Chitosan, a deacetylated product of an abundant naturally occurring biopolymer chitin, has been used in a range of applications, particularly in food and health areas, as an antimicrobial agent. In the work reported here Pinus radiata wood was impregnated with chitosan as an environmentally compatible organic biocide. It is important to understand micro-distribution of bioprotectants in impregnated wood products to properly evaluate their performance. We developed a novel microscopic techniques to visualise impregnated chitosan within wood tissues using light microscope and field emission scanning electron microscope (FE-SEM) in combination with a dye that specifically stained wood cell walls and a heavy metal stain that chemically react with chitosan. Sections were viewed with the light microscope without staining with a dye as well as after staining with the dye toluidine blue. Light microscopy was also undertaken on sections that had been stained with 1% aqueous osmium tetroxide (OsO4). For SEM observations, the sections were treated with OsO4 and then examined with the FE-SEM, first in the secondary electron imaging mode (SEI) and then in the backscattered electron imaging (BEI) mode, imaging the same areas of a section in both SEI and BEI modes. The preparation techniques employed and the combined use of light and scanning electron microscopy provided valuable complementary information, revealing that chitosan had penetrated into the cavities (cell lumens, intercellular spaces) of all sizes present within wood tissues and had also impregnated early wood cell walls.

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