Reprint from "AMERICAN MIDLAND NATURALIST" Vol. 50, No. 2, pp. 511-512

Nembutal as a Relaxing Agent for Mollusks*

With the increasing need for studies of the anatomy and comparative morphology of mollusks, means for relaxing specimens prior to fixation have proven to be of prime ;n4i.D.tance. In the Vadc-Mecum, Lee includes cl number of agents which have proven useful in narcotization. Among them the more widely known include: menthol, nicotine, chloroform, ether, alcohol, chloretone, chloral hydrate, magnesium chloride or sulphate (Epsom Salts), and others. Often asphyxiation is employed to attain the same end. Among freshwater mollusks menthol has been used with considerable success by E. J. Allen at Plymouth (see Lee, Vade-Mecum, p. 10); E. G. Berry (1943); E. Abciel- Malek (1951; thesis, 1952); and others. Although excellent relaxation can be attained with menthol, it has proven often to be too unpredictable. As a consequence, alternate methods are desirable. For example, a colleague who collected aquatic pulmonates in Alaska last summer reported that of the batches of snails prepared for fixation with menthol only, some were satisfactory. His results were summarized in his own words as follows: "Some look pretty good to me and others pretty poor as you will see." For the past several months our work has been concerned with field and laboratory studies of amnicolid snails in the genus . As the were brought from the field an attempt was made to obtain series of well relaxed specimens. At first menthol was used in this process, but in spite of the fact that the generally accepted technique was employed by two individuals independently there were too many unrelaxed specimens in each batch to warrant the exclusive use of that method. In searching for another anes- thetic, nembutal was tested. The idea was first suggested during a field trip with Normah Hartweg who used it to good advantage in relaxing amphibians and reptiles by injection. He told me that it was possible to apply the needle to a rattlesnake while the was still in the collecting bag. The nembutal worked so well that all danger of handling poisonous reptiles was avoided. During that same field trip the drug was used on aquatic mollusks. The concentrations used at that time were too high, consequently those series did not relax well. Later the nembutal method was suggested to Carl Obrecht of the University of Detroit. He found that by diluting the stock solution to one-tenth good results could be obtained. In our work with small amnicolid snails of the genus Pomatiopsis, the following schedule yielded good results: Fifty to one hundred animals were placed in a finger bowl with about 80 cc of tap water. A few drops of a concentrated alcohol were added to the water. When the animals were quite active, 1 cc of 1/10th stock solution of veter- inary sodium nembutal (60 mg. per cc.) was introduced. At intervals of one to two hours the diluted (1,/10th) solution was added in doses of 1 to 3 cc. After 12 to 24 hours the animals so treated were well relaxed and ready for fixation. It was of interest to find that there was a constant difference between the two of Pomatiopsis studied. The larger species, Pomatiopsis lapidaria, on the average took twice as long in this relaxa- tion process as did the smaller species, Pomatiopsis cincinnatiensis. Whether this difference in the time required for complete relaxation is merely a reflection of the difference of size between these forms or whether it i% due to a specific difference in reaction to the driAg remains an open question. The addition of a small amount of alcohol prior to the application of the nembutal seen:0cl to irritate or stimulate rctivity. While they are thms ,c-ive the d,ug is applied. It is not clear whether the use of alcohol is advantageous. In these procedures the method is entirely empirical. Information is needed as to how either nembutal or menthol act to bring about the kind of relaxation needed for proper fixation. Lee Worrell of our School of Pharmacy suggested that he could understand why nembutal would serve to relax the animals but in terms of the nature of the substance he felt it would be difficult to appre-

* Support for this work was provided in part by the Horace H. Rackham School of Graduate Studies of the University of Michigan. 512 THE AMERICAN MIDLAND NATURALIST 50 (2) date in what manner the menthol caused relaxation. In this connection it is interesting to note that with nembutal one does not have any difficulty due to moving a container or jarring one with snails that are being relaxed. The specimens can even be manipulated without causing contraction. It is generally conceded that, with menthol, it is ruinous to good relaxation to disturb in any way the animals being relaxed.—HENRY VAN DER SCHAUB, University of Michigan, Museum of Zoology, Ann Arbor.

REFER& N CES

ABDEL-MALEK, Emma 1951—Menthol relaxation of helminths before fixation. J. Parasit. 37:321. 1952—Morphology, Bionomics and host-parasite relations of Planorbiciae (Mol- lusca: Pulmonata) Thesis, University of Michigan--Microfilm Service. BERRY, E. G. 1943—The Anmicolidae of Michigan; distribution, ecology, and . Mus. Zool., Univ. Mich. Misc. Pap. No. 57. LEE, BOLLES (edited by J. B. Gatenby and T. S. Painter) 1937—The Microtomises Vade-Mecum. Tenth Edition, P. Blakiston's Son & Co., Inc., Phila.