Morphological reconstruction during cell regeneration in the ciliate Spirostomum ambiguum.

When the ciliate Spirostomum ambiguum is transected into two pieces, both fragments regenerate and proliferate. In the anterior fragments, which have lost their contractile vacuoles due to transection, new contractile vacuoles were formed at their posterior ends in a few minutes. When the cells were cut into three pieces, new contractile vacuoles were formed in the anterior and middle fragments, both at their posterior ends. Thus, the anterior-posterior axis of S. ambiguum was maintained after transection. Morphological repair, including the formation of the contractile vacuole, was also observed when only the anteriormost portion was transected to cut out a small fragment that did not contain part of the macronucleus. Scanning electron microscopy was performed to observe changes in the shape of the cleavage surface of S. ambiguum during the wound healing process. Within minutes after cutting, the cut surface was covered with a cilia-free membrane, preventing leakage of cytoplasmic contents. The surface of the cut area then rounded with time and was covered with cilia, completing the repair of the cut area in about one day.

SEM observation of non-fixed and water freeze-dried Spirostomum ambiguum.

We aimed to freeze-dry the ciliate Spirostomum ambiguum, obtained from water, without fixation and observe it using scanning electron microscopy (SEM). Living cells were placed on a specimen stub and frozen upon contact with a Cu block kept at either -80 °C or -100 °C. Samples were then freeze-dried and observed by SEM. In most cases, no damage to the specimen due to ice crystal formation was observed. Because of the instantaneous freezing, the metachronal wave of cilia on the body surface of the ciliate was well preserved. Approximately 70-80% of cells were preserved in the contracted state due to inevitable exposure to mechanical vibration immediately before freezing. The remaining samples were preserved in a fully-extended state. Morphometric measurements of the cell surface showed that in the extended state, ciliary rows were almost parallel to the long axis of the cell, whereas in the contracted state, they were twisted in a left-handed helix at an angle of 45-65°. The distance between adjacent cilia along a ciliary row was 1.88 ± 0.43 µm in the extended state and 1.32 ± 0.41 µm in the contracted state (mean ± SD). However, the spacing between adjacent ciliary rows remained unchanged.