Objectives of this lab:

The objectives of this lab were to continue searching for soil ciliates as well as use the soil collected to find the soil texture percentages and what type of soil it is classified as.

Procedure:

1. Using a ruler measure the amount of sand slit and clay in the test tube.
2. Use the total measurement of soil to find the percentage of sand slit and clay by using this equation:

measurement of sand, slit or clay (cm)/total measurement of soil (cm)   x 100

3.  Continue looking for soil ciliates using soil from non-flooded plate and a compound microscope.

4. Drop 4, 5 microliters of soil onto glass slide and look through compound microscope at 4x and 10x.

Observations:

For the soil texture my soil measured in total 2 cm. It contained 40% sand ( .7 cm), 20% slit (.5cm) and 20% clay (.5cm). Using a soil texture calculator it was found that the soil was classified as “loam”.

Using my soil I found one ciliate. I used methyl cellulose to slow it down, however it was very fast and very small and I eventually lost sight of it. My group mate found a ciliate using the soil form Bermuda grass provided to use by the lab.

In Future Labs:

Going forward the ciliate and soil texture found will be used for the final lab presentation as Lab 13 was our last actual lab.

Storage: 

Glass slides were washed off, micropipettes were put back up and the soil was stored in the plastic bin.

Objectives of this lab:

The objectives of this lab were to continue searching for soil ciliates as well as use the soil collected to find the soil texture percentages and what type of soil it is classified as.

Procedure:

1. Using a ruler measure the amount of sand slit and clay in the test tube.
2. Use the total measurement of soil to find the percentage of sand slit and clay by using this equation:

measurement of sand, slit or clay (cm)/total measurement of soil (cm)   x 100

3.  Continue looking for soil ciliates using soil from non-flooded plate and a compound microscope.

4. Drop 4, 5 microliters of soil onto glass slide and look through compound microscope at 4x and 10x.

Observations:

For the soil texture my soil measured in total 2 cm. It contained 40% sand ( .7 cm), 20% slit (.5cm) and 20% clay (.5cm). Using a soil texture calculator it was found that the soil was classified as “loam”.

Using my soil I found one ciliate. I used methyl cellulose to slow it down, however it was very fast and very small and I eventually lost sight of it. My group mate found a ciliate using the soil form Bermuda grass provided to use by the lab.

In Future Labs:

Going forward the ciliate and soil texture found will be used for the final lab presentation as Lab 13 was our last actual lab.

Storage: 

Glass slides were washed off, micropipettes were put back up and the soil was stored in the plastic bin.

Objectives of this Lab:

The main objective of this lab was to collect a soil sample to use for the Metadata as well as to continue looking for soil ciliates.

Procedure:

1. Obtain 4 mL of soil from plastic bag and put into tube.
2. Add water up to 10 mL of tube.
3. Mix contents together with the vertex.
4. Add a drop of dispersing agent and re-mix.
5. Observe tube after 30 seconds.
6. Let tube sit undistributed overnight.
7. Use ruler to measure % of sand, slit and clay.
8. Like lab 11, use soil from plate to continue looking for ciliates.

Observations:

It was easy to see the sand particles separate after about 3o seconds. I did not calculate the % yet as we have to let it sit overnight.

For finding ciliates I obtained 6 drops of soil from my plate and placed them on a concavity slide. Using an objective microscope I viewed the drops at 10x. My soil samples were very dense and I had to dilute the drops with water. I added 5 mL of water to the soil plate. Then I added 5 uL drop of soil into a concavity slide. I added 2 uL drops of water to the drop to dilute the sample however it was still hard to see anything so I diluted it again using the same procedure. Even after diluting my drops I still did not find anything. This includes ciliates or any other organisms.

In Future Labs:

In future labs I will have a better idea of what to look for when trying to discover ciliates based on this lab’s pre lab and what we talked about in class (morphology) prior to beginning the lab.

Objectives of this lab:

The main objective of this lab was to gain knowledge on soil ciliates to use in trying to find ciliates within our soil.

Procedure:

1. Review powerpoint on ciliates and our plan for using the soil we collected.
2. Find water percent of soil using the equation wet soil-dry/wet soil x 100.
3. Put 500 ul of soil and water into centrifuge to let all soil sink to bottom.
4. Take 15o ul of water contents out and put on lid of clean petri dish.
5. Take a strip of PH paper and put over 150 ul drop.
6. Wait for PH paper to change color.
7. Observe soil under dissecting microscope and search for ciliates.
8. Once you see a ciliate use a micropipettor to collect ciliate and transfer it to 500 ul of media in a clean 24 well plate.

Observations:

The PH level of my drop of soil ciliates was neither basic or acidic it was at 7.

I did not find any ciliates in my soil using the dissecting microscope. In addition the water run off was very muddy making it more difficult to search for them.

I transferred 10 ul of the run off and put it on a slide to observe under the compound microscope at 10x and 4x to see if I could find any movement but I did not. I think it was in part it was because of how muddy the run-off ended up being. I tried multiple methods to separate out the soil including a pipette tip and a glass slide. I also removed 2100 ul of water from the soil plate to see if that would help.

In Future Labs:

In future labs I will now have knowledge on ciliates and how they act which will help me in finding them. I will also have more time to search for them as I will know the procedure.

Objective of this lab:

The objective of this lab was to focus on reading data and transferring that data to Excel using the graph tools provided.

Purpose:

The purpose of this lab was to use the data found from the control and treatment groups of Tetrahymena to create graph figures in Excel that correlate to the results. These graphs will be of use in future labs and in the process of writing the results portion of our research paper.

Procedure:

1. Open Excel.
2. For the cell count: Choose the mean data from both the control and treatment group (found in descriptive statistics).
3. Go to inset and click “recommended charts”. Option for different visual will appear using the data you selected.
4. Choose the chart that best represents your data.
5. Label the axises. Y will be Cells (ml) and X will be control and treatment.
6. Next you must add error bars.
7. To do this click on the graph and click on “Add chart element”. Go to the “Error Bar” tab and select “more error bar options”.
8. Go to “custom” and click “specify value button”.
9. Use the standard errors from the control and treatment group (found in descriptive statistics).
10. Be sure to use 2 cells next to each other.
11. Select positive error value and negative error value as those 2 cells.
12. Repeat steps for the different assays as well as the optical density (subtract treatment number from PPT+Twine Extract number) data.

Observations:

These graphs include the data for the cell reproduction, direction assay, vacuole formation assay, speed assay and also the Optical Density Assay.

In Future Labs:

In future labs time proficiency is very important while preparing to write a paper. Having gained more knowledge on excel I can use that to my advantage in future labs.

Objectives of this Lab:

The main objective for this lab was to demonstrate knowledge on how to use Excel to perform statistical analysis.

Purpose of Lab:

The purpose of this lab was to gain statistics on the experimental assays performed.

Procedure:

1. For Control Cell Count do descriptive statistics and a histogram.
2. Repeat for Treatment Cell Count.
3. Use Control and Treatment numbers to perform the F-Test and the T-Test.
4. Do descriptive analysis of Control Vacuole Formation ground at 5 and 15 minutes.
5. Repeat this with the Treatment Vacuole formation group.
6. Create Histogram for Control Vacuole formation at 5 and 15 minutes.
7. Repeat with the Treatment vacuole formation group.
8. Using both the Control and Treatment groups (5 MINUTES) create an F-Test and a T-Test.
9. Repeat for the Control and Treatment Vacuole Formation groups at 15 minutes.

Observations:

This assay was the vacuole formation. My partner performed the experiment using the treatment and I used the control group. However the observations contain the data for both the treatment and control for the whole class. It also includes the cell count statistics for the whole class that were calculated.

In Future Labs:

In future labs it will be important to make sure you fully understand the topic before coming into lab. Because of confusion using excel and how to perform all the different tests I ran out of time. To prevent this it is good to make sure you fully understand the concepts in the pre-lab.

Objectives of this Lab:

The objective of this lab was to recognize the procedure for gaining data for each of our experimental assay’s.

Purpose of this lab:

The purpose of this lab was to correctly retrieve data for this experiment while following the procedures. Another purpose of this lab was to practice using Serological Pipettes rather than traditional ones to obtain our stock culture.

Procedure:

1. Obtain 5 ml of the treated Tetrahymena and the control group using a serological pipette. The two 5 ml should be placed in separate tubes and labeled.
2. Use the spectrophotometer to measure the optical density of both the treated and untreated Tetrahymena.
3.  Transmit to “0” and switch to absorbency.
4.  Add twine juice with no Tetrahymena. Record absorbency.
5. Repeat this with the control group and the treatment group. Make sure to “0” it out before each measurement.
6. Using three drops of 2 ul of Tetrahymena and 1 ul of Iodine take a picture and count the number of cells in each drop.
7.  Next is the Vacuole Formation Assay (My partner used the treated Tetrahymena and I used the control group. Both data will be discussed in the observations).
8. The first step for this Assay is to place 1o ul on a flat slide.
9. Add 2 ul of India Ink and pipette up and down to mix the solution.
10. Add 2 ul of methyl cellulose and place cover slide over the flat slide.
11. At 100x and 400x record the number of Vacuoles at 5 minutes, 15 minutes and 3o minutes.

Observations:

These were the pictures taken of the cell counts of 3 drops:

The cell counts were 43,9 and 19 and my partners were 10, 34 and 39.

For the control group vacuole count these were the results:

Vacuoles started to appear slowly but there were only a few major differences between the counts. For instance cell #10 and cell #5. For my assay the compound microscope would not focus at 100x and 400x so I had to look through the 40x.

Due to time constraints my partner was not able to finish the count for 30 minutes, however these were the results recorded:

This was seen at 400x magnification.

For the Spectrophotometer we measured 3 things:  Twine juice before inoculation, the control media with Tetrahymena and the Twine juice with Tetrahymena.

The Twine Juice absorbency: 0.058

The Control media with Tetrahymena: 0.039

The Twine juice with Tetrahymena: 0.155

In future labs:

We can use our time more efficiently so we have less time constraints as well as better organize our procedures within the experiment.

Objectives of this lab:

The objectives of this lab were to prepare Baling twine to be used later in lab in our experiments. Another objective was to continue practicing micropipetting and calculating cell counts.

Purpose of the lab:

The purpose of this lab was to prepare us for data collection when we begin our experiments as well as give us more practice using the lab techniques we will utilize throughout our experiment.

Procedure:

1. Prepare microjuice
2. Take .5 grams of polypropylene twine and mix with 50 ml of protease-peptone-tryptone.
3. Boil and stir/vortex
4. Keep in microwave for an hour and at 55 degrees water bath overnight.
5. In open-lab this will be autoclaved and 5 um ( the right amount ingestible for tetrahymena).
6. For the second part of lab practice cell count.
7. Add 2o ul  of TH to 5 ul of Iodine on a Petri Plate Lid.
8. Mix up and down by pipetting.
9. Add 3 separate 5 ul drops to a slide.
10. TMTC was over 50 so dilute 1:10.
11. Add 5 ul of first drop to 45 ul of PPT media.
12. Record count and report average.
13. 3d part of lab is protocols.
14. Lysosomal Assay:
15. Place 2o ul Tetrahymena culture on a concavity slide.
16. Add 2 ul of India ink to drop of cells and mix cells ink.
17. Place cover slide over and put on 400x magnification on compound microscope and start stop watch.
18. Scan slide for 10 cells and count vacuoles seen.
19. Repeat at 1o, 20 and 3o minutes.

Observations:

For the cell count:

1st drop: 240

2nd drop: 212

3d drop: 152

I had to dilute the solution because the cell count was still over 5o.

Cell count after dilution:

1st drop: 92

2nd drop: 76

3d drop: 80

I calculated the average by taking the diluted count of each drop dividing by 5 and then multiplying by 50 (because of the dilution factor) and then by 1000 to change the units. The average ended up being 826,667.

Because of time constraints I was not able to finish the lysosomal assay. I got counts for 0, 10, and 20 minutes and was only able to do 5 cells.

Objective of this lab:

The objectives of this lab were to gather a soil sample to be used later in lab. Another objective was to form teams for designing an experiment (also another objective). Another objective was to practice micropippeting Serial Dilution and using a P-1000 and from that being able to calculate concentrations.

Purpose of the lab:

The purpose of this lab was to prepare us for designing our own experiment as well as giving us more practice use micropippetors because this is important when calculating data and recording it.

Procedure:

1. First part of lab we weighed an empty dish and then filled it with soil. Then we weighed it with the soil. We covered the dishes and put them under the fume hood to be used later in the semester.
2. Next we move on to serial dilution and cell count.
3. First we observed the Tetrahymena already filled in the “stock well” on the plate. We used a objective microscope to do this.
4. Next using a micropippetor we pick up 4 900 uL of the Tetrahymena Stock Culture to 4 separate wells.
5. Add 100 uL of the undiluted stock culture to the first well.
6. Add 100 uL of the 10^-1 culture to the ^-2 culture.
7. Add 1oo uL of the 10^-2 culture to the ^-3 culture.
8. Add 100 uL of the 10^-3 culture to the ^-4 culture.
9. Using a dissecting scope we observe each diluted plate.
10. Pick one of the dilutions to use for 3 trials.
11. Using the objective scope using the dilution picked, pick up 5 uL using a .5-20 micropippetor (making sure it has the right size tip).
12. Observe under microscope for changes in cell count.
13. Use the formula for calculating cell concentration to calculate cell concentration.
14. After this we went to the computer lab.
15. Within research group, outline the experiment (hypothesis, variables, type of microplastic using, procedure)

Observations:
In the undiluted culture the cell count was very high. After progressively diluting the concentration I chose the 1:100 dilution to take a cell count of. In the second culture there were around 30 cells which after dilution evolved to just 4. Due to time constraint we only did one trial. That was the cell count in 5 uL. The cell count per uL in the drop was 4/5. The cells/uL in the undiluted sample was 160 uL and the cells/mL in the undiluted sample was 1600.

This was the diluted 10^-2 I used.

These were my calculations:

Storage:

The solutions were left in the well plate and the concavity slide used was washed and set out to dry. All the tips of the micropipettors were disposed of.

Future Goals:

In future labs my micropippeting skills will be more accurate from the practice. In addition, my goals for future labs is to be able to better manage my time and be able to finish the lab using more proficient lab skills.

Objective of this lab:

There were multiple objectives for this lab including learning more about Tetrahymena and practicing our microscope skills. In addition to these two, another objective was that after this lab we should be able to correctly use a Micropipettor.

Purpose of this lab:

The purpose of this lab was to be able to correctly utilize a Micropipettor including, how to measure the amount of liquid and which is the proper micropipettor to use.

Procedure:

1. Go over the different sizes of micropippetes and how the measuring and pippetting works.
2. Practice pipetting using a plate and regular water.
3. Practice by pressing to first stop, put the tip (with cover on it) in water, release slowly, then go to first stop on plate. After this go to second plate to let air out. I tried this using a 1-20 micropipette and also a 20-200 one.
4. Using a dissecting microscope, and a measurement of 100 microliters, look at the ciliates in the plate using a black light.
5. Then using a micropippetor absorb a measurement of 5 microliters of the ciliates and place on a concavity plate (using a P-10 micropipettor).
6. Place concavity plate under compound microscope and observe at 4x and 10x.

Observations:

At the 4x magnification on the compound miroscope the ciliates were fast moving and had a transparent look to them. They were very small and it looked like there about 50 0f them in my range of view, which at 4x was the whole drop of ciliates. I did 3 trials using 4x and my results were as follows:

I also did three trials using the 10x magnification and my results were as follows:

To estimate the number of ciliates I counted how many there were in a quadrant and multiplied that by four.

Storage: The micropipettes are put onto a rack where they hang. The tips are disposed of and the concavity plate is washed and left out to dry.

Future goals: In the future in labs similar I can come up with a more effective way to estimate the ciliate count through the microscope. I can also apply my knowledge of miropipettes as we will be using them a lot in future labs.