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.

• Title: Meeting Tetrahymena Activity
• Rationale: The main objective of this lab was to familiarize ourselves with Tetrahymena, a soil ciliate. We furthered our experience and fluency in using compound and dissecting microscopes in order to do this. Next, we had to learn and get accustomed to using micropipettes, in order to accurately drop a certain amount of the ciliate. Finally, we had to familiarize ourselves with Primary articles, obtained through the Baylor library and Pubmed, in order to do the proper research needed to learn more about Tetrahymena and it’s characteristics.
• Materials:

1. Micropipette
2. Compound Microscope
3. Dissecting Microscope
4. 24-Well plate
5. Light Defracttor(For Dissecting Microscope)
6. Concavity slide
7. Cover Slip

• Procedure –

MICORPIPPETTES

1. Make sure correct measurement wanted is on the micropipette.
2. Apply the tip to the micropipette. It is vital to not cross contaminate it, so if one gets dirty, simply discard and replace it.
3. Push the pipette down to the first level before placing it into the water. This will prevent air bubbles from getting into the sample.
4. Place the pipette tip in water and release, allowing for the intake of the substance.
5. While placing the specimen onto the concave slide, push it all the way down to both levels, releasing the entire droplet on to the glass.
6. Practice transferring 20,100, and 150 microliters.

FOR VIEWING TETRAHYMENA

1. Transfer 100 μL of Tetrahymena, using the correct micropipette.
2. Under the dissecting microscope, observe the Tetrahymena in the well plate itself.
3. While looking through the dissecting microscope pick 5 μL of cells from the will with a p10 micropipette
4. Use a coverslip when viewing at 40x
5. record how many cells observed and attempt to come up with the diameter of one tetrahymena cell, using FOV measurements.
6. Record all info.

OBSERVATION:

• STORAGE:

1. I discarded the tips of the micropipettes and placed them where I found them
2. I Cleaned the Concavity Slides and Cover Slip with normal and DI water and placed them in the back of the class.
3. I Placed the lid back onto the 24-well plate and placed it where I found it.
4. I put the cover back onto both the dissecting and light microscopes, respectfully, unplugged them and turned the lights off, and placed them where I found them.

• Conclusion:

1. In conclusion, I furthered my microscope using skills in this lab by focusing on Tetrahymena. I learned a lot about the species as well. For example, it has seven different sexes and can reproduce in about 21 different ways. Along with this, I learned that Tetrahymena is being affected  by microplastics in soil.  I also learned of the importance of preciseness, through using the micropipette. It is extremely vital for the results of the experiment to use precisely the amount of specimen required by the experimental procedure.

• Future Goals:

In the future, I plan on using the coverslip on high power as I should’ve done. Lucky for me, the specimen was not disturbed when I put it on high power, however, there was some tetrahymena that I was not able to see, because they gravitated towards the bottom layer of the water. I only saw a “shadow-like-image” of the specimen.

August 30th, 2018

Ciliate Challenge

• Rationale – We compared specimens of Ciliates in order to familiarize ourselves with using materials in a lab setting, such as a dissecting microscopes, pipettes, and wells. We also performed the experiment in order to observe different behaviors of Ciliates and to classify them.
• Procedure –

1.) Obtain Clean 24 mL Plate.

2.) Pick up each individual specimen with a separate pipette and place them into their separate wells.

3.) Make sure not to cross contaminate any of the wells, and not to mistakingly reuse a pipette from one sample to the other. (This will Result in flaws of the experiment!)

4.) Observe each unknown sample under a microscope and record the results in your lab notebook.

5.) Record the Species’ number, shape, relative size, movement, location in media, other characteristics, and draw a sketch.

• Observations –

• Conclusion – This lab exposed us to using various tools, common in a biology lab setting, such as a dissecting microscopes, pipettes, and wells, while also exposing us tomultiple ciliate cultures. Along with this, we analyzed ciliates in order to identify them. I found the biodiversity of all the ciliates extremely fascinating. They are so small, yet

  there’re still numerous kinds that have traits. I also found it interesting how each group of ciliate developed different traits in order to thrive in their individual environments.