09/20/18

I. TITLE Serial Dilutions and Cell Count

II. RATIONALE

The purpose of this experiment was to curate an accurate method in which one would determine the concentration of Tetrahymena in a stock culture. This involved proficiency and mastery of how to perform a serial dilution, as increasing the skills necessary in order to use a micropipettor. Furthermore, one was able to analyze the effect that a substance had on altering a population of Tetrahymena and use that information to generate an hypothesis for future research.

III. MATERIALS

Dissecting Microscope

Compound Microscope

Concavity slide

Micropipettors

24-Well Plate

Tetrahymena culture media

IV. PROCEDURE

1. Observe the Tetrahymena in the stock well of the 24-well plate with a dissecting microscope
2. Record observations regarding the Tetrahymena in lab notebook

Performing a 10-fold serial dilution

1. Label 4 wells of the 24-well plate as 10^-1, 10^-2, 10^-3, and 10^-4 in a column-like sequence
2. Add 900 μL of the Tetrahymena culture media into each of the 4 wells
3. Add 100 μL of 10^0 stock culture to into the 10^0 tube. Mix by quickly pipetting up and down. Change micropipettor tip.
4. Add 100μL of 10^-1 sample to 10^-2 well and mix by pipetting up and down. Change micropipettor tip.
5. Add 100μL of 10^-2 sample to 10^-3 well and mix by pipetting up and down. Change micropipettor tip.
6. Add 100μL of 10^-3 sample to 10^-4 well and mix by pipetting up and down. Change micropipettor tip.

Cell Counts

1. Observe the 24-well plate under a dissecting microscope and determine which well has a countable concentration. Record the optimal dilution.
2. Transfer 5μL of the well with countable concentration onto a clean concavity slide.
3. Count cells under 10X objective.
4. Add 5μL of methyl cellulose onto the Tetrahymena to aid in counting cells if they are moving too quickly.

V. DATA & OBSERVATIONS

Observations of Tetrahymena:

10^0: Living, inconsistent and erratic movement patterns, concentrated

10^-1: Noticeable amount of population died off, inconsistent and erratic movement patterns, diluted

10^-2: Increased amount of population died off, slow movement patterns, more diluted

10^-3: No seen Tetrahymena, highly diluted

10^-4: No seen Tetrahymena, most diluted

Tetrahymena viewed at 10x objective.

Note: I ran short on time to finish the following two trials.

Math used for data:

cell/ml = (#of cells/volume of drop µl) x ( 1000µl/ml )x ( dilution factor )

= (7 cells / 5µL ) x ( 1000µL/mL ) ( 100 )

=  140,000 cells/mL

VII. STORAGE
The 24-well plates were returned to the lab instructors at the end of the lab for proper disposal of the Tetrahymena culture media and stock culture. The concavity slides were carefully cleaned off with water and bleach, then dried off for future use.

VII. CONCLUSION

Overrall, learning how to perform serial dilutions on Tetrahymena thusly reminded myself how important it is to maintain a level of accuracy when it comes to pipetting measurements in a lab. When it came to diluting the samples on the well plate, there were a series of steps I had to take to ensure that my measurements were correct, such as making sure I utilized the correct micropipettor for the amount of culture I took out. For instance, using a .5-10µL micropipettor to suction out 5µL of the optimally diluted sample onto the concavity slide. Referencing the data, it was interesting to note that as the 10^0 Tetrahymena culture became more diluted, the amount of cilia in the sample decreased. This could provide insight into how Tertrahymena could be impacted by a change in their living environments.

VIII. FUTURE STEPS

Moving forward, I hope to become more comfortable with using microscopes to analyze ciliates. I had a bit of trouble in my attempts to view the Tetrahymena under the dissecting microscope, so I hope that through more practice, I am able to view samples in an efficient manner. On the other hand, my goal is to enhance my critical analysis skills by reading more scientific literature. This would be greatly beneficial for determining proper methodology for my research project and increase my creativity when it comes to curating my approach.