Materials and Methods

COLLECTION OF ANIMALS: The animals were collected from Houghton Bay, Wellington, N.Z. by removing them from rocks at low water. A cold chisel and hammer were used to fracture the rock on which the animals were resting, in order to obtain the specimens attached to small pieces of rock. The animals were then placed in quart jars filled with sea water and were transported immediately to the laboratory. Here they were set in aquaria (10 animals to a tank measuring 27 × 17 × 12 cms.). The time lapse between removal from the sea and placement in aquaria was about 1½ hrs. The water in aquaria was replaced after 12 hrs., and thereafter every 2 weeks. No aeration of tanks was necessary. The animals were fed chopped mussel (Mytilus) every two weeks, 12 hrs. before the water was changed.

ANAESTHETIZATION: The animals proved difficult to anaesthetize in an extended condition. The best narcotics of the several used were menthol, chloral hydrate-menthol, and isotonic magnesium chloride in sea water. The chloral hydrate-menthol mixture consisted of 4 g. menthol 6 g. chloral hydrate and sufficient water to make a thick paste. This mixture was added drop by drop, over a period of 2-3 hrs. to a beaker of sea water containing an expanded anemone (Le Thi, unpublished thesis). After this time the tentacles no longer responded to touch, and the beaker water was pumped into the coelenteron with a rubber bulb pipette. This served to complete anaesthetization, and to expand the animal slightly. The menthol method of narcotization used was that of Lee (1924), and the method using magnesium chloride in sea water was applied according to Batham, Pantin, and Robson (1960).

It must be noted that none of the methods used was entirely satisfactory with I. Olivacea, and resulted, generally, in the oral disc and tentacles being somewhat retracted inside a slightly overinflated column. The results with any one method also varied with different individuals.

FIXATION: When anaesthetized, the animals were either removed to buffered 4% formaldehyde (Pease, 1964), or formalin was added to a beaker of fresh sea water containing the animal to make a concentration of 10% formalin. In both cases fixative was repeatedly pippetted down the throat of the animals for several minutes to ensure adequate fixation of internal parts. Fixatives were allowed to act for at least 12 hrs.

HISTOLOGICAL METHODS: For study of sections, fixed animals were embedded in paraffin and sectioned longitudinally and transversally at 5-6μ. Some animals were embedded in 20% gelatin and sectioned on a freeze microtome. The following histological staining methods were employed: page 3 1. Heidenhain's iron haematoxylin-orange G; 2. Delafield's haematoxylin-eosin-fast green FCF (Wineera, 1968); 3. Mallory's triple stain (Gray, 1954); 4. Mallory/Azan method (Gurr, 1962); 5. The Falg technique (Gurr, 1962); 6. Gordon and Sweets' method for reticulin (Pearse, 1960); 7. Fullmer and Lillie's method for elastic fibres (Fullmer and Lillie, 1956); 8. The Allochrome procedure (Lillie, 1951).

Histochemical methods used were:

A.	Polysaccharides: 1. PAS reaction with and without diastase digestion and pyridine extraction (Pearse, 1960). The Schiff solution was prepared according to Coleman's method (cited in Gray, 1954); 2. Mowry's method (1958) for acidic polysaccharides; 3. Toluidine blue staining (5 min. in 0.1% toluidine blue in 30% ethanol). Sections were examined in water prior to dehydration and mounting; 4. Low temperature sulphation (Moore and Schoenberg, 1957) followed by toluidine blue staining as in (3) above.
B.	Proteins: 1. Mercury/bromphenol blue method (Pearse, 1960); 2. Naphthol yellow S staining (Deitch, 1955); 3. The Sakaguchi reaction for arginine (Pearse, 1960); 4. The Millon reaction for tyrosine (Casselman, 1959); 5. The DMAB-nitrate method for tryptophan (Pearse, 1960).
C.	Nucleic Acids: 1. The Feulgen reaction (Pearse, 1960); 2. Methyl green-pyronin staining. Chloroform washed methyl green and pyronin Y made up according to Kurnick (cited in Pearse, 1960) were used in the proportions 10 parts of methyl green solution to 8 parts of pyronin Y solution to 27 parts of distilled water. The staining time was 6 min. Ribonuclease digestion (the enzyme was from Sigma) by a solution of glass distilled water containing 1 mg. enzyme/ml. water was performed for 3 hrs. on some slides before staining.
D.	Lipids: 1. Staining with oil red O in 60% isopropyl alcohol (Casselman, 1959) (frozen gelatin sections used). Some slides were treated with pyridine at 60°C for 24 hrs. before staining. One slide was stained with oil red O and the positions of positive staining sites were recorded using a microscope stage micrometer. The slide was then extracted in hot pyridine (as stated above), restained in oil red O and examined.
E.	Cambination Methods: 1. Himes and Moriber's triple stain (1956) for DNA, protein, and polysaccharide; 2. Mowry colloidal iron-PAS; 3. Mowry colloidal iron-PAS-naphthol yellow S (Mowry/PAS/NYS) with and without prior treatment in pepsin (2 mg./ml. in 0.02 N HCl-acetate buffer at 37°C for 2 hrs.). Slides not treated with pepsin were placed in buffer solution at 37°C for a similar time.

Additional Methods: 1. Small pieces of body wall were post fixed in 1% OsO₄ in distilled water, and embedded in araldite according to Richardson, Jarett, and Finke (1960). Sections were cut at 0.5 and 1.0μ on a LKB ultrotome, and placed on albuminized slides. Staining methods used were the methylene blue-Azure II procedure of Richardson et al (1960), and the PAS test. 2. Dissociation preparations of the body wall page 4 were prepared using the maceration technique of Goodrich (1942) and from trypsin digestions (the enzyme used at 0.5% in sea water at room temperature for 4 hrs.) on fixed and fresh tissue. "Milton" and "Janola" (commercial solutions of sodium hypochlorite) were also used as dissociating agents, both on fresh and fixed tissue. The cells obtained by these techniques were examined by phase contrast microscopy. Ectoderm and endoderm layers of the body wall were isolated after partial dissociation by scraping one or other from the surface. 3. Determination of ciliary beats. Particles of charcoal were placed on the column, oral disc, tentacles, and the pharynx surface with the aid of a binocular microscope. The animal was then studied to determine whether or not the particles were moved over these body surfaces. 4. Staining with reduced methylene blue for nervous elements of the body wall. Pieces of body wall were cut from well expanded anaesthetized animals and stained, vitally, according to the method of Batham, Pantin and Robson (1960). The reduced methylene blue was prepared according to Pantin (1948). Tissue was examined in sea water under a cover glass prior to fixing in 5 % ammonium molybdate and the preparation of permanent whole mounts or paraffin sections.