General Background
Caenorhabditis elegans, or C. elegans are a common worm-like organism that live in the soil of temperate climates. They are eukaryotic, and are often used in biological research as they are very easily characterized and are able to be bred inexpensively and easily. They have two sexes: hermaphrodites which have 956 somatic cells, and males, which have 1031 somatic cells (Department of Molecular, Cellular, and Biomedical Sciences, 2018). Like humans, nematodes have complex systems and specialized cells including nerve cells, muscle cells, a digestive and excretory system, and a reproductive system. Their neural structures consist of sense organs in the head that control response to taste, smell, temperature, and touch (Department of Molecular, Cellular, and Biomedical Sciences, 2018). The proteins that nematodes use to respond to chemicals are located in the plasma membrane of the neuron cells and are used to sense them. These chemicals usually only interact with a single chemical, or ligand, or with chemicals that have a similar structure. A receptor and its ligand act as a “lock-and-key” (Department of Molecular, Cellular, and Biomedical Sciences, 2018). The receptor changes shape to fit the ligand, and this causes the movement of the nematode towards or away from a substance. This nematode feeds mainly on bacteria found on rotting vegetation, so it is essential for them to be able to sense them and elicit a response (Department of Molecular, Cellular, and Biomedical Sciences, 2018). This also helps them avoid toxic conditions that could potentially be in this environment. Also, like humans, nematodes use senses to feel out their environment. They use olfactory (smell) and gustatory (taste) receptors to get a sense of what is around them as well as any potential danger that could be coming up (Department of Molecular, Cellular, and Biomedical Sciences, 2018).
Biological Process in Question
The biological process being investigated is chemotaxis. Chemotaxis is the movement of cells in response to a stimulus, either towards or away from it. Organisms use this to find food or steer clear of poisonous substances. C. elegans are able to use both olfactory and gustatory senses to respond to their environment. Approximately 30% of the C. elegans’ cells are neuron cells, and 10% of these are used specifically for chemotaxis.
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Initial Observations
To further investigate the chemotaxis of C. elegans, they had to be observed in a neutral environment to get a general idea of their responses. The first step was to observe the C. elegans in a neutral environment. They were placed onto an agar plate and observed under the microscope. They were then compared to each other by the way they moved and how big they were. The next step was to count the number that were on the plate which gave a better idea of how to carefully count them when conducting the experiment. Then the C. elegans were observed in an environment with a substance that would elicit a response, and a neutral substance. Wilton’s Butter was used as the attractant, and water as the neutral substance. Then four agar plates had to be set up to test these attractants. The bottoms were labeled with a dot on one side labeled “A” and a dot on the other labeled “B”, and a line drawn down the center with a circle in the middle. In groups of four, one person from each group did one trial, which created four in the end. From these four trials, it was concluded that the C. elegans were attracted to the Wilton’s butter far more than they were the water. This is shown by the average chemotaxis index and the standard deviation. A chemotaxis index of 1.00 shows a complete attraction of the nematode to the substance, while 0 corresponds to no response. For this experiment, the chemotaxis index was 0.675. This showed that all of the nematodes weren’t attracted to the Wilton’s butter, but a lot of them were. Standard deviation tells us how far away from the expected value (1.00) it was. The standard deviation was 0.2794. This showed that the nematodes weren’t too far off from complete attraction. Since the nematodes were so attracted to the Wilton’s butter, a question that arose was if the saltiness came from the butter.
Rationale and Hypothesis
The treatment being looked at was Sodium Chloride. Since the nematodes were attracted to Wilton’s Butter in the previous experiment, the question arose if other salty substances would attract them. Since sodium chloride is table salt, it is possible that it could be an attractant (Hwang, H. et al. 2015). It affected the biological process because it provided stimulus for the C. elegans to either move toward or move away from. In other words, it generated a chemotaxis response. This gave way to the hypothesis, if nematodes are exposed to an environment with Sodium Chloride and water, then the nematodes will be more attracted to the sodium chloride than the water.
Experimental Design and Methods
The first step in the experiment was to put on PPE. The following items were needed: lab goat, gloves, and safety glasses. Next, all materials needed to be gathered: an Olympus VMZ Stereo Microscope, nine agar plates, a 20 µL pipette, Sodium Chloride, C. elegans, Wilton’s Butter, Sodium Azide, and water.
The agar plates were then split into three groups of three. One for the negative control, one for the positive control, and one for the experimental group. The negative control that was tested was water vs water. To set up the plates, the bottoms had to be labeled with a dot on one side marked “A”, and a dot on the opposite side marked “B”, and a line through the middle with a circle halfway down to mark where the C. elegans would be placed. The Sodium Azide was under the fume hood, and 2 µL of this had to be placed on dot “A” and then repeated for dot “B”. The plates were then brought back to the station and 2 µL of water was pipetted onto dot “A” and the same on dot “B”. The final step was to pipette 2 µL of C. elegans into the center marked on the agar plates. Fifteen to twenty worms were then counted by looking under the microscope, and then the lids were placed on the plates and a timer was set of thirty minutes.
The next step was to set up the positive control, which was Wilton’s Butter vs water. The steps above were repeated, but instead of water on the “A” dot, 2 µL of Wilton’s Butter was placed there instead. Dot “B” was kept the same as well as the 2 µL of C. elegans in the center. Fifteen to twenty worms were counted, and the lids were placed on, and a timer set for thirty minutes.
The next step was setting up the experimental group. For this, sodium chloride vs. water was tested. The steps above were repeated, except instead of Wilton’s Butter being placed on dot “A”, 2 µL of sodium chloride was placed there. Like above, dot “B” remained 2 µL of water. 2 µL of C. elegans was pipetted in the center, fifteen to twenty worms were counted, and a timer was set for thirty minutes after the lids were placed on.
For every experiment, there needs to be independent variables and dependent variables. The independent variable in this experiment was the chemical on dot “A” of the agar plates (water, Wilton’s Butter, sodium chloride). The dependent variable was the chemotaxis response of C. elegans to the different chemicals. In each experiment, there are also variables that are attempted to be controlled. For this experiment, these variables were the number of C. elegans on each plate, the time waited to observe them, and the amount of each substance used. They were controlled the best that they could because if any of them were different, it could throw off the results of the experiment and lead to conclusions that could possibly be incorrect and not match up with those that would have been yielded if a more controlled experiment were performed.
To get the correct data was a little difficult due to the unforeseeable number of C. elegans in each 2 µL sample, so some steps to make sure the data was as accurate as possible were put into place. First, the plates were observed by each individual person in a partnership to get an individual count, and then compared with the partner. Then, the partnership across the table counted to double check the numbers. Then the chemotaxis index was calculated for all the trails and averaged, as well as the standard deviation. To calculate the chemotaxis index, the following equation was used: chemotaxis index = (# nematodes on A side - # nematodes on B side)(# nematodes on A side + # nematodes on B side)
This equation was used for each of the three trials and then the indexes were added and divided by three to get the average chemotaxis index.
Discussions
The objective of this experiment was to observe C. elegans’ chemotaxis response to sodium chloride. To do this, an experiment was designed using a negative control of water vs water, a positive control of Wilton’s Butter vs water, and an experimental group of sodium chloride vs water.
The hypothesis was that if C. elegans were introduced to an environment with both water and sodium chloride, they would move toward the sodium chloride more times than the water.
The results did support the hypothesis. Using the data that was found to make calculations, values of standard deviations were found. When using these values, they did not overlap, so the results were significant. The graph shows that although the sodium chloride was less of an attractant than Wilton’s Butter, it was more of an attractant than water.
There were some trends in the date that was produced. The more neutral the substance, the less of a chemotaxis index there is, the more salty the substance, the higher the chemotaxis index. The standard deviations were quite low compared to the results yielded from the experiment in week one, but they were around the same for the substances that attracted the nematodes, and significantly lower for the negative control which was to be expected.
This experiment all goes back to the biological process of chemotaxis. Nematodes will go toward something that they find attractive, like a good taste or smell, and will stay away from something that is toxic. The salty substances of both the Wilton’s Butter and the sodium chloride attracted the nematodes, which meant that they liked both substances and preferred them over water, mainly because it had a stronger taste.
Errors are always possible in an experiment, sometimes they are easy to pick out and other times they are not. Some errors that could have occurred are the miscounting of nematodes, inaccurate measurement of substances, change in temperature, and waiting too little time before observing. Each of these could have a significant effect on the outcome of the experiment. If there was a miscount, then the date would have been off, if the measurements were inaccurate, the nematodes could have moved a different way because there was too much or too little. If there was a change in temperature, it could have changed the mood of the nematodes and they could have completely changed their direction of movement, and if too little time had passed before observing, the nematodes could have either not moved or not finished moving toward their desired substance, and the results would have been off. To improve this, the experiment should be carried out more carefully and in a better controlled environment. Triple checking counts, making sure measurements are taken carefully, and setting a timer exactly when the agar plate was finished setting up are some precautions that could be taken to improve this.
A new experiment that could be conducted is a test on other water soluble compounds. In the two experiments done above with the Wilton’s Butter and sodium chloride, they were tested due to a question that came up about the saltiness of the substances. Going further off of this, a new question could arise about the attractiveness of water soluble compounds. In a research article about this question, it is found that in C. elegans, a soluble and olfactory attractant is NH4Ac (Frøkjær-Jensen, Christian, et al. 2008). This created a new hypothesis: If nematodes are placed in an environment with water and soluble compounds, then they will be more attracted to the soluble compounds than the water.
Literature Cited
- Department of Molecular, Cellular, and Biomedical Sciences (2018). Biology 411 Lab Manual. Durham NH.
- Hwang, H. et al. 2015, “A Sensitive C. Elegans Chemotaxis Assay Using Microfluidic Device Generating a Linear Gradient of Chemoaffectors.” Bulletin of the Korean Chemical Society. Volume 36. Pages 1096-1099.
- Frøkjær-Jensen, Christian, et al. “Ammonium-Acetate Is Sensed by Gustatory and Olfactory Neurons in Caenorhabditis Elegans.” PLOS ONE, Public Library of Science, 18 June 2008.
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