Zoology Publications from Victoria University of Wellington—Nos. 58 to 61
Material and Methods
Material and Methods
COLLECTION AND CULTURE OF ANIMALS: Specimens of S. tenella were obtained from Kau Bay, Wellington, N.Z. In this region the animals are found in association with Obelia geniculata colonies growing attached to fronds of the bladder kelp Macrocystis pyrifera. Fronds of the kelp bearing dense populations of Obelia were transported to the laboratory in buckets of sea water where they were examined for colonies of Syncoryne. The Syncoryne colonies were then carefully cut from the kelp fronds with sharp scissors. Care was taken that as little kelp as possible was left attached to the colonies, which were then placed in finger-bowls 4″ in diameter × 1½″ deep containing filtered sea water. The filtered sea water was beaten vigorously with a glass rod to stir air into it before it was used in the culture dishes. Aeration by a mechanical pump hindered attachment of colonies of S. tenella to the culture dishes. It was necessary to change the water in the dishes every day until the remaining pieces of kelp attached to the colonies had disintegrated. After this time the water was changed every two or three days.
The animals were fed twice a week on brine shrimp (Artemia) newly hatched from sea water. For the first week, when colonies were still attaching to the culture dishes, brine shrimp were presented to hydranths individually, on the end of a probe. When colonies had become attached however, the method of feeding was to pipette 20-30cc sea water containing a dense population of brine shrimp into the culture dishes. The dishes were then left for about half an hour, with occasional stirring of the water or rotation of the dishes to ensure an even distribution of brine shrimp to the colonies. During this time most hydranths had ingested 3-4 brine shrimp and some had ingested as many as 8 or 10. The number ingested was determined by counting the eyespots visible in the coelenteron. After this period of time each culture dish was emptied and any brine shrimp adhering to the bottom or sides of the dishes were removed by swilling fresh sea water around in the dishes, and filtered sea water was placed in them once more. The water was replaced again after 12-18 hours to remove digestive wastes expelled by the polyps.
Colonies of S. tenella kept in this way for three months remained healthy, and grew to a larger size than had been observed in their natural page 3 habitat. Usually hydranths grow from short, unbranched caulomes in their natural habitat but when the hydroid is cultured in bowls tall, branched hydrocauli are often produced. Gonophores were produced only rarely on cultured animals.
While culturing S. tenella in this way it was found that small pieces of Obelia geniculata colonies, if obtained free of bladder kelp, would survive a considerable time in the culture dishes. One small colony bearing 3 hydranths survived for one month. Little growth of this colony occurred, however, and the hydranths required feeding more often than those of S. tenella.
ANAESTHETIZATION & FIXATION. For the study of live individuals, hydranths were detached from the colony with a sharp probe, at the junction of hydrocaulus and stolon. The animals were transported from culture dishes to petri dishes or cavity slides with pipettes.
Fixatives employed were 10% formalin in sea water, Baker's formol/calcium (Pearse, 1960) and Lillie's FAA (Pearse, 1960). All fixatives were allowed to act overnight at room temperature. Specimens fixed in FAA were then removed to 70 % ethanol for storage, but those fixed in the other fluids were stored in their respective fixatives.
Some animals were anaesthetized prior to fixation by placing them in sea water containing magnesium chloride in the proportions 5 sea water: 1 of 7% aqu. MgCl2. 6H2O. After approximately 15 mins. the animals were rendered insensible in an extended position, and were placed into fixative. Some specimens were starved for one week prior to fixation, and others were fixed 12-18 hrs. following feeding.
HISTOLOGICAL & HISTOCHEMICAL METHODS: Most specimens were embedded in paraffin wax and sectioned at 5μ thickness on a Cambridge rocking microtome. Serial transverse and longitudinal sections were prepared.
Some of the formol/calcium fixed specimens were embedded in 15% gelatin and cut at 10μ thickness on a freezing microtome, and some of the FAA fixed animals were embedded in celloidin and sectioned at 15μ.
The staining methods employed have been described elsewhere (Wineera, 1971). An additional method used was periodic acid-Schiff followed by alcian blue staining followed by naphthol yellow S staining. This method demonstrates neutral mucopolysaccharides (and other PAS positive substances), acidic mucopolysaccharides, and protein in the one preparation. It is particularly useful because the naphthol yellow S stains nuclei as well as various protein aggregations. Hereafter it will be termed the PAS/AB/NYS technique. Periodic acid oxidation was for 5 min. in 0.5% HIO4, and staining in Schiff's solution (prepared according to Coleman (cited in Gray, 1954)) was for 20 min. The alcian blue was used as a 0.1% solution in acetic acid at pH 2.6 for 20 min. and the naphthol yellow S was employed according to Deitch (1955). Control slides bearing serial sections were treated either in 0.02N HCl containing 2 mg. pepsin/ml for 3 hrs. at 37°C, or in 0.05 m. borate buffer containing 0.2 mg. trypsin/ml for 30 min. at 37°C prior to the staining schedule. Test slides were placed in 0.02N HCl or in 0.05 m. borate buffer without enzymes at 37°C for a similar time before staining.page 4
Dissociated cells were obtained using the maceration techniques of Goodrich (1942) and the Hertwigs (1879, cited in Lee 1924). These were examined by phase contrast microscopy.