Lab 5: Serial Dilutions
Objectives/Goals: The objectives of this lab is to learn how to do serial dilutions, observe the amount of tetrahymena in each dilution, improve our skills in micropipetting, and storing our collected soil samples.
Materials:
- Petri dish
- p-1000 micropipettor
- well plate
- concavity slide
- stock solution
- compound microscope
- dissecting microscope
- collected soil
Procedure:
Collecting Soil-
- Collect soil from tree
- Weigh bottom of the petri dish with and without soil
- Transfer soil from bag to Petri dish
- Label top and bottom of Petri dish with name and soil identify
- Record soil weight
- Cover Petri dish and place in fume hood
10-fold Serial Dilution-
- Observe tetrahymena in the stock well plate using a dissecting microscope
- Choose a vertical column in the 24 well plate
- Label 4 wells of the 24-well plate: 10^-1, 10^-2, 10^-3, 10^-4.
- Add 900 μl of the tetrahymena culture media to the 4 wells
- Add 100 μl of 10^0 (undiluted) stock culture to the -1 well, mix by pipetting up and down, change tips with each dilution.
- Add 100 μl of 10^-1 sample to -2 well, mix
- Add 100 μl of 10^-2 sample to -3 well, mix
- Add 100 μl of 10^-3 sample to -4 well, mix
- Observe each well under the dissecting microscope and count the number of ciliates you see.
- Choose a well and transfer three 5 μl drops to a concavity slide
- Observe the 3 drops under the compound microscope and count the ciliates from each drop, record the number of tetrahymena you see.
- Determine the average count between all 3 drops and record in data table.
Computer Lab-
- Come up with various questions regarding ciliates and microplastics
- Hypothesize
- Methods of treatment
- Research how to do an experiment based on your hypothesis.
Data:
| Mass of dish | 5.8 g |
| Mass of dish with soil | 18.9 g |
| Mass of soil | 13.1 |
| Trial | Dilution: Which Dilution did you use to make your counts? | Cell count in 5µl | Cells per µl in the drop (x/5) | Cells/µl in the undiluted sample (x dilution factor) | Cells/ml in the undiluted sample (x 1000 µl/ml) |
| 1 | 10^-2 | 11 | 2.2 | 220 | 220000 |
| 2 | 10^-3 | 6 | 1.2 | 1200 | 1200000 |
| 3 | 10^-4 | 0 | 0 | 0 | 0 |
| Average | – | – | – | – | 710000 |
Observations:
The more we diluted the wells the less tetrahymena were observed. 10^-2 dilution was the most countable well plate.
Storage:
The Petri dishes with soil inside were put under the fume hoods to dry out and allow the ciliates to become cysts. The concavity slides were rinsed with the cleaning solution and water and were placed on paper towels to dry. The microscopes were covered. The used micropipettor tips were discarded, and the micropipettors were hung on their racks. The stock solution, Tetrahymena media, and well plates were left on the tables for the instructors to handle.
Conclusion/Goals:
In this lab I was able to further my skills in micropipetting and using lab tools. My goal was to learn how to properly execute a 10-fold serial dilution which I was able to complete. I also was able to gain a deeper understanding of where ciliates are found due to me getting my dirt. The computer lab enabled us to think outside the box on how the waste we put into the environment has an effect on the organisms around it.