Title: Serial Dilution and Research for Experimental Design

Rationale: The main purposes of this lab was to learn serial dilution, as well as its importance, coming up with a cell count through the use of light and dissecting microscopes, to further our experience with usinand to do scientific research in order to come up with an experiment viable for the class regarding the effect of microplastics on Tetrahymena

Materials:

• Micropippettors (1000 ul, 200 ul, 20 ul)
• Dissecting Microscope
• 24 well plate
• Culture Media
• Concavity Slide
• Cover Slip
• Light Defractor
• Dissecting Microscope
• Light Microscope
• Stock Sample

Procedure:

• Soil Collection

1. For Soil collection label both sides of the petri dish and put the soil in. (Make sure to not overfill the petri dish, because we will eventually need to put water in it)
2. Store the petri dish towards the back of the class with the others.
3. Put excess soil, from the zip-lock bag, in the back of the class with the other ones.

• Dilution Observation of Tetrahymena

1. View the tetrahymena culture, directly from the 24 well-plate, under the dissecting microscope and observe the specimen.
2. Add 900 ul of culture media to 4 well plates.
3. Will 200 ul pipette, take 100 ul from stock culture and transfer it to the first well with the culture media.
4. Change tip on the pipette.
5. Transfer 100 ul of the first well solution to the second.
6. Change tip on the pipette.
7. Transfer 100 ul of the second well to the third.
8. Change tip on the pipette.
9. Transfer 100 ul of the third well to the fourth well.
10. Choose a diluted well that has a good amount of specimen. It should not be too concentrated nor too empty; it should have a good amount of species to observe.
11. With a 20 uL pipette, transfer 5 ul of the culture, that you chose, to a concavity slide. (Since there are 3 trials, apply 3 drops)
12. Once set up, place the concavity slide on the light microscope and observe the droplet.
13. In table record, the dilution that you used, the cell count observed through the 5 ul droplets, the number of cells per ul in the droplet, the number of cells that would be in the undiluted sample(x dilution factor), and the cells/ml in the undiluted sample.
14. Record observations in the table, and clean up the area.

• Experimental Design

1. Work with team members to come up with an experimental question regarding tetrahymena and microplastics. Use computers and research sites to aid you in the process if needed.
2. Make a hypothesis for your question
3. Come up with an experimental design and treatment plan in order to replicate the experiment. (Create a gameplan).
4. Included in the treatment plan, explain how you would set up the experiment using a 24 well plate.

• Observation

• Storage:

1. I stored my labeled soil sample in the back right side of the room where they were designated to be stored in the fume hood.
2. I stored the remaining of my soil, from the ziplock bag, by the fume hood where In the designated area
3. After every time I used a micropipettor, I discarded the tip in the designated area, told by my professor, and I stored the micropipettes where I originally found them for the next class to use.
4. I unplugged the microscopes, after use, put their covers/protection on them, and put them where I found them for the next class to use.
5. I put the cover of the 24 well plate back on to the well plate and placed it where I originally found it for the next class to use.
6. I properly cleansed the concave disk, and the coverslip, with water, D.I water, dried them and put them in the back of the classroom where I was designated to store them.

• Conclusion

-In conclusion, I furthered my experience working with different micropipettors, light microscopes, and dissecting microscopes.  I also learned the importance and/-or use of diluting a solution in order to find a proper amount of ciliate to observe when necessary. For me in this experiment the 1:10 ratio of dilution worked the best for me to get the right amount of ciliate to use. Along with this, I furthered my knowledge of coming up with research ideas and a proper scientific method.

• Future Goals

– In the future, I plan on using the results of my experiment today to make me more efficient. For example, I WILL continue to dilute samples in order to get the right amount of cells in the sample. I also plan on practicing using pipettes, because when I used the 1:100 ratio and the 1:1000 ratio, there were extremley small amounts of cilliated visible, therefore there is a possibility I did not pipette as accurately as possible.