Title: Ludox Protocol Revised 2/8

Materials:

• soil
• Ludox
• Glutaraldehyde
• serological pipette
• centrifuge

Procedure:

• (pre lab: we practiced using a micropipette and a serological pipette)
• Collect 5 grams of soil and add 10 ml of water. Mix for 5-10 minutes.
• Allow the vial to rest for 1-2 minutes.
• Transfer 3.68 ml of soil water into a glass tube.
• Add 368 µl of glutaraldehyde (this should yield a 2% solution).
• vortex the tube for 1 minute.
• Inject 4 ml of the sample into a Ludox tube. (place p1000 at the 2 ml line)
• Add the colored layer of water to the top of the Ludox layer
• Place in a centrifuge at 4300xg for 15 minutes).
• Extract the layer of cells from the Ludox tube.
• Place the pellet of cells in a small, clean tube.

Mistakes:

• Not adding the water carefully enough to the top layer
• accidentally mixing the pellet layer with the water layer and Ludox layer

Results:

We were not able to finish the procedure so there are no results to report.

Data:

Mass of the Ludox test tube= 40.8 grams

Conclusion:

The next step would be to take out a sample from our tube and count the number of cells on a slide. Then we can begin the process for analyzing DNA. The method we used seemed to really help produce a clear and distinct layer of cells to analyze. I was not expecting to find such a pronounced layer. I also surprised to discover how many cells we had in our sample. We filled a total of 4 tubes with the pellet. The samples are labeled CPLMKC and they are on a rack.

2/1/2018, Ludox gradient centrifugation protocol

Purpose: The purpose of this lab was to revise our Ludox protocol to make it more efficient to yield better results. We will be using the Ludox method to extract cells for DNA analysis.

Materials:

• Ludox
• Gluteraldehyde
• Grassland soil
• centrifuge

Procedure:

• Place 10 grams of soil and 20 grams of water in a baby food jar.
• Mix for 5 minutes and let settle for 1 minute.
• Add 900 µl (3 times) of soil juice into the Ludox at the 5 mL mark.
• Add 2 mL of food colored water to the top layer of the mixture.
• Weigh the tube on a scale (make sure that the weight matches the other group in your table!)
• Centrifuge on 4300 g for 15 minutes.
• Place in the refrigerator.
• Draw 2 mL of the organic matter from the tube (pull from the top).
• Take 5, 2 µl drops of the mixture in the tube and count the cells.
• Calculate the efficiency.
• Pellet out the cells in the microfuge tube by spinning at 1200 g for 1 minute.

Data: the weight of the Ludox tube was 23.1 grams

Results:

We were not able to complete the process so there are no results to analyze yet. Sources of error could come from not ensuring that the Ludox tubes at each table were the same weight. My groups Ludox tube did not match the weight of the other groups Ludox tube, so we had to add more water. This means that we added more water than the protocol initially called for.

Conclusion:

Since we have not finished the procedure, there is nothing to conclude yet. The next step would be to place our Ludox tubes in the centrifuge for 15 minutes, and then we will extract the cells for counting. We have also decided on a method to establish our control. We will add extra Tetrahymena to the same grassland soil sample we are using and repeat the Ludox protocol on this control group.

Where is it stored:

Our tube is labelled “CKL” on the side and it has our weight (23.1 g) listed on the cap. It is stored on a tube holder in the lab.

Ludox Centrifugation, 1/25/2018

Purpose: Can we get cells out of the soil and if so, can this procedure apply to our protocol?

Materials: Ludox (wear gloves!), high power centrifuge, lower power centrifuge

Procedure:

1.Add 8ml Ludox HS 40 to a 15 ml conical tube.

2. Add 2 ml of liquid from the soil samples in the jars into the Ludox using a p1000.

3. Add 2 ml of distilled water (with red food coloring) to the top of the Ludox mixture.

4. Weigh test tubes (both at the lab table).

5.Centrifuge in a swinging bucket rotor for 15 min at 4300 x g.

6. Use a pipette to remove 3-5 ml layer of cells.

7. Place three drops of cells onto a concavity slide and observe under the microscope.

8.Transfer cells to a clean 15 ml comical tube.

9.Dilute with buffer to 10 ml and spin at 4300 x g for 10 minutes to wash and pellet the cells.

10.Remove the supernatant and store the pellet in the freezer.

Mistakes: Not adding the water carefully to the Ludox and accidentally mixing the water into the Ludox.

Results:

Our sample contained a couple of cells. I personally was not able to find a ciliate, but Camille and Katy each found one small ciliate (using a high magnification).This proved that our method for extracting cells was successful. Future steps would be to use this protocol when attempting to extract cells for our major experiment with classifying ciliates based on DNA sequencing. An unexpected finding would be the ciliate Dr. Adair showed us, which had formed a protective layer to remain dormant during the cool weeks. A source of error would be not being able to solely extract the cells from the test tube with the Ludox in it. There was no way to ensure that only cells were taken up by the micropipette. This may explain why some samples taken with the micropipette had ciliates, but others did not.

Storage:

Our sample is labeled CKL and has a smiley face drawn on the cap.

Date/title:

1/11/2018, Soil diversity

Purpose: The purpose of this experiment was to observe the ciliate diversity in soil. We discussed the importance of soil diversity and the role soil organisms play in their environment. Soil diversity is important because it preserves nutrients, which is beneficial for agriculture.

Procedure:

1. Take 10 μ of the liquid from the Burmuda grass using a micropipetter.
2. Place the liquid on a concave slide.
3. Place the slide under the microscope and search for various organisms.
4.  Record your findings in a notebook.

Data:

I found one large ciliate. It moved sporadically and was in the shape of a peanut. I found about 20 tiny, round ciliates that moved very quickly.

Conclusion:

There was plenty of biodiversity in the sample, as expected. We already knew that their was a large population of ciliates, nematodes, and other organisms in the Burmuda Grass sample, but this experiment proved that this statement is still correct. Ciliates of various shapes and sizes were discovered. I was able to find other organisms, such as amoebas in the sample I took as well. As a whole, diversity enables various groups of organisms to thrive in an environment. A potential source of error could have been not measuring the proper amount of liquid taken from the Burmuda Grass sample.

Storage:

No sample was kept.

1/18 research article presentations

Purpose:  To explore the primary literature concerning challenges in metabarcoding soil ciliates. The purpose was also to start brainstorming protocol ideas.

Procedure (ideas for protocol):

1. collect a soil sample (NOT from a flower bed/an area with a lot of artificial fertilizer)
2. Dry out the soil.
3. Rehydrate soil via a flooded plate.
4. Isolate the ciliates in your sample (using a micropipette).
5. Place the isolated ciliates in the isopycnic centriguation to separate the ciliates.
6. Select a primer (rDNA?)
7. Sequence the DNA using a kit (such as NextGen Sequencing)
8. Amplify segments of DNA (via Amplicon)

Results (protocol):

1. Compare results with other information in the data base and attempt to classify your ciliates!

Group presentations:

Group 1- The authors looked at 5 estuaries in Australia. They focused on sequencing the DNA of the protists in each estuary. An application we can use for our experiment would be the pyrosequencing because it utilizes a lot of data at one time. The Molecular Operational Taxonomic Unit could be used to cluster DNA into groups. Some challenges these authors ran into, would be the limitation of DNA based monitoring and nonproportional data.

Group 2- We can use eDNA to eliminate potentially inactive or irrelevant DNA found. Amplicon is a way for us to see similarities in the chunks of the genome in the data base.

Group 3: The protists in various places throughout Europe were sequenced in order to get a picture of the biodiversity. The challenge with this experiment was that the authors were not able to make a standard sample. Each sample was too different from the next one.

Group 4: A gene named Cox 1 was specifically singled out to depict genetic diversity between closely related protists. When a population was separated via the founder effect, the authors noticed a large variation in the Cox 1 gene. This is because the new population had its own separate gene pool. The DNA was extracted using a master purification kit which we could potentially use for our experiment.

Group 5: Several organisms (protists and ciliates) were used in this experiment. The goal was to see if the  D1-D2 region was an ideal threshold for ciliates in their identification. An issue with this, is there may not be enough genetic material to work with. This article highlighted application of primers that we could use for our protocol.

Group 6: The authors of this article wanted to examine the effects of land use intensity on protists. I learned that both eDNA and rRNA are good ways to avoid obtaining unwanted inactive DNA. We can use the NextGen sequencing for our experiment as well. T-RFLP could be used after NextGen, to distinguish unique characteristics of ciliates.

Group 7: HTS (High-Throughput Sequencing) was used to sequence DNA. Dr.Adair informed us that this will not be applicable to our class because of the complexity of HTS. The issue with HTS was finding protists that matched one another.

Group 8: UniEuk is a metabarcoding system that is used to identify ciliates. The goal is to create a uniform taxonomy. Dr. Adair suggested that we start with B4 like the authors of this article.

Conclusions:

Protocols can be developed properly now that we have an idea of what to look for. We can learn from the challenges that the authors of the articles faced to make our protocol more successful. The class has developed a frame for what the protocol will look like and we have also familiarized ourselves with the terms for the equipment we will need.

There was no sample stored.