Calcium oxalate crystals in the green alga Spirogyra hatillensis (Zygnematales, Chlorophyta)

Vegetative cells of the freshwater green alga Spirogyra hatillensis Transeau contain numerous cruciate inclusions that are up to 34 mum long. A crystalline nature was shown by birefringence in polarized light. Rather than occurring free in the large central vacuole, these inclusions were associated with cytoplasmic strands that suspended the nucleus in a cytoplasmic embayment of the central vacuole and with the parietal cytoplasm, which contains the spiral chloroplasts. Some crystals moved directionally along the cytoplasmic strands, and their movement was arrested by cytochalasin B, indicating that actin microfilaments had a role in crystal movement. Solubility tests showed that the inclusions were composed of calcium oxalate; they were not soluble in concentrated acetic acid or sodium hypochlorite, but they dissolved rapidly without effervescence in weak hydrochloric acid. The Yasue cytochemical test for calcium oxalate stained the crystals intensely. The crystals were associated with a water-soluble organic matrix that retained the shape of the crystal after demineralization. Scanning electron microscopy showed crystals to have arms with tapered tips and rhombohedral cross sections or with truncated ends and rectangular sections. Crystal growth in the Z-axis occurred at the intersection of the axes as well as at other sites along the arms. Transmission electron microscopy showed the presence of bounding membranes around the mineral inclusions. A colorimetric enzymatic test for oxalate was used to quantify this compound in S. hatillensis, and it was also used in conjunction with microscopic examination. Neutral red was found to accumulate in numerous small vacuoles, up to 2 mum in diameter, that were present in the parietal cytoplasm and in the cytoplasmic strands supporting the nucleus. In filaments undergoing conjugation as part of sexual reproduction, unfused cells contained crystals, but zygotes did not.