Kristin Hookassian
9/20/18
Lab 5: Experimental Designs and Serial Dilution
Objectives:
The objective of this lab is to practice micropipetting, creating an experimental design, becoming familiar with scientific literature, and to become familiar with Tetrahymena, a ciliate that we will be using for the rest of the semester. This lab allowed us to become familiar with more advanced equipment, like the P-1000 micropipettor. However, the overall purpose of the lab was to familiarize ourselves with Tetrahymena, explore the possible applications of it regarding our research through scientific articles, create a practical experimental design, and practice micropipetting.
Procedure:
- Using the micro pipettor available to you and a jar of water, become familiar with using a micro pipettor by taking different samples sizes and transferring them to a dish.
- Once you’re done, place the jar of water and the micropipette back in it designated place, making sure to properly dispose of the tips.
- Find a dissecting microscope, uncover it, plug it in, and turn it on making sure it is in the correct initial stage position when starting the lab.
- Take the prepared 24-well sample and place it on the stage, placing the well with the Tetrahymena sample in the center of the stage. Adjust the light, stage position, magnification, fine adjustment knobs as needed. Using the black lower light cover is highly suggested to observe the Tetrahymena.
- Record the observation you make of the Tetrahymena in your lab notebook.
- Once you have received a confident understanding of the Tetrahymena, take the 24-well plate off the microscope and properly turn off, clean, wrap up, and cover your dissecting microscope.
- Then take the compound microscope, uncover it, plug it in, and turn it on, making sure it is on the lowest objective lens, x4, when beginning.
- Using the P-10, .5-10µm micro pipettor, take a sample of 5.00µm of the Tetrahymena from the 24-well you previously observed them in.
- Place the sample in the center of a concavity slide. Use a slide cover if available, however, it is not required. Then place the prepared slide on the stage. Adjust the light, stage position, magnification, fine adjustment knobs as needed until you receive a clear image of the sample. Place the micro pipettor back in its desired area, making sure to dispose of the tip in the proper area.
- Record your observations of the Tetrahymena on the x4 objective lens in your lab notebook and answer the questions/fill out the chart affiliated with the CILI CURE binder for this lab activity. During this time, you will need to refer to the field of view (FOV) measurements in µm to calculate the diameter of the Tetrahymena. The equation for the FOV is stated below, however you can use last week’s calculations as well. Repeat this step and the preceding step with the x10 and x40 objective lens. Take pictures as desired. Go to open lab if you are unable to complete the needed observations.
- Once you are done making your observations, take the slide off the stage and wash it off with a water in the sink then place it in the proper place on a paper towel to dry.
- Properly turn off, clean, wrap up, and cover your compound microscope. Clean your work area and lab partners areas as needed.
- Next, go to a computer, specifically a computer lab, and pull up the article you found prior to the lab regarding microplastics and its effects on soil organisms. This will serve as a foundation for the information you will be attempting to find that could be applicable to your original research you will be conducting later in the semester.
- Using the scientific databases available, specially PubMed, find articles relating to microplastics and soil organisms and start to create an experimental design that could be performed involved Tetrahymena in the lab. Discuss what you find with your lab partner(s) to receive their feedback and develop a way that can include both what you are interested in and what they are interested in. Record these articles and ideas in both your lab notebook and on the QTM sheet.
- Properly log out of the computer.
Using the compound and dissecting microscopes allowed us to view the Tetrahymena in different ways. This way, we were able to become more familiar with its characteristics. It was interesting to see them underneath each scope, as they allowed us to view them in different ways as we zoomed in on the sample.
Observations:
Trial |
Dilution: which dilution did you use to make your counts? |
Cell count in 5ul |
Cells per ul in the Drop (divide by 5) |
Cells/ul in the undiluted sample (x dilution factor) |
Cells/ml in the undiluted sample (x 1000 ul/ml) |
1 |
10e-1 |
20 |
4 |
40 |
40,000 |
2 |
10e-1 |
30 |
6 |
60 |
60,000 |
3 |
10e-1 |
50 |
10 |
100 |
100,000 |
Average |
– |
– |
– |
– |
66,666.66 |
Experimental Design Ideas:
a.) Question: How does different concentrations of microplastics effect the pH of a Tetrahymena’s surroundings.
b.) Hypothesis: An increase in microplastic concentration will lower the pH of a Tetrahymena’s environment, therefore making it more acidic.
Storage:
We placed the 24-well plates in the desired stop on the lab bench. The sample slides we prepared were cleaned with water and then left to dry for later use. The micropipettor tips were disposed in the plastic cups. The microscopes were properly turned off, cleaned, wrapped up, and covered with the stage lowered completely. The work areas were wiped down. In the computer lab, we logged out of our accounts before leaving.
Conclusion:
In conclusion, the number of ciliates varied between each trial, but were relatively close to one another (refer to table). This lab let us to practice the compound and dissecting microscopes and see the direct differences between two microscopes with the same sample. Additionally, it allowed us to practice the micro pipetting, preparing slides, and time management with different activities. By observing the Tetrahymena in the lab let us to take those characteristics and apply them to real experiments we could be able to conduct in the lab. In the computer lab, I was able to better by teamwork skills by comparing ideas on experiments, data collection, and results to form a a conclusion.