The use of chloretone as an anesthetic for paramecium.

Abstract

The described methods for quieting paramecia by using formaldehyde, quince seed jelly, cotton fibers, etc., are by no means satisfactory, if observations are to extend over more than a few minutes. They are of no use at all for observing a single animal, or a particular group of animals, over a period of several hours or days. Experiments have been made with chloretone∗ (trichlortertiarybutyl alcohol), and have shown it to be especially valuable for quieting paramecia for long periods of time, up to 8 days, with no undesirable effects.

The paramecia used were extracted from wild cultures, and grown in hay infusion of known constant concentration. New cultures were started from time to time by adding a pipette full of the old to fresh infusions. These cultures contained a few of the smaller rotifers, many of the smaller ciliates, and a preponderance of Paramecium caudatum, a mixed population which seemed to be favorable for their growth and reproduction. It was found that a solution containing about 0.06 percent chloretone by weight would anesthetize paramecia in a few minutes, and keep them anesthetized for varying periods of time. The animals would recover if placed in fresh culture fluid previous to cytolysis.

The technique consisted of placing 1 drop of culture fluid, densely populated with paramecia, in a shallow glass chamber on a slide, and then adding 1 drop of 0.12 percent chloretone solution. After measuring the drops of the two fluids from their respective pipettes it was found that such a mixture contained 0.056 percent chloretone. In all subsequent tests the same two pipettes were used. The size of the drops can be adjusted so as to yield just enough fluid to fill the chamber. The latter was then sealed with a cover glass using a mixture of bee's wax and white vaseline, the consistency of which just allowed of its easy spreading at 20° C. (approximately 1 part of wax to 3 parts of vaseline). Gentle heat applied to the cover insured an air-tight seal. Vaseline alone did not give good results, since the animals always recovered within a few hours (from 2 to 24), a fact so far unexplained satisfactorily. It was noted that a few individuals are sometimes killed immediately upon the addition of the chloretone, due probably to the fact that they come in contact with the chloretone before it has been diluted with the culture fluid.