Countercurrent Separation: A New Method for Studying Behavior of Small Aquatic Organisms

Abstract

A new method for the analysis of behavior in small free-swimming aquatic organisms is described. In this procedure, called countercurrent separation, a dense solution flows down along the bottom of an inclined chamber while a light solution flows in the opposite direction, upward along the top of the chamber. The attraction of animals (injected into the center of the chamber) to one solution or the other is then determined by observing the proportion of animals that emerges from the chamber in that solution. When used with the nematode Caenorhabditis elegans, it is estimated that the apparatus is equivalent to at least nine theoretical plates.There is growing interest in studying behavior and its biological basis in many types of small organisms, especially by the use of genetic techniques (1). This interest stems from two basic advantages of small organisms. First, their size generally coincides with relative simplicity, and second, they reproduce relatively rapidly, a property that greatly facilitates genetic investigations. In order to take full advantage of these characteristics it is desirable to have methods for analyzing behavior in large populations. Such methods allow behavioral parameters to be determined with a minimum of error due to individual variation and, in addition, should provide mechanisms for isolating behavioral mutants. The technique of countercurrent distribution as applied to Drosophila (2) is a good example of this kind of method. However, it is not easily applied to aquatic organisms, and many of the most interesting organisms are aquatic, e.g., bacteria, protozoa, and nematodes. In this report, I describe a new technique called countercurrent separation that is similar to countercurrent distribution but is somewhat simpler in its operation and is appropriate for aquatic organisms.The new method is based on analogous techniques for the extraction of molecules, organelles, or cells from one phase to another immiscible phase (3-5). The simplest countercurrent technique, used in the petroleum industry (6), uses a column in which a dense phase continuously flows down through a rising light phase. However, because of the limited rate of diffusion, for high efficiency it is usually necessary to intersperse phase-mixing operations with phase-separating operations (7). This process is commonly accomplished in one of two ways: (i) a continuous-flow column comprising alternate mixing and separating stages (7), or (ii) a series of chambers in which the phases are mixed, separated, and transferred in temporally discreet operations (3). In applying these concepts to studies of behavior in aquatic organisms it seemed that the relatively high rate of movement of a swimming animal, compared to the rate of diffusion of a molecule, would make a simple continuous method feasible.In the method of countercurrent separation developed here for nematodes, two solutions are made to flow through a chamber in opposite directions toward separate collection vessels. Conditions are designed so that the solu...

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Selective attraction of marine bacterivorous nematodes to their bacterial food

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