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Chemotaxis and Phototaxis in Physarum

Introduction

This exercise will allow developing and testing hypotheses about movement in the plasmodial slime mold, Physarum polycephalum.

Physarum.JPG (370126 bytes) Physarum polycephalum

Ecology and Movement

The plasmodial slime mold is found in dark, moist environments such as those found on a forest floor. The vegetative form of this organism, called a plasmodium, is a multinucleate mass that slowly creeps along the leaf litter and decaying logs, consuming dead organic material and microorganisms. As the plasmodium moves, it typically takes on a net-like appearance with numerous interconnections.

The plasmodium moves in response to chemical stimuli (called chemotaxis) and in response to light (called phototaxis). Negative phototaxis refers to movement away from light; positive phototaxis is movement toward light.

Life Cycle

When environmental conditions are unfavorable such as when sufficient food or moisture are unavailable, sporangia form, and spores are produced by meiosis. Spores are resistant to environmental extremes and germinate when environmental conditions become favorable. They germinate to produce haploid cells that are either biflagellate (two flagella) or amoeboid. These cells can act as gametes, fusing to produce a diploid zygote that matures into the plasmodium.

Laboratory Exercise

Day 1

In this exercise you will observe movement of the plasmodium in response to the presence of four different substances and also in response to light. The test substance will be placed on one side of a sterile agar plate and a control (distilled water) will be placed on the other side. The center of the plate will be inoculated with Physarum as indicated in the diagram below. The test substances are: glucose, salt (NaCl), banana, oatmeal flakes. Movement in response to light will be determined by wrapping 1/2 of the plate with aluminum foil. The other half will be exposed to light.

Developing Hypotheses

Before beginning, you should hypothesize how Physarum will move in response to each of the conditions described above. A  hypothesis should be a statement, not a question. A statement can be tested but a question cannot. One possible hypothesis is: Physarum will move away from paper disks soaked with a 20 mM NaCl solution. This hypothesis can be tested. It is either true or false. The data collected will either support the hypothesis or will show that it is false. 

The following hypothesis is neither true nor false because it is a question: Does Physarum move away from paper disks soaked with a 20 mM NaCl solution? It is not possible to design an experiment to show if this hypothesis  is true or false.

Procedure

Examine one of the stock cultures of Physarum and notice the web-like structure of the plasmodium. Examine the plasmodium under a dissecting microscope and notice the movement of the cytoplasm. Can you detect movement of the plasmodium?

Obtain 5 sterile agar plates and invert them so that the agar side is up. Obtain a wax pencil and draw a dividing line down the center of each plate so that it is divided in half. Be sure that the line is on the portion of the plate that contains the agar, not on the cover. Number the plates 1 through 5. Write the letter "C" (for control) on one side of the line. It is better to write the letter "C" on the bottom of the plate than on the lid because the lid might accidentally become twisted so that it identifies the incorrect side. After drawing the line, numbering the plate, and writing the letter C on one side of the line, turn the plates over so that the agar side (bottom) remains down for the rest of the experiment. These plates will be used to test movement of the plasmodium as shown in the diagram above.

Cut the agar in the stock culture plate into 1 cm square blocks. You will need five blocks, one for each plate that you inoculate. Transfer a single block containing a piece of plasmodium to the center of each of your test plates. The block should be inverted so that the plasmodium lies between the block and the test plate agar.

Add the control substance (distilled water) to the first plate by dipping a sterile paper disk into distilled water and placing it on the side of the plate that contains the letter "C." Place the disk approximately 1 cm from the edge. Repeat this procedure for plates 2 through 4 but not for plate 5. For each plate, the wet disk should be placed on the side indicated by the letter "C."

Place test substances on plates 1 through 4 using the following procedures described below. The test substance should be placed on the opposite side of the plate that contains the distilled water (control). See the diagram above.

Plate 1 - Dip a sterile paper disk in 100 mM glucose solution and place it on the side of the plate opposite to the control.  

Plate 2 - Dip a sterile paper disk in 20 mM NaCl and place it on the plate.

Plate 3 - The test substance for this plate is a piece of apple or banana. Use a piece of apple or banana that is approximately the same diameter as the paper disk and approximately the same thickness as the agar block.

Plate 4 - Use an oatmeal flake as the test substance.

Seal the plates using tape and place them in a dark area until the next laboratory class. Do not turn the plates over; the agar side should remain down.

Plate 5 will be used to test for movement in response to light. Sprinkle oatmeal flakes on both sides of the plate. Seal the plate with tape and cover one side with aluminum foil. Place this plate with the other plates while the plasmodium grows. 

Day 2

Data Collection

Examine each of the plates used in the experiment and record the side of the plate that had the most growth. Use the table below to record your data. Your data will be pooled with the class data for statistical analysis.

Test Substance
or Condition
Side with most growth
Glucose 
NaCl 
Banana 
Oat flakes 
Light/Dark 

Enter the total numbers for your class in the table below.

Glucose__________      Control__________
NaCl__________Control__________
Banana__________Control__________
Oat flakes__________Control__________
Light__________Dark__________

Statistical Analysis

Statistical tests can be used to determine if data collected are different than what are expected. For example, suppose that you flip a coin 10 times and get 8 heads and 2 tails. This could be due to chance. It could also be due to one side of the coin being heavier than the other. If the coin is balanced, you would expect 5 heads and 5 tails because the chance of getting either heads or tails is 1/2. Statistical analysis can be used to determine if 8 heads and 2 tails is significantly different than 5 heads and 5 tails. In the chemotaxis experiment, if the test substance had no effect, then we would expect half of the plates to show growth primarily on the side of the test substance and the other half of the plates to have growth primarily on the side with the control (distilled water).

What is the chance of getting 8 heads and 2 tails if you expect 1/2 of the coin tosses to be heads? Statistical tests help us determine whether differences in the data are real differences or whether they are due to chance. In this example above, we will calculate the probability of obtaining 8 heads and 2 tails. The difference might due to chance. The alternative hypothesis is that the 8 heads and 2 tails is not due to chance; it is a real difference, perhaps due to one side of the coin being heavier. The statistical test gives the probability that the observed outcome (8 and 2) could be due to chance. Suppose that we calculated that the probability of it being due to chance is 1 out of 1,000,000. We would conclude that it is not due to chance, that the coin is biased or something is causing it to come up heads. Suppose that we calculated the probability of 8 and 2 being due to chance is 0.25 (one out of 4). We would conclude that this could happen, that the coin is not biased. What do we use as a cut-off probability? Scientists often use 1 out of 20 which equals 0.05. If the probability that the difference is due to chance is less than 0.05, then we conclude that the difference is real. If the probability is greater than 0.05, we conclude that the difference is not significant, it could be due to chance. 

We will perform a binomial test to determine the chance of getting 8 and 2 if the probability of getting heads or tails is 0.5. Click here to open an Excel worksheet for performing the binomial test. Enter either the number of heads or the number of tails in cell B2. Enter the total number of coin tosses in cell B3. The probability of obtaining one success is entered in cell B4. For a coin toss, this is 0.5 because the chance of getting a heads or a tails is 1/2. The spreadsheet prints the calculated probability of obtaining 8 heads out of 10 coin tosses in cell E5. Because this probability is less than 0.05, we conclude that the coin toss is biased. The coin is either heavier on one side or something caused more heads than tails.

We will use a binomial test to test if Physarum grows toward or away from a test substance. If Physarum does not have a preference, we expect that on average, half of the plates will have more growth on the side of the plate with the test substance and half of the plates will have more growth on the side with the control (water).