Foliose lichen thallus rhizines
Cross section of the thallus of a foliose lichen found on a dead tree. The Fast Green has stained the upper cortex (right) green. Below this layer in the medulla are the algae (Safranin-O stained round cells) interspersed with the fungal hyphae (Fast Green) which predominate further below the surface. The lower cortex (left) has stained very dark. Extending further left are the rhizines which anchor the thallus on the ventral side to the substrate. These are bundles of hyphae. Specimen from the edge of the Dowagiac River (5/15). The source specimen is shown in: www.flickr.com/photos/14643312@N02/17758847712/ and www.flickr.com/photos/14643312@N02/17573214550.
Lichen are reported to be difficult to adhere to the slide. The hyphae in the medulla did not survive this staining protocol as well as Hematoxylin - Erythrosin B with everything else remaining constant. The following protocol for fixation is largely based on Chamberlain, "Methods in Plant Histology, 5th ed., 1905. Staining was per Johansen. Fixed 24 hours in aqueous chromic acid-acetic acid (3.7 g potassium dichromate/l + 10 ml glacial acetic acid/l). (The substation of potassium dichromate for chromic acid based on equivalent chromate conc. is reported to work as long as the pH is less than 3.4). Washed 24 hours in flowing water. Dehydrated in 10, 35, 50, 75, 85, 95, 99 % IPA in water, 6 hours each min. Infiltrated in xylene saturated with Paraplast for 2 days, followed by 2 changes of melted Praplast for 2 hours each. Embedded in Paraplast. Sectioned on a Spencer 820 microtome at 11 micron. Cleared in Xylene 2X, 10 min each. Rehydrated 99 (10 min), 95, 85, 70 % IPA, 2 min. each. Stained in Johansen's Safranin-O, Fast Green (24 hours in Safranin-O, 15 sec. in fast green). Cleared 2X xyene 5 min each. Mounted with DEPEX.
Photographed on a Spencer 42 petrographic polarizing microscope using an original magnification of 200X, using a Sony NEX 5N with a Leica MIKAS 1/3X adapter.
Foliose lichen thallus rhizines
Cross section of the thallus of a foliose lichen found on a dead tree. The Fast Green has stained the upper cortex (right) green. Below this layer in the medulla are the algae (Safranin-O stained round cells) interspersed with the fungal hyphae (Fast Green) which predominate further below the surface. The lower cortex (left) has stained very dark. Extending further left are the rhizines which anchor the thallus on the ventral side to the substrate. These are bundles of hyphae. Specimen from the edge of the Dowagiac River (5/15). The source specimen is shown in: www.flickr.com/photos/14643312@N02/17758847712/ and www.flickr.com/photos/14643312@N02/17573214550.
Lichen are reported to be difficult to adhere to the slide. The hyphae in the medulla did not survive this staining protocol as well as Hematoxylin - Erythrosin B with everything else remaining constant. The following protocol for fixation is largely based on Chamberlain, "Methods in Plant Histology, 5th ed., 1905. Staining was per Johansen. Fixed 24 hours in aqueous chromic acid-acetic acid (3.7 g potassium dichromate/l + 10 ml glacial acetic acid/l). (The substation of potassium dichromate for chromic acid based on equivalent chromate conc. is reported to work as long as the pH is less than 3.4). Washed 24 hours in flowing water. Dehydrated in 10, 35, 50, 75, 85, 95, 99 % IPA in water, 6 hours each min. Infiltrated in xylene saturated with Paraplast for 2 days, followed by 2 changes of melted Praplast for 2 hours each. Embedded in Paraplast. Sectioned on a Spencer 820 microtome at 11 micron. Cleared in Xylene 2X, 10 min each. Rehydrated 99 (10 min), 95, 85, 70 % IPA, 2 min. each. Stained in Johansen's Safranin-O, Fast Green (24 hours in Safranin-O, 15 sec. in fast green). Cleared 2X xyene 5 min each. Mounted with DEPEX.
Photographed on a Spencer 42 petrographic polarizing microscope using an original magnification of 200X, using a Sony NEX 5N with a Leica MIKAS 1/3X adapter.