Lab 5: Preparation of Soil Experiment, Dilution Practice, Experimental Design
Christina Clark
September 20, 2018
Lab 5: Preparation of Soil Experiment, Dilution Practice, Experimental Design
Preparation of Soil Experiment
Objective
The objective of this experiment in lab 5 was to dry out the soil, in order that the ciliates go dormant. Later, one will rehydrate the ciliates and observe them further within the experiment.The objective of the dilution practice in lab 5 was to become more familiar with serial dilution. In order to count the number of cells in a culture easier, on may use serial dilution. This will allow one to be able to estimate the number of cells in a solution which concentration is too high. The objective of the experimental design is to help student brainstorm a testable experiment to further the knowledge of ciliates. By doing this, students become more familiar with the thought process of a scientist and are able to create an experiment that has a falsifiable hypothesis and procedures the are able to be replicated for accurate results.
Procedure
- Obtain a plastic bag and some sort of scooping tool.
- Go out to one of the four pre-marked areas along the river.
- With the scooping tool, collect soil no deeper than 3-4 centimeters, as ciliates need air.
- Fill the bag approximately halfway up.
- Label the bag with full name and soil identifier (initials, section number, and semester).
- Obtain a petri dish and write the same thing on the top and bottom of the dish.
- Weigh the bottom of the dish. Record the mass.
- Fill the dish with soil; enough to cover the bottom. Weigh the bottom of the dish and soil combined. Record mass.
- Place the top back on the petri dish and place under fume hood. Take the left over soil (reseal the bag) and place it in the provided bucket.
- Go back to the lab station and turn on the dissecting microscope.
- Begin by observing the concentration of cells in the stock culture using the dissecting microscope.
- Label four wells of the 24-well plate: 10-1,10-2,10-3, and 10-4. (Easiest of one chooses a vertical column.
- Add 900µl of Tetrahymena culture media to the four wells.
- Add 100µl of the undiluted stock culture to the 10-1 Mix slowly by pipetting up and down. Change tips.
- Add 100µl of the 10-1sample to the well marked 10-2. Mix slowly by pipetting up and down. Change tips.
- Add 100µl of the 10-2sample to the well marked 10-3. Mix slowly by pipetting up and down. Change tips.
- Add 100µl of the 10-3sample to the well marked 10-4. Mix slowly by pipetting up and down. Change tips.
- Observe the well plate under a dissecting microscope and estimate which well has a countable concentration.
- Record the optimal dilution for cell counting and transfer 5µl to a clean concavity slide.
- Using the compound microscope, count the cells using the 4x objective. Repeat 3 times.
- Calculate the average cells/ml in the stock solution.
- Clean up the lab station and go to the computer lab.
- Work with team members a come up with a question.
- Based off the question, create a hypothesis (must me falsifiable).
- Create a procedure for the experiment in a way that another student could set up and replicate the experiment.
Data
| Bottom of Petri Dish | Bottom of Petri Dish and Soil | |
| Mass (g) | 5.7g | 31.9g |
| Trial | Dilution | Cell count in 5µl | Cells per µl in the drop (divide by 5) | Cells/µl in the undiluted sample (x dilution factor) | Cells/ml in the undiluted sample (x1000 µl/ml) |
| 1 | 10-1 | 7 | 1.4 | 140 | 140,000 |
| 2 | 10-1 | 4 | 0.8 | 80 | 80,000 |
| 3 | 10-1 | 3 | 0.6 | 60 | 60,000 |
| Average | 10-1 | 4.6 | 0.933 | 93.33 | 93,333.33 |
Question: Do microplastics affect the survival rate of Tetrahymena?
Hypothesis: If microplastics affects the survival rate of Tetrahymena, then the higher the concentration of the microplastics will decrease the survival rate of the Tetrahymena.
Methods:
- Add .4ml of Tetrahymena to 20ml of PPT.
- Obtain 900µl of media using a P-1000 and transfer to the A column in the 24 well plate.
- Add 100µl of microplastics to the well marked A1. Mix by slowly pipetting up and down within the well. Change tips.
- Add 100µl of the A1 sample to the well marked B1. Mix by slowly pipetting up and down within the well. Change tips.
- Add 100µl of the B1 sample to the well marked C1. Mix by slowly pipetting up and down within the well. Change tips.
- Add 100µl of the C1 sample to the well marked D1. Mix by slowly pipetting up and down within the well. Change tips.
- Allow to grow for 168 hours. (1 week)
- Repeat steps (2-6) three more times in separate columns.
Storage
The PPT was kept in a sealed container. Stock culture was already in the well. The well plate was covered.
Conclusion
By preparing the soil today in order for it to dry out, we are able to successfully perform its correlating experiment in about 4 weeks. This shows student that experiments take time and patience. Also, today we worked on serial diluting. By serial diluting the stock culture student were successfully able to estimate the amount of cells in the original stock culture. This method is helpful because it would be nearly impossible for one to count the number of cells at such high concentration. By serial diluting students are able to produce accurate estimations. The last hour of the lab we worked on setting up our own experiment. It is good for students to begin to train their brain to think like scientist. Once students start to learn this thought process, the labs will become easier in the future.
Future Goals
In the future I would like to repeat the serial dilution experiment again in different columns instead of just extracting 5µl three times onto a concavity slide. I think this would better confirm the results. I would also like to become more efficient with pipetting as it is an essential skill in micro-biology.