# Atlas of Micromorphological Degradation of Archaeological Birch Bark
## 2 Materials and Methods
### Description of Embedding and Sectioning  
Samples 1-3, 6-8 and 12 were wet, while the others were already air-dried. They were all documented and cut with a razor blade to a rectangular size of about 7 mm \(\times\) 3 mm. The cutting provided information about the brittleness and risk of delamination. First attempts showed that non-embedded sectioning did not allow for a thorough study of deterioration due to the separation of the layers during cutting. Further, the lumen content of the thin-walled cells obscured the cell wall study and had to be removed [25]. After test staining with safranine aniline blue, toluidine blue, a polychromatic stain commonly used to highlight fungal structures in plant cells walls [70] or to examine erosion bacteria degradation in waterlogged wood [71], was used.  
Figure 6: **Left**: birch bark fragments from Lake Moosee (521 m a.s.l) were recovered during an excavation in 2011 from a waterlogged sediment. Small, waterlogged fragments floating on water retain phloem, indicating that they were not harvested from the tree but detached naturally. During cutting, many fractures occurred, and small pieces broke off. **Right**: sample 12 does not have original white colour apart from a fresh fracture with splintered surface. Inner bark has dark-brown colour and is very brittle, and lenticels are not well defined and have frayed edges.  
Figure 7: Sample 13 was extracted from a birch bark fragment recovered in the Kosackenberg cave. Inner and outer surfaces have reddish-brown overlay with white blooming. Normally alternating white and reddish-brown layers are not present, as reddish-brown colouring of thick-walled cells is completely lost. Lentcels have also lost their normally dark colour and are hardly distinguishable from normal phlem. Fragment is very light and extremely fragile; handling leads to fragmentation and loss.  
Thin sections were obtained by immersing the samples in 70, 80 and 96% ethanol (Alcosuisse, Switzerland) and twice in 100% ethanol (Merck, Darmstadt, Germany) for at least 3 h each at room temperature. The samples were then immersed twice in 100% acetone (Merck, Darmstadt, Germany) for at least 2.5 h at room temperature and in increasing concentrations of Epon/acetone solution (1:2, 1:1, 2:1). Finally, they were embedded in 100% Epon (Fluka, Switzerland) and left to harden at 60 \({}^{\circ}\)C for 5 days.  
Thin sections of 1 \(\upmu\)m were produced for LM with a UC6 ultramicrotome (Leica Microsystems, Vienna, Austria) and put on glass slides with 1 mL of distilled water. The glass slides were placed on a hot plate with a temperature of 55 \({}^{\circ}\)C to stretch the sections. After evaporating the water, they were stained with an aqueous solution of 0.5% toluidine blue O (\(w/v\)) (Merck, Darmstadt, Germany) for 3 min at 55 \({}^{\circ}\)C. They were then washed in water, dried on the hot plate and mounted with Entellan(Merck, Darmstadt, Germany).  
Ultrathin sections (75 nm) were cut from the same embedded samples for electron microscopy (TEM). The sections, mounted on 200 mesh copper grids, were stained with Uranyless (Electron Microscopy Sciences, Hatfield, PA, USA) and lead citrate (Leica Microsystems, Vienna, Austria) with an ultrastainer (Leica Microsystems, Vienna, Austria).