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Confocal Laser Scanning Microscopy (CLSM) of decayed wood
1998 - IRG/WP 98-10273
Confocal laser scanning microscopy (CLSM) is currently being used to examine biodegraded wood and is proving to be a useful new technique in this area of work. Non-invasive optical sectioning within a thick specimen (20 - 50 ┬Ám), coupled with post image processing techniques allows manipulation of images and 3-D reconstruction from serial sections. Glutaraldehyde can cause cell autofluorescence, and since wood cell components do not react with it, a relatively simple technique for localising fungal hyphae, using glutaraldehyde as a fixative, was developed. Subsequent use of probes specific for chitin provided superior images of fungal hyphae in wood. High resolution, sequential, 2-D images can be produced to determine the mode of fungal attack within a thick wood specimen in a dynamic way. Voxel 3-D reconstruction of a series of image stacks enabled stereo viewing of objects.
Ying Xiao, R N Wakeling, A P Singh

Visualising Bacteria in Wood Using Confocal Laser Scanning Microscopy (CLSM)
1998 - IRG/WP 98-10272
A fluorescent phospholipid probe was used in conjunction with confocal laser scanning microscopy (CLSM), to visualise bacteria which inhabit in radiata pine wood and degrade pit membranes. CLSM has the ability to collect fluorescent images through different emission filters at the same time, so it is possible to distinguish gram-positive and gram-negative bacteria in infected wood by counterstaining wood sections with specific fluorescent stains. Images obtained using CLSM were compared with those acquired using light microscopy (LM) and scanning electron microscopy (SEM). Strong fluorescence of the phospholipid probe made it possible to visualise bacteria in wood even when present in numbers too small to detect by LM or SEM.
Ying Xiao, A P Singh, R N Wakeling

Quantification of methylene bis thiocyanate in wood and its effect on a sapstain fungus Ophiostoma floccosum
2006 - IRG/WP 06-10591
The mobility and concentrations of MBT at different depths of wood billets (200 mm long, 40 mm in diameter) were determined using NMR spectroscopy and ICP-ASE, and then correlated the effect of wood MBT concentrations on growth of O. floccosum in a bioassay using stereomicroscopy and confocal laser scanning microscopy (CLSM). The 13C NMR spectra showed the presence or absence of MBT in wood but was unable to detect small concentrations. ICP-ASE however, produced quantitative data across the depth (40 mm) of the wood billets tested. Within 7 days of storage, MBT penetrated from surface to pith but showed significantly higher concentrations of MBT in surface wood compared to pith wood. Present study highlighted the benefit of using CLSM for fungal detection in wood. The information obtained from the ICP-ASE and the CLSM analysis suggested that to inhibit growth of O. floccosum, the MBT concentration needs to be greater than 55 ?g/g of dry wood. Due to its high detection capacity, ICP-ASE is identified as an excellent tool for MBT detection and quantification in wood.
T Singh