Lab 5: Serial Dilutions and Cell Count

Sheridan Mikhail

9/21/18

Objective: The objective of Lab 5 was to allow students to practice sample collection, preform serial dilution, and experience experimental procedure set up.

Purpose: Soil samples were collected for future use. The purpose of the Serial Dilution and Cell Count lab was to give students another tool to use in the experiments they would design in testing microplastics and Tetrahymena. Specifically the ability to preform a serial dilution in order to count Tetrahymena.  The computer lab time was used to finalize experiment process and create a feasible materials list.

Soil Collection:

Materials

• Plastic Bag
• Spoon
• Soil
• Phone
• Petri dish
• Balance

Procedure

1. Students collect soil from designated areas
2. Using a spoon to remove the top layer of dirt students filled 1/2 of a plastic bag.
3. The plastic bag was then labeled with designated ID number SGM32F18
4. In class, a petri dish was placed on a balance and the mass was recorded in grams.
5. Then the petri dish bottom and allocated amount of soil was placed on the balance and mass recorded in the Table 1 below.

Table 1:

Serial Dilution:

Materials

• P-10 Micropipettor
• P-200 Micropipettor
• P-1000 Micropipettor
• 24 Well plate
• Concavity slides
• Compound Microscope
• Dissecting Microscope

Procedure

1. Students were given a 24 well plate with a preprepared stock solution with Tetrahymena in well 4C
2. The stock solution was observed under a dissecting microscope, the solution looked like moving dust particles and slightly cloudy.
3. Students then chose a column of wells to preform a 10-fold serial dilution. Fig.2 
4. 900µl of Tetrahymena culture media was placed in the wells of the column using a P-1000 micropipettor
5. 100µl of 10⁰ Tetrahymena stock culture was added to well  3A with a P-100 micropipettor and mixed by pipetting up and down 5 times and the tip was disposed of making a 10^-1 dilution. Fig.1  
6. Then 100µl of 10-¹ was added to 3B, mixed by pipetting up and down 5 times making a dilution of 10^-2 and the tip was then disposed of (see Fig. 1) 
7. 100µl of 10^-2 was then added to 3C, mixed, and the tip was disposed of. 
8. 100µl of 10^-3 was then added to 3D, mixed, and the tip was disposed of. 
9. After the serial dilution was complete the different dilutions were observed under a dissecting microscope. The well with a reasonable amount of cells, 30-40, was chosen to prepare a wet mount.
10. Using concavity slides, students placed 3 5µl drops onto the slide with a P-10 micropipettor. Then they counted how many cells were present under the compound microscope. Table 2 shows cell count.

Figure 1:

Figure 2:

Table 2:

Practice Problems for Serial Dilution and Cell Count

1. 0.5 ml= 500µl
2. There would be 5000 Tetrahymena in a 10^-3 mL of the 20mL water sample.
3. .1 g of India ink should be added to 10mL of water to make a 1% solution.
4. 4 mL of cells and 16 mL of cell culture media would make
5. The solution of 100% ethanol diluted 1:10 would be 9.09% ethanol
6. To make a 1/10^6 dilution one would have to preform 6 1/10 serial dilutions (Figure 1).
7. 4700 cells per 1mL would be found in a sample of 10µl of culture with 42 cells.

Computer Lab:

Procedure:

*See below for proposed experiment

1. Students used pre-prepared questions to come up with a falsifiable hypothesis
2. Students finalized their hypothesis for their experiment
3. Students also identified their realistic reason for the type of microplastic chosen
4. A list of materials was created for the experiment

Data:

Question:

What effects do microplastics have on Tetrahymena survival rates?

Hypothesis: Increased amount of microplastics will show decreased survival rates in Tetrahymena.

Reason for microplastic chosen: Polyethelene Microbeads have been used as an exfoliant in face wash and body wash. Microplastics between 1-5µl can be eaten by Tetrahymena therefore the microplastics are ensured to be eaten by the ciliates.

Experimental procedure:

1. 4 treatments would be preformed by 2 groups each.
2. Groups 1-2 would add 0.025g of Polyethelene Microbeads to 900µl of media and 100µl of 10⁰ stock solution.
3. After 24 hours, the each person in the group would preform 1 10-fold serial dilution and count the surviving cells.
4. The same procedure would be preformed for the remaining groups however the amount of pollutant added would change.
5. Group 7 would preform a serial dilution and cell count on a non polluted sample which would act as the control.

Conclusion: The serial dilution and cell count portion of this lab allowed students to develop another tool for their experimental design.  This lab allowed students to develop real life experimental procedure. Using knowledge gained from the pre-lab about experimental procedure, students proposed a hypothesis and experiment on Tetrahymena and Microplastics uses.

Storage: Students stored collected soil samples in petri dishes under the fume hood. Wet mounts made in the serial dilution experiment were bleached and laid out to dry. All information for possible experimental procedure was saved to Google Documents.

Future goals: The future goals of this lab is to have students develop and preform their own experiments using the techniques taught.