Purpose:

The purpose of lab 4 was to introduce us to primary literature and how to find it and use it to look at questions and ideas we may have for our future Tetrahymena experiment.  Through looking at primary literature we can get a better idea of how Tetrahymena are used in research.

Procedure:

1. Place a drop of the Tetrahymena sample on a concave slide.  Place a cover slip over it.
2. Observe this sample under 4x, 10x, and 40x magnification on a compound microscope.  Record your observations.
3. To slow Tetrahymena movement, take the concave slide and put a drop of methylcellulose in your sample.  Record any new observations.
4. After recording, rinse slide and put all materials away.
5. Open up your labtop or go to the computer lab and download Zotero to keep all of your articles.
6. Research topics for Tetrahymena experimentation and come up with a hypothesis you want to test.  Save your articles in Zotero.

Observations:

4x- Tetrahymena are very small and move very quickly

10x- About the same as the 4x observations

40x- Not able to see Tetrahymena but can see individual organelles.

Primary and Review Article:

Article Analysis: http://www.sciencedirect.com/science/article/pii/S0278691515000289

1. Central question:  The central question of this primary article is questioning what the effects of melamine, a raw material, will have on Tetrahymena.
2. Introduction Information:  Melamine is an organic compound that contains a large amount of nitrogen.  It is commonly found in plastics, decoration, paint, and paper industries, but it is banned in food.  Melamine is believed to be slightly toxic and can cause chronic toxicity.  Tetrahymena is a unicellular eukaryote that is often used as a study representative for protozoa and unicellular organisms.  Tetrahymena react very similarly to pollutants in the environment; therefore, it is commonly used to study toxicological issues.
3. Design to Experiment:  1.  The Tetrahymena were first separated at 3500rpm for 20 minutes.  The sample of Tetrahymena was then introduced into a medium made of 15g tryptone, 5g yeast extract, and 1g glucose in 1000mL and cultured at 30 degrees Celsius for 72 hours.  2. Melamine was placed under ultraviolet germicidal irradiation an hour prior to use to make sure there was no microorganism contamination.  3. The medium is dispensed into 5 test tubes and each test tube was given 0, 0.005, 0.010, 0.015, and 0.020g of melamine respectively. The Tetrahymena strain  was then suspended at the density of 2×10^5 cells/mL.  100 uL of the suspended cell were inoculated into each test tube. The tubes were then cultured for 20 hours at 30 degrees Celsius while being constantly shaken at 220 rpm.  Another 5 test tubes were created as a control group with no melamine present.  4.  The Tetrahymena were counted under a microscope after the 20 hours using a hemocytometer.  The half maximal inhibitory concentration was calculated, and the experiment was repeated three more times.
4. Questions about Results: Does the medium they were placed in have any effect on the proliferation rate? The results were that the number of Tetrahymena decreased as the amount of melamine present in the sample increased.  This shows that Tetrahymena are highly sensitive to melamine-induced damage. For mating purposes, this experiment showed that low concentrations of melamine (1-2 g/L) were mating at an increasing rate, but concentrations of melamine higher than 2 g/L caused a declining mating rate.
5. Discussion:  The probable cause of why Tetrahymena’s mating rates increased with low dosages of melamine is because the melamine slightly damaged the genome of the Tetrahymena, so to compensate they mated quickly to makeup for the damaged genes.  Under high concentrations though, the genome was too damaged and the mating genes were also damaged so they were not able to repair the damage done. It is believed that the damage done to the genome was only applicable in high concentrations of melamine, and low concentrations of melamine showed negligible damage. The Tetrahymena’s self-repair capacity made up for the small amounts of damage done under low concentrations.

Review Article: https://www.ncbi.nlm.nih.gov/pubmed/28846184

1. Central question:  How do poikilothermic organisms (ex. bacteria, fungi, etc.) maintain homeostasis in cold weather?
2. Primary Findings:  These organisms adapt the lipid composition in their membranes to adapt to the weather change and maintain their membrane fluidity.  This is known as homeoviscous adaptation, and the main component of it is the composition of a acyl chain.
3. Questions:  Is this way of maintaining homeostasis more effective than controlling our own body temperature?  What exactly are acyl chains?

Conclusion:

This lab gave me a good understanding of Tetrahymena, and I also got further practice using the microscopes.  This lab also showed me how to properly find and analyze primary literature, which was something very useful because I haven’t had much experience doing this.