Biological Bulletin Publications, Classic Resources:  H. D. RUSSELL, 1963: pp 68-70

SOME RECENT METHODS FOR NARCOTIZATION, KILLING, FIXATION, AND PRESERVATION OF MARINE ORGANISMS.

Appendix I.
(1) Propylene Phenoxetol. (footnote 8)
(2) Sevin and Rapid Freezing .............. M. R. Carriker (footnote 9)

(1) Propylene phenoxetol: for many bivalves, particularly those in which the valves do not close tightly; for gastropods in which the shell is wanting; and for some tunicates. It is important that the narcotic have access to the soft parts; in the case of tightly-closing bivalves this can be facilitated by pegging the valves open with slivers of wood while the animals are siphoning actively.

Narcotization may be accomplished by either of the following two ways: (a) Shake 5 ml of propylene phenoxetol in 15-20 ml of seawater to produce a fine emulsion and add this to a small quantity of still seawater containing the actively siphoning specimens. Within a half hour or so animals should be completely relaxed and may be fixed in this condition without causing any contraction of the tissues. (b) Add the phenoxetol (a quantity not to exceed 1% of the volume of seawater present) gently to the vessel of still seawater containing the animals, so that it collects as a globule on the bottom of the container. Organisms may be left in the solution overnight and should be well relaxed at the end of this period. Histological sections of animals treated in this way show no deterioration of the tissues and no loss of staining properties; and narcotized animals if washed and placed in fresh seawater, recover and appear to function normally (Owen 1955; footnote 8).

Killing, fixation, and preservation: Kill and fix narcotized animals in 10% neutralized formalin (200 g of hexamine per liter of commercial formalin: Smith, 1947; footnote 8) for 4 to 24 hours, depending upon the size of the specimens and inject the formalin into the body cavity of larger animals to insure good fixation and to help distend them. Wash preserved animals thoroughly in running tap water for 6 to 12 hours, depending on size and preserve them permanently in 1% propylene phenoxetol and 5-10% glycerol. Particular advantages of propylene phenoxetol as a preservative: is colorless, practically odorless, color retention in specimens is good, and tissues remain soft and flexible and suitable for dissection (Owen & Steedman, 1958; footnote 8). The method may also be usefuI for other invertebrates lacking an exoskeleton and is worth attempting.

(2) Sevin and rapid freezing: For full narcotization and killing of muricid and naticid gastropods. This is the only known method available to date which makes possible killing and preservation of fully relaxed muricids.

Prepare a stock solution of Sevin (l-naphthyl N-methyl carbamate, available from Union Carbide Chemicals Co.) as follows and store in refrigerator in tightly stoppered glass bottle. Add 0.1 g Sevin crystals to 15 ml (11.6g) acetone. To prepare a solution of 10 ppm. of Sevin add 1.16 ml of stock solution of Sevin to one liter of seawater and mix thoroughly. This must be prepared daily before use as it does not keep.

Narcotization: Place animals in the Sevin solution, 10 ppm. at room temperature for 1 hour, preferably keeping animals out of touch with each other and in the case of gastroods, upside down, and in a depth of fluid at least thrice the height of the specimens. After 1 hour transfer the animals to a fresh solution of Sevin and leave them in it for 3 hours. Employ a salinity of seawater in which the animals normally live. Slightly better narcotization is sometimes obtained if the second change of Sevin is held in 1 atmosphere of CO₂, and if a salinity some 30/00 below the environmental salinity is employed.

Killing: Remove relaxed animals from the Sevin solution one at a time and place the extended soft parts (if possess exoskeleton), or the maximum flat surface of non-shelled forms, against the surface of a block of dry ice held in a deep freezer or insulated chamber. Insulate the preparation and leave for 24 hours or more. Adding chipped dry ice around specimens accelerates freezing, although this is not always necessary. Many animals have to be frozen for at least 24 hours to entirely eliminate reaction to the preservative. Inject larger specimens for fuller distension. Specimens may be held in the frozen state for long periods provided they are not allowed to desiccate.

Freshly thawed, relaxed, unpreserved snails are ideal for detailed anatomical study since the organs retain color, texture and pliability characteristic of living relaxed tissues and take aqueous stains readily. Prior to freezing, specimens relaxed in Sevin recover from the treatment with either Sevin and Sevin CO₂ when returned to running seawater. Deep freezing is required to kill the animals in a relaxed condition as treatment with Sevin does not insure complete insensibility. This method of narcotization and killing, as well as slow and/or rapid deep freezing alone, may be useful for other highly refractory organisms and should be tested on them.

Fixation and preservation: Animals may be fixed in formalin, washed and stored in 1% proplene phenoxetol-5% glycerol, or may be fixed and preserved in 70% alcohol.