Lab #5: Experimental Design and Serial Dilutions

09/20/18

Haiden Jordal

Objectives:

There were three parts to this lab. First the purpose of obtaining the soil samples from the soil around the trees is to later be used in research of soil ciliates. The second part, was serial dilutions. The objective of this part was to teach students to use micropipettes to dilute a solution in order to calculate a concentration of cells when there is an overwhelming amount of cells to count in an original solution. The last part in the computer lab allowed students to record their results from in the lab on a class wide spreadsheet. It was also necessary to brainstorm and identify an experimental design to go along with an experimental question and hypothesis. All of which should be testable and ready to replicate in lab.

Procedure:

-Soil Collection

1. Choose an area of soil within the rhizosphere of a bald cypress tree by the creek to collect a soil sample.
2. Bring the soil sample to lab for labeling and record the mass of the soil.
3. Place sample inside a petri dish under the vent hood for later use.

-Practicing with Pipettes

• Use water to practice picking up and dispersing different amounts with the newly introduced 1000 ul pipette.

-Serial Dilutions

1. Identify the stock solution of the 24-well plate and observe the activity of the Tetrahymena under the dissecting microscope.
2. Transfer 900 ul of media to each of four wells in a single column using the 1000 ul micropipette.
3. Transfer 100 ul of the Tetrahymena stock to the first well and label it 10^-1.
4. Mix around the solution by pipetting it up and down. Then change tips.
5. Transfer 100 ul of the solution of the first well into the second well and mix. Change tips.
6. Repeat this process for the remaining wells.
7. Determine the well that has a countable concentration and transfer 5 ul of it to a concavity slide.
8. Observe the slide under the compound microscope at 4x magnification and then at 10x and record the number of cells transferred.
9. Repeat this twice with two more wells.
10. Calculate the concentration of cells in cells/ml.
11. Formula-    cells/ml = (# of cells/ volume of drop ul)x(1000 ul/ml)x(dilution factor)
12. In the computer lab, transcribe the results from lab onto the spreadsheet provided by Dr. Adair for class data.

-Experimental Design

1. Utilize the computer lab to find research and brainstorm an experimental question.
2. Based on the question, create a falsifiable hypothesis.
3. Design the experiment, explain the methods including the set up making sure to list the control and tested variable.

Observations:

Soil:

Serial Dilutions:

Experimental Design:

• Experimental question: How will the introduction of propylene microplastics effect the population of living Tetrahymena ciliates?
• Hypothesis: The propylene will decrease the survival rate of tetrahymena in the contaminated populations.
• Experimental design: Using 24 well-plates, there will be two rows of observation: one with six control groups and the other with six treated groups. There will be media placed in the control groups to maintain the single variable of the microplastics. This is done in order to reduce error. The original population size will be recorded and then recorded again once a week for three weeks to observe the continual growth or decay of populations of all twelve wells.

Storage:

The micropipettes were put back on the rack after use while all of the pipette tips were disposed into the tip waste cups. The compound microscope was put back in the center of the lab table and covered after being set to 4x magnification and lowered to lowest stage setting. The dissecting microscope was turned off and placed back in the middle as well. The 24 well-plate and the media used in the dilution were both stored towards the side of the table, while the concavity slide was washed and placed on the counter to dry. The unused tips were put back in the lab drawer to the side, and the lab instructions went back in the drawer in the center of the table. In the computer lab the computers were properly logged off of.

Conclusion: 

In lab, I learned the valuable skill of serial diluting. It is essential in determine the concentration of microscopic cells when there are far too many cells to count in a single sample. Diluting the sample to keeps the concentration constant while the number of visible cells reduces. This makes counting them and calculating the average number of cells per much easier and accurate. In addition, we began our first steps to creating our own scientific literature by using the framework of other sources and a guide to come up with our experimental design and method. It is evident that continuing to use scientific literature in our research will be key to effectively producing our own publishable experiment. In lab today, we came up with the idea to use propylene as our microplastic toxin as it is one of the most abundant microplastics in the world.

Future Steps:

In the future with our new groups, we will be starting our development of our experiment. Further research will be needed to understand the habits of Tetraahymena over time. Also while we decided upon propylene, it could be useful to research other types of potential microplastics that can be hazardous to soil ciliates like tetrahymena and whether or not we are using the best one for observation.