Inside a Sporophyte
Longitudinal cross section through the centre of a developing sporophyte capsule from a moss. The Calyptra is at the left and the stem (not shown) connecting it to the plant is at the right. The dark staining cells surrounding the columella in the center contain the developing spores. A photo of the moss from which the sporophytes were obtained is at www.flickr.com/photos/14643312@N02/17164418555/. Stained with Flemming's Safranin, Gentian Violet, Orange Stain, substituting Crystal Violet for Gentian Violet. This specimen was sectioned at 7 micron. In hindsight, this was too thin. My next attempt will be at 12 micron.
The following protocol is largely based on Chamberlain, "Methods in Plant Histology, 5th ed., 1905. 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, min 6 hours each. Cleared in IPA:Xylene at 3:1, 2:1, 1:3,1 hour each and pure xylene for 10 min. Infiltrated in xylene saturated with Paraplast for 5 days, followed by 3 changes of melted Praplast for 2 hours each. Embedded in Paraplast. Sectioned on a Spencer 820 microtome at 7 micron. Applied to a charged slide from a tissue bath. Cleared in Xylene 2X, 10 min each. Rehydrated 99, 95 % IPA,10 min. each. Stained 1% Safranin in 50% IPA 24 hr. Differentiated 50 % IPA, 10 min. Stained 1 % aq Crystal Violet 10 min. Dip 4X in water. Stain 1 % Orange B 1 min. Dip 4X 95 % IPA, agitate 5 sec in 99 % IPA. Rinse off IPA with Eugenol (clove oil), wick off. Pour Eugenol over slide. Cleared 2X Xylene 2.5 min each. Mounted with DEPEX.
Bright field image at an original magnification of 20X on a Nikon MS invetted microscope using a Sony A7R with a Leica MIKAS 1/3X adapter.
Inside a Sporophyte
Longitudinal cross section through the centre of a developing sporophyte capsule from a moss. The Calyptra is at the left and the stem (not shown) connecting it to the plant is at the right. The dark staining cells surrounding the columella in the center contain the developing spores. A photo of the moss from which the sporophytes were obtained is at www.flickr.com/photos/14643312@N02/17164418555/. Stained with Flemming's Safranin, Gentian Violet, Orange Stain, substituting Crystal Violet for Gentian Violet. This specimen was sectioned at 7 micron. In hindsight, this was too thin. My next attempt will be at 12 micron.
The following protocol is largely based on Chamberlain, "Methods in Plant Histology, 5th ed., 1905. 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, min 6 hours each. Cleared in IPA:Xylene at 3:1, 2:1, 1:3,1 hour each and pure xylene for 10 min. Infiltrated in xylene saturated with Paraplast for 5 days, followed by 3 changes of melted Praplast for 2 hours each. Embedded in Paraplast. Sectioned on a Spencer 820 microtome at 7 micron. Applied to a charged slide from a tissue bath. Cleared in Xylene 2X, 10 min each. Rehydrated 99, 95 % IPA,10 min. each. Stained 1% Safranin in 50% IPA 24 hr. Differentiated 50 % IPA, 10 min. Stained 1 % aq Crystal Violet 10 min. Dip 4X in water. Stain 1 % Orange B 1 min. Dip 4X 95 % IPA, agitate 5 sec in 99 % IPA. Rinse off IPA with Eugenol (clove oil), wick off. Pour Eugenol over slide. Cleared 2X Xylene 2.5 min each. Mounted with DEPEX.
Bright field image at an original magnification of 20X on a Nikon MS invetted microscope using a Sony A7R with a Leica MIKAS 1/3X adapter.