First we'll investigate cellular movement, that is movement of the whole cell from one location to another. Cells, like larger multicellular organisms, move to go to a more favorable environment, or move away from an unfavorable environment. For cells, the environmental clues they sense can be chemical compounds, temperature, or light etc. Chemicals that attractant can be a food source, or a sexual attractant. This process of moving toward a chemical is termed positive chemotaxis. Movement away from a chemical is termed negative chemotaxis. Similarly terms like phototaxis and thermotaxis is used. We'll study the movement toward a chemoattractant by bacteria and slime molds.
Bacterial chemotaxis has been well studied. A typical Escherichia coli swims around by rotating its bundle of flagella. Rotation in one direction causes the cell to swim straight. Rotation in the oppposite direction causes the cell to tumble. Tumbles typically occur roughly every second or two and results in a random change the direction of swim. On the bacterial cell surface there are receptors that can bind to chemicals like sugars and amino acids. The binding of these chemicals sends a signal inside the cell to change the direction of rotation of the flagella. When the cell happens to swim from a low concentration of a chemoattractant to a higher concentration, more receptors bind chemoattractant and a signal is sent to continue swimming straight for a bit longer. Conversely, when the cell happens to swim from a high concentration to a lower concentration, another signal is sent to tumble more quickly. This program of behavior causes the cell to move toward an area of higher concentration.
Bacterial Swarm Plate Assay is a simple way to visualize bacterial chemotaxis. In this assay, agar plates are poured with a particular concentration of chemical attractant which is consumed by the bacteria since its also a nutrient. As the bacteria consumes the nutrient, they produce a concentration gradient of the nutrient, lowest where they are and higher towards the periphery of the plate. The bacteria respond to this gradient by moving towards area of higher concentration. In this process, a ring of bacteria move outward toward the edge of the plate.
Slime molds typically grow in dark moist environment of forest detritus. The slime mold we will use is Physarum polycephalum, acellular slime mold. The whole large yellow vegetative mass is one huge cell with many nuclei. This growing vegetative form of the slime mold crawls over material and engulfs decaying matter and bacteria. It favors nutritous, damp areas, away from light and moves towards those environments by negative phototaxis (away from light), and positive chemotaxis (towards food). Its fairly large size, and its very active cytoplasmic streaming makes it a very favorable organism for studying cellular movement and chemotaxis.
This is a very simple plate with 1.5 - 2% agar in distilled water. Gently heat the agar water mixture to melt the agar, and then slowly bring it to a boil. Boil for a few minutes, then pour enough agar into sterile petri plates to cover the bottom of the plate. Let sit for 30 minutes or so for the agar to cool and harden.
Since we'll use sugars as chemoattracts which do not survive boiling well, the components for the plates have been sterilized individually and we will mix them together sterily, and then pour the plates.
Each group will pour 8-10 plates with either no sugar or one of the sugars.
|1 mL||1M||KPO4 pH 7.0|
|0.1 mL||1M||Mg SO4|
This plate has very low percentage (0.25%) agar to allow the bacteria to swim so handle with care. Note the label on the bacterial swarm plates; one should have no chemoattractant, and others have 3 different sugars. Divide the plate into 3 equal quarters. Place one strain in the center of each quarter, noting on the bottom without tipping it upside down which quarter has which strain. Innoculate cells in a shape of x with an innoculating loop. in the center of the plate. Wrap the plates with saran wrap and incubate right side up at room temperature (actually 37°C incubator would be ok, but Princeton in the summer time without air conditioning is about right).
Observe cytoplasmic streaming under the dissecting microscope. You may want to include some observation of cytoplasmic streaming for your class, posing such questions as: What function might cytoplasmic streaming serve? Does it behavior work in serving the function you propose? Are the streaming directional? Does it change direction? Does the change in direction occur randomly or periodically?
Start the slime mold assay. The slime mold is growing on similar water agar plates which had been sprinkled with oatmeal. The growing part of the slime mold is towards the thick yellow edge.
Now we want to try to test the hypothesis that slime molds will display chemotaxis towards some material and not others, and prefer some material over others. You may use any of several material as test substance. We will set up each plate with two choices for the slime mold. Since you have 3 plates, and testing two choices, how would you set up the experiment? Before setting up the experiment, it is important to label it on the bottom, which is easier to do when you can turn the plate over before you put anything on the agar on the plates. On the bottom of each plate, note on one edge the substance to be tested and the other edge the other item to be tested, and draw a line straight down the middle between them. Then turn the plate right side up and place the substance you chose by the label on one edge of the agar plate and another item on the opposite edge. Do this for each of your 3 plates. Then cut a 1 cm2 agar piece with a thick piece of plasmodium out of the culture and place it in the center of the agar test plate with the plasmodium side down. Be sure to not include pieces of oatmeal that is on the culture plate. Then wrap the dishes in aluminum foil and incubate right side up at room temperature. We will examine these plates also tomorrow morning.
Look at your plates and note if there is a ring of bacteria. How large is the ring? Are the edges of the rink sharp? Did any of your rings cross each other? Can you distinguish the differences between the strains in their ability to form chemotactic rings, and thus their ability to chemotax toward each of the chemoattractants?
Slime mold assay. Look at each of your plates. Note toward which substance the slime mold moved. Do this objectively by noting whether or not the slime mold went out even a little way into one half or the other. Of course 3 plates is not enough to get statistically significant results. Compare your results with others which tested the same substance, and determine whether the slime mold exhibited chemotaxis against it.
We use Escherichia coli, but most motile bacteria are probably capable of exhibiting chemotaxis. For example, chemotaxis has been studied in Salmonella typhimurium, Bacillus subtilus, Caulobacter and Pseudomonas among others.
|1M KPO4 pH 7.0||11.3g dibasic + 4.8 g monobasic / 100 mL|
|1M Mg SO4||12.3g / 50 mL|
|1M (NH4)2SO4||6.6g / 50 mL|
|2.5% agar in distlled water|
|50 mM galactose||0.045 g/ 5 mL|
|100 mM ribose||0.075 g/ 5 mL|
Physarum plasmodium can be obtained as sclerotium stage, a resting structure dried on a filter paper from Carolina Biological. They can be made to grow by putting a piece of the filter paper with the sclerotium upside down on 1.5-2% agar plates sprinkled with oatmeal flakes then wetting the filter paper with sterile water. Wrap the plate with plastic to keep it moist, then with aluminum foil to keep out light. They will grow out and will need to be transfered 3-4 days if left at room temperature, but once grown out, they can be kept in refrigerator for weeks.
Substances to be tested easily by putting chunks of food down. Students have successfully tested various candies like gummi bears. They should recognize that the chucks of food should be roughly similar in size. The test can be made more uniform by making an agar block with known concentration of single chemicals like sugars, alternatively by soaking a filter paper punched out with paper punch into a chemical solution, but may not be as fun.
Adler, J. 1976. The sensing of chemicals by bacteria. Scientific American Apr 234(4):40-47.
Bozzone, Donna M. & Martin, Denise A. 1998. Chemotaxis in the Plasmodial Slime Mold, Physarum polycephalum. An Experimental System for Student Exploration & Investigation. The American Biology Teacher 60:59-62