Abel Thomas

1/31/19

Purpose: Today in class we talked more about the benefits of metabarcoding. We also tested our soil’s water % content, retesting the pH, we made sure we prepared our test tubes for culture and we looked for ciliates.

Materials:

Compound microscope

Light microscope

NFP with your soil

small plate with your soil

test tube with your soil

Dispersing agent

vortex

micropippeters

slides

scale

Procedure:

1. Grab your containers with soil
2. test water % content by weighing the small plate again using the scale and subtracting your original weight by the new weight you found and then dividing by the original weight and then multiplying by a 100
3. retest the pH level if not done in last lab
4. use your NFP and put it under the light microscope to see if you see any movement
5. micropippete from either NFP or test tube and put it on a slide
6. use compound microscope to zoom in on the images
7. record any ciliates found by taking a picture or drawing it
8. put one drop of dispersing agent in test tube after done using it as a way to find ciliates
9. put the tube on top of the vortex and hold it still as it mixes it thoroughly
10. put everything in it’s respective place

Clean Up:

After all the procedure were complete, students made sure that their microscopes were unplugged, their slides were clean and put away, and their soil was put back to where it belonged.

Conclusion/Future Goals:

Today we found that my water % content was 9.72% and my pH was 6.0. I was able to find a couple of ciliates but I was not able to take a picture before it ran away. I was able to draw a picture of it. It was long and thin and oval shaped. I could see the cilia on it and many nuclei. My goal for next lab is to hopefully capture an image of the ciliate and be able to put that image on a presentation or paper.

Abel Thomas

1/24/19

Purpose: Today’s lab, we presented our articles and talked about the benefits of metabarcoding. We also made our NFP and tested our pH levels.

materials:

• Dissecting microscope
• Big plate
• Small plate
• Plastic
• pH paper
• scale
• water

Procedure:

Today we were able to give our presentations to the group about each of our metabarcoding articles. After our presentations we were able to look more deeply into the soil we collected. We found that the tree we collected from was an oak. Our soil was taken from the rhizosphere of the tree. We were able to measure the circumference of the tree to be about 255cm. The region the tree was found was at Region R.

In Lab Procedure: We took our soil from the tree and then put it into a NFP and made sure to leave enough soil so we could find ciliates. After that we were able to put 3 mL of soil into a falcon tube and filled the rest of the tube with water. We then mixed the falcon tub and tested its pH levels.

Clean up: We made sure we unplugged our microscopes and also placed the plates and the bags in their respective spots.

Conclusion/ Future goals: The pH levels were found to be around 6.0. Our goals for after this lab is to ensure we save the ciliates and make sure we are able to find some this semester using our NFP. We hope to find sufficient information this semester to be able to write our research papers.

Abel Thomas

1/17/19

Purpose: The purpose of this lab was to introduce students back into the mindset of ciliates by giving them more information on how to determine which ciliates are which through metabarcoding. The purpose is to fully understand the concept by giving a presentation the week after over metabarcoding and ciliate discovery.

Summary of Lab: Today in lab we had to bring our computers so we could work on the powerpoint presentation that we will be presenting to the class next week. My partners are Bassam and Paul and our article is over the COI gene and how that could help determine the diversity of the ciliates. Before starting on the powerpoint, Dr. Adair talked to us about the importance of metabarcoding and how it is different from regular barcoding. Through her talk, it was easier to see what we were looking for in our article. After her talk, my partners and I were able to find most of our key information from the article, we used our time wisely so we won’t have to do much work outside of the classroom.

Goals for this semester: My goal for this semester is to truly dive into the world of ciliates and learn more about this fascinating microscopic world. I also want to make sure I stay on top of my classwork better in this class and make sure I can get an A. This course has already taught me so much and I am so excited to learn more, especially about the sequencing aspect of ciliates. Through this class I want to have a better understanding of the world of biology and a greater appreciation for the soil and world around me.

Purpose/Objective: Today’s lab let us observe the soil texture and approximate the percentages and identify what type of soil we ad. Along with that we did more with our ciliate observation.

Materials:

Falcon tube containing your soil

NFP with soil

Compound microscope

Dissecting microscope

Slide

Micropippetor

Ruler

Procedure:

Soil Texture:

1. Take your soil texture falcon tube
2. Take a ruler
3. use centimeter side of ruler and put it at the bottom of the tube
4. measure the total amount of material in the tube
5. Observe the difference between the materials and measure that (sand is on the bottom, then silt, then clay)

Ciliate Observation

1. Take NFP and put it under the dissecting microscope to see if you see any movement
2. Take micropippetor and take 10 uL drops of soil and put it on the slide
3. Observe the ciliates you find from the samples

Data: The soil texture was 45% sand, 45% silt and 10% clay

I was able to observe two different types of ciliates in my observation but I was not able to take any pictures but I will find the ciliates using the observations I wrote down about the ciliates

Storage: After our work is done we made sure all our equipment was safely stored and put back in it’s correct positions and we made sure to clean our lab area as well.

Conclusion/Future Goals: Even though I was unable to get pictures of the ciliates, that does not mean my research was a bust. For my presentation I will be able to talk about why I was not able to take pictures of the ciliates and I will say what things I should do to improve on that.

Objective/Purpose: The objective and the purpose of this lab is to understand the concept of evolution and how life is all related. Along with this, we should become more and more comfortable with identifying ciliates and their morphologies. Then we also will be calculating soil texture and percentages which we will see next week.

Materials:

Falcon Tube

Compound Microscope

Dissecting Microscope

Micropippetors

Soil Sample from NFP

Extra soil from the baggy

slide

Procedure:

Soil Texture

1. Take 4 mL of the soil from the baggy and put it in your falcon tube
2. Add 6 mL of water and put it in the falcon tube
3. Add one drop of 2% dispersing agent into the tube
4. Vortex the tube for 10 seconds
5. Put it on the rack

Ciliate Observation

1. Take the NFP and put it on your dissecting microscope. Look for any movement in the soil or water
2. Micropippete some of the liquid and put it on the slide so you can observe it on the compound microscope
3. Continue to do this process until you see a ciliate or some movement

During this lab time I was unable to observe a ciliate moving around even though I took dozens of drops in varying regions of my sample. I know there are ciliates in my soil because I was able to observe one last week.

Storage: We made sure to put our baggies and our plates back in the white tubs that they belong in and to unplug and take out the microscopes and cover them up properly. After that, we made sure our lab areas were clean and to clean and properly put away the slides.

Conclusion/Future Goals: I was unable to find any ciliates today but I was able to have my soil texture ready for next week. I had to do 2 runs of this but it just helped me understand how to do the process. My future goals is to observe the ciliates that are in my soil and being able to identify it.

Objective/Purpose: The purpose of this lab was to observe and understand the diversity that the soil we obtained  earlier this semester contains.

Materials:

Lab Notebook

Pipettors

Compound microscope

Dissecting microscope

Soil Sample

Slides

Centrifuge

Microfuge tube

pH paper

Water Content %:

1. Obtain soil sample
2. Calculate the weight of the sample by looking at the lid (Determine if you calculated with the lid or without the lid
3. Retrieve your sample weight when you first weighed it many weeks ago
4. Calculate water content % by taking the mass weight of the soil from the original and subtract that by the weight on the lid of the sample (make sure you do not include the weight of the container) and then divide by the original weight of the soil. After this you multiply the number found by 100

Found to be 18.18%

Determining pH:

1. Take 1 mL of liquid from the soil onto a microfuge tube.
2. Place the tube into the centrifuge and spin it for 1 minute
3. Use the pippetor and pippete some of the liquid onto the lid of the container
4. Put pH paper on top and observe the pH after waiting a minute

Found the pH to be 6.8- making it slightly acidic

Observing the soil and look for ciliates:

For this section we would take our soil samples and put them under the dissecting microscope. We moved our soil to one side and had the water put on another. What we are looking for was the middle section where the water meets the soil. If no movements or ciliates could be found in that observation, then students would pippete a drop of the water from the soil into a slide to observe it underneath a compound microscope. You keep doing this process until you are able to see a ciliate or some type of organism in the soil.

Strorage:

We cleaned up the lab area and made sure that everything was put back into place except for the soil samples which Kaitlyn took. We ensured that the lab area was clean and organized and to dispose of all the pipettes used and to clean the slides we used.

Conclusion/Future Goals: I was able to observe a ciliate moving which ensure that my soil choice was good. Next week, hopefully I will be able to get to see more ciliates and make more observations of other lifeforms to understand the biodiversity of the soil.

Objective/Purpose: The purpose of this lab is to make figures over the data that your table group of 3 found. We looked at the mean values of the control and treatment levels of cell counts as well as vacuole count, swim speed, and direction changes.

Materials:

Laptop

Data from experiment

Procedure:

1. Log onto computers and opened up excel
2. Share a single excel folder so group can add their own data
3. Input the mean of the control for cell counts and optical density
4. Input the mean of the treatment for cell counts and optical density
5. Select data from both treatment and control for cell counts
6. Make bar graph from recommended charts tab
7. Click on chart designs and then add chart elements so you can add error bars
8. Make sure title and axes are correctly labelled
9. Do steps 5-8 for optical density, and vacuole formation
10. Save all graphs as JPEGs

Figure 1

Figure 2

Figure 3

Figure 4

Storage: we made sure the area was all cleaned up and that all the computers were logged off.

Conclusion/Future Goals: We created our graphs and these will be included in our rough draft that is due next week. The graphs help show a visual representation of the data that was found. Through these graphs, it helps the reader understand the implications that polypropylene have on tetrahymena cultures.

Objective/Purpose: The object/purpose of this lab was to analyze and interpret our data using Excel.

Materials:

Laptop

Data from experiment

Procedure:

1. Login to computer
2. Obtain data from each student and put it in a single column. One for control and one for experimental
3. Get data from vacuole formation for 5 minutes and make a column for the control group
4. Repeat step 3 but do it for the experimental group
5. Get data from vacuole formation for 15 and 30 minutes for control and combine them in one column
6. Repeat step 5 but for the experimental group
7. Take the Dimensional Statistics for all of the columns to help find mean, standard deviation, range and the minimum and maximum values
8. After taking the Dimensional Statistics, make your bin range which is the range from your smallest number to your largest number so they will be all inclusive to your histogram. Do this for every column
9. Take the histogram of each column with its corresponding bin range and place the histogram graph next to data
10. Take an f-test between the control column and the experimental column for each set point to find the variance between the two
11. If the variances have a significant difference between them, use a t-test with unequal variances, if they do not take a t- test with equal variances
12. Determine if the p-value from the t-test is greater or less than .05. If it is less than you reject the Null Hypothesis. If it is greater than you accept the Null Hypothesis.

Storage: I made sure my laptop and computer were off and that I logged off before I left.

Conclusion/Future Goals: The conclusion is that we finished analyzing our data and have significant data that proves that the polypropylene has an effect on the Tetrahymena. The next step is to use this information in writing a paper about how people need to be more wary about their plastics.

Purpose/Objective: The purpose and objective of today’s lab was to do our experimentation on how polypropylene from bailing twine would effect Tetrahymena. My experiment was over the vacuole formation and cell count so I had to count for that data at periodic intervals.

Materials:

Experimental Tetrahymena solution

Control Tetrahymena solution

compound microscope

2 flat slides

1 cover slip

2 uL of India Ink

2 uL of Methyl Cellulose

Timer

1 uL of Iodine

Spectrophotometer

micropippetors

lab notebook

Data from Spectrophotometer:

• Black: .060
• Control: .017
• Treatment: .141

Procedure for Group:

1. drop 3  individual drops of 2 uL of Tetrahymena (either control or experimental) on slide
2. mix 1 uL of iodine into each sample of Tetrahymena and mix thoroughly through pippeting up and down
3. observe count of Tetrahymena in each drop and write in the lab notebook
4. Compare with another student that did the opposite Tetrahymena that you did… if you did experimental, find someone that did the control

Procedure for Vacuole Assay:

1. Place 10 uL of Tetrahymena culture on flat slide
2. Add 2 uL of India INk to the drop of cells and pippete up and down to mix cells and the ink
3. Place 2 uL of methyl cellulose in the center of the drop
4. Place a cover slide on the drop and focus the slide on 100x-400x magnification on compoun microscope
5. After 5 minutes scan slide for cells and count the number of vacuoles
6. after 15 minutes, count vacuoles for 10 more cells
7. Repeat at 30 minutes

The experimental Tetrahymena solution was not able to be found in the correct increments so only one data time was found

Storage: I made sure I cleaned up my lab area and cleaned my slides and cover slip using water and bleach. I also made sure that with every micropippete I would throw the used pippete to make sure I was not mixing unneeded materials.

Conclusion/Future Goals: We found that through the inclusion of polypropylene into the Tetrahymena that the Tetrahymena will have an increase of vacuoles compared to the control solution. My future goal is to see how microplastics and polypropylene affect this model organism further more and how it can affect the environment.

Purpose/Objective: The purpose and objective of this lab was to allow us to see what we will be working with for the rest of the semester which is Bailing twine which is a microplastic used to hold the bales of hay together. Through this lab we made sure that we have a sample ready for use for next week and also we observed more effects of Tetrahymena to different substances and how different things other than cell count might be altered.

Materials:

.5 g of black polypropylene (PP)

Sterile glass container

50 mL of sterile proteose-peptone-tryptone (PPT) media (more if needed to dilute)

40 uL of Tetrahymena (more if needed)

5 uL of Iodine

concavity slide

compound microscope

lab notebook

micropippetors

coverslip

1-5 uL of India Ink

Timer

Procedure for PP Microplastic production:

1. measure .5 g of polypropylene (PP) and cut it up into the smallest pieces you can with scissors
2. add the PP into a sterile glass
3. Add 50 mL of PPT media
4. boil and stir for entire class

Procedure for Ciliate Count Challenge:

1. Add 20 uL f cells to 5 uL of Iodine
2. Add 3-5 uL drops to a slide and count the stained cells

Average = 32,667 cells/mL

Procedure for Lysosome Formation Assay:

1. Place 20 uL of Tetrahymena culture on a concavity slide
2. Add 1-5 uL of India Ink to the drop of cells and pipette up and down to mix the cells and the ink
3. Place the coverslip and focus the slide on 400x magnification on the compound microscope and start timeR
4. Scan the side for the 10 cells and count any lysosomes. This is time 0.
5. After 10 minutes, count the number of lysosomes for 10 or more cells
6. Repeat the procedure for 20 minutes and 30 minutes.

I was unable to get the 30 minute markings because I took too long to focus the microscope and have it all set up

Storage: We made sure that we would dispose of caps for the micropipettors after we used them, we washed, bleached, and dried all the concavity slides, petri dishes and cover slips before putting them back in their original place, and we also made sure that we put the microscopes back into their places and covered them.

Conclusion/Future Goals: We concluded that with the exposure of India ink that the Tetrahymena would increase its number of lysosomes. Also we were able to finish up our introductory section of the PP microplastic production. My future goal would be to master serial dilution and using a compound microscope at higher powers. Another goal is to be able to understand the basic concepts of the many effects that microplastics have on this world.