Zoe
Learned behavior in Physarum
Goal. In Figure 1, the yellow stuff on the petri dish is a physarum (or slime mold). Despite their size, physarum are single celled organisms with multiple nuclei. As they grow, they spread towards valuable nutrients (they especially like to eat oats). Because of this ability to find the shortest path towards a food source (see a slime mold completing a maze in figure 1, Physarum are considered a form of intelligence. To explore the bounds of this ability I wanted to investigate if slime molds, like mammals, could learn.
Setup. Physarum really hate salt, so in order to test their capacity to learn, I grew two sets of organisms on an agar gel of water and oats; then added table salt (NaCl) to one of the gel mixtures. After culturing physarum on the two gels, I tested the extent of each group's salt avoidance, I presented them with a salty path with an oat reward at the end. Although Physarum usually move away from salt, I hypothesized that individuals raised to associate salt with oats, would be more willing to traverse a salt covered path in order to reach a food source.
Picture 2. Final plate setup.
Picture 3. Maze setup for experimental group. Control mazes had no salt.
The Process.
I started with a single fresh physarum from Carolina Biological. It was fed with sterile oats, and it happily filled a petri dish over the course of a few days (Pictured above). In order to grow my population of test subjects, I prepared several additional plates of 2% agar gel, then transferred 1 square cm pieces of the original organism onto the empty plates. These subcultured physarum were then also fed with sterile oats.
In order to raise physarum on salt along with oats, I needed to force salt into the food source. This meant grinding up the oats directly into a gel of water and agar. In a preliminary test, I tried culturing physarum onto plates full of 10% oat mixture. This high concentration of oats made an incredibly viscous agar which introduced uncertainty in the concentration (because the oats settled to the bottom of the melted agar) and made a big mess in the microwave. Unlike the first two sets of physarum, the individuals grown on the very Oaty plates did not spread out. This led to very dense, circular individuals which could not be easily subcultured (there was not enough area to transfer to the mazes). At this point, I switched to feeding each plate of physarum with three small discs of oat-gel (with or without salt) on the periphery of the plate (Picture 2). For the salt fed group, I began with 100 mM of NaCl, and 5% Oats by mass (for the sake of the microwave).
After a few days of growth, there were visible differences in the exploration patterns of the two groups. While the five control physarum (fed only on oats) had each colonized large portions of the plate, reaching at least two of the three food sources, the salt fed individuals had each concentrated on top of a single disc of food. They did not seem to be growing or exploring, and all of the tissue outside the bounds of the oat disc had been abandoned completely. Contrary to most research which indicates that salt should increase the rate of exploration (3), the salt cultured physarum seemed too weak to grow or spread out. In order to allow the new subcultures to be large enough to transfer to the mazes, I gave them an additional week to grow.
To make the mazes, I (with a lot of help from Mr. Edgar) cut t-shaped walls out of a white plastic grid. I sterilized these mazes with ethanol and placed them into plates of 2% Agar before the gel solidified. In each maze, I also embedded a rectangle of 5% oat gel into the agar plate, on one side of the T. In half of the mazes, I added a square of 200 mM NaCl agar between the intersection of the T and the food source in order to create a bridge which would force physarum to cross salt in order to reach the food source (Picture 3).
Results. The results were quite inconclusive (Figure 1). Major uncertainty was introduced by the fact that the experimental group had expanded very little on their plates by the time I subcultured them, so the starting organisms were likely much smaller than the control group (Picture 5). In a future iteration of this experiment, the time allotted to each subculturing should be expanded, and a small food source should be included at the starting point of the maze in order to strengthen physarum's ability to grow.
Picture 4. Magnified image of salt cultured physarum concentrated on a single food source.
Conclusion. In this first attempt at observing physarum cultured with salt, I learned mostly about more general physarum behaviour. Although I saw a couple examples of salt cultured physarum crossing an NaCl bridge, the lack of consistency in these results, and the overall lack of exploration from the salt cultured physarum do not point to a conclusive answer. In the future, culturing the slat physarum with much lower concentrations of NaCl, and including salt not only in the food source, but also in the surrounding agar may result in more exploration on the subcultured plates. Additionally, a small quantity of oat agar at the starting point of the maze may allow for exploration due to the somewhat less dire condition. More mature physarum, at every stage of the protocol would likely have improved results.
Sources. 1. ( Maze Clip) https://www.youtube.com/watch?v=olCEGsKWQ3c&ab_channel=bioGraphicMagazine
2. (Maze image) doi: 10.1126/article.37268
3. https://www.nature.com/articles/s41598-019-50872-z#Fig2