate that fibrous materials can dissolve to a significant extent in fixatives, and that formalin buffer is more aggressive to fibers than Karnovsky's fixative. Caution should therefore be used in the selection of fixatives and in the storage of lung tissues from fiber inhalation studies, especially when those tissues will be used for fiber recovery and analysis.