Since coralline algal cells contain calcium carbonate, they must be decalcified prior to sectioning. Before specimens are decalcified for sectioning, however, some preliminary preparation must be carried out. A fairly large specimen should be examined under a dissecting microscope, and an area chosen from whcih to remove a piece for further study. Thoroughly study as much material as possible under the dissection microscope before you begin taking out pieces for decalcification. Try to remove a small piece of a margin so that details of the medulla can be seen in the section. Look for different sizes and stages of conceptacle development so that you stand a reasonable chance of obtaining more than one stage of the life cycle in sections. Pieces should be excised and cut to not more than 5 mm diameter, preferably less if possible. For material being sectioned to study conceptacles, the lower part of the thallus can be carefully chipped away to reduce the thickness of the material and decrease the chance of damage from incomplete decalcification. Epiphytic species on fleshy algae or seagrasses can be decalcified while still attached to the host.
The solution which should be used for decalcification will depend on the type of sections to be cut, and it may be necessary to experiment with different solutions to obtain the best results with your material. The folowing decalcification solutions have been found useful for various purposes during this work:
10% nitric acid
This solution gives the quickest results, but the large bubbles that form during decalcification can severely distrupt cells. It works best with relatively thin specimens, up to 500 mm in thickness, but can distort cellular relationships in larger, thicker specimens. Nevertheless, I use it routinely when I need to make sections quickly. Specimens up to 500 um thickness can be decalcified in 0.5-2 hours, while very thick specimens may require up to 6 hours or more for complete decalcification. Decalcify until no bubbles are visible when the material is examined under a dissection microscope, but check thicker specimens thoroughly as a still calcified core may remain after all bubbles cease. After declacification is complete, the material should be placed in 80% ethanol, changed after an hour to prevent acid damage to the microtome blade (unless disposable blades are being used), and stored for at least two hours prior to being sectioned.
Perenyi's fluid
This consists of a solution of 4 parts 10% nitric acid, 3 parts 90% ethanol, and 3 parts of 0.5% chromic acid. It is almost as quick as plain 10% nitric acid, but smaller bubbles cause less damage to the material, and it sections tend to cut better on a freezing microtome. Process as per 10% nitric acid.
1% nitric acid (in 5% formalin-seawater)
This can be used to leave specimens overnight for decalcification. It seems to cause less damage to material than either of the above solutions, but is rather slow for thicker specimens. Process as per 10% nitric acid.
5% acetic acid (in 5% formalin-seawater)
The results obtained with this solution are generally better than those obtained with Perenyi's fluid, but decalcification takes much longer. The smaller bubbles produced by this solution cause even less damage to the material, but thick specimens can require up to three days and several changes of solution for effective decalcification. Process as per 10% nitric acid. If there is still calcium carbonate left after 1-2 days, the material can be transferred to Perenyi’s fluid for further decalcification.
EDTA
This is the preferred solution for decalcifying material for transmission electron microscopy. Specimens are decalcified over several days in a refrigerator, and the medium is changed several times. See TEM preparation for more information.