A Micro-Bioassay Method for Acetylcholine. ∗

One of the most specific and sensitive tests for acetylcholine in body fluids is the leech muscle suspended in a saline bath containing eserine (Fuehner, Minz 1 , 2 ). When only small quantities of blood are available they must be diluted with sufficient saline to fill the bath. This procedure naturally decreases the sensitiveness of the test. Moreover, in smaller animals, the repeated drawing of several cc. of blood is often detrimental to the carrying out of a series of tests. The method presented here eliminates these disadvantages by suspending the leech in foam from small quantities of blood.

The leech is attached to a hook at the bottom of a glass bath (2×6 cm.), the other end leading to a writing lever. A narrow opening at the funnel-shaped bottom of the bath is connected with an air tank by means of rubber tubing, so that air is allowed to enter at the desired rate. A wire loop serves as a guide to prevent the blood soaked thread from adhering to the wall of the tube. The leech is prepared in the usual manner and is suspended in eserinized saline until it is relaxed. The saline is then drained completely and the blood which is to be tested is placed in the bottom of the tube. The air passing through the blood creates a foam, which passes over the muscle as it is carried upward. If there is acetylcholine present in the blood, the contraction of the muscle then starts immediately. (See Fig. 1.) In order to obtain uniform results, it is necessary to keep the amount of air entering the bath constant. After the foam has exerted its effect on the leech, the bath is washed with saline, with which it is left filled, until the muscle is relaxed and ready for another test.