January 26

Lab Week 3 Post – Ludox Centrifugation and Ciliate Extraction (01-25-18)

Task, Rationale, and Purpose: The task of this week’s lab was to begin the protocol designed from last week. The purpose or rationale is to begin the learning process of DNA extraction and identification through this lab. In order for future collaborations and research, the students need to understand how the process of polymerase chain reaction (PCR), centrifugation, and sequence comparison occurs. Additionally, the lecture portion of the lab is designed to review the indicators of soil health and how ciliates are vital components for determining it.

Procedure:

  1. Acquire a p1000 tip and cut a few centimeters off the end.
  2. Using the p1000 with the modified tip, pipette 2 ml of the soil sample into the 8 ml of Ludox HS 40.
  3. Apply 2 ml of distilled water very slowly to create a film layer of water.
  4. Record the weight in grams.
  5. Using the swinging bucket rotor, centrifuge the sample for 15 minutes at 4300 x g making sure that the weight is evenly distributed on both sides of the bucket.
  6. Using a dropper, remove the second layer, cell layer, from the tube (layer under the distilled water column) and place in a microfuge tube
  7. Transfer 7 microliters of the cell layer fluid onto a concavity slide and observe the sample under 100x magnification using a compound microscope.
  8. Record your observations.
  9. Centrifuge the cell layer fluid further to isolate the ciliates from the prokaryotes.
  10. Remove the remaining fluid in the microfuge to isolate the pellet created from the centrifuge.
  11. Freeze the ciliates.

Data/Results:

Weight: 21.3 grams

 

Storage:

The microfuge was stored in the freezer.

Conclusion:

The lab was successful and the procedure was completed with plenty of time to spare. However, the process of observing the samples under the compound microscope was lengthy. The group was unsure if any organisms were discovered, but the lab tour and the process of observations was exciting and very informative. For future experiments, the protocol for PCR should be conducted with metabarcoding following it. Additionally, this lab could be repeated in order to test the authenticity of our results.

Category: Angelo Wong, BIO 1105 31 | LEAVE A COMMENT
January 26

Lab 3: Ludox Gradient Centrifugation

1-25-2018

Purpose: To test the ludox centrifugation protocol developed for isolating ciliates. To isolate ciliates from a pre-gathered soil sample and observe them using microscopy.

Procedure:

  1. Add 8 mL ludox HS 40 to a 15 mL conical tube
  2. Quickly inject 2 mL of liquid from soil sample into ludox tube with p1000
  3. Add 2 mL of water to the top of the ludox mixture
  4. Centrifuge in a swinging bucket rotor for 15 minutes at 4300 g
  5. Carefully remove the tube and use a pipette to remove the cell layer from the tube
  6. Transfer the organic matter to a set of clean microfuge tubes
  7. Transfer 3 10 uL drops of the organic matter to a concavity slide and count the cells using a compound microscope under 100x magnification
  8. Centrifuge the organic matter once again
  9. Remove any remaining water from the tube while preserving the pellet using micropipette
  10. Freeze and store pellet

Results:

The ludox centrifugation protocol developed worked very well for separating soil ciliates and organic matter from their surroundings. The density gradient created by the centrifugation technique made it very easy to extract the organic matter. Our group found one live ciliate in a drop of organic matter we observed under the microscope. Its morphology was similar to that of tetrahymena. As such, we speculate there may be more ciliates within our sample to be observed later. To conclude, our hypothesis surrounding ludox centrifugation as it pertains to cell isolation was correct.

Next Steps:

As the ludox centrifugation was successful and ciliates were located, our next step is to observe and isolate more ciliates in our sample. As such, we will rehydrate and thaw the pellet of organic matter we stored and attempt to isolate ciliates within it. After, we will begin with the application of our developed meta-barcoding procedure. So far, everything has been going according to our calculations. Denaturation will be the first step of PCR meta-barcoding we start on next time.

 

Category: Austin Medley | LEAVE A COMMENT
January 26

Lab 3: Ludox Centrifugation

1/24/2018

Purpose: The purpose of today`s lab was to test the protocol introduced to us by Dr. Adair as we prepared a sample of ciliates for metabarcoding. We learned how to isolate ciliates with Ludox by integrating density and centrifugation. Today’s lab was important in that it’s going to serve as a building block for the rest of the semester as we design the best protocol for our experiment.

 

Procedures:

  1. Place 8 ml of Ludox HS 40 into a 15 mL conical test tube. (You may cut the tip of the micropette to avoid getting grass in your sample).
  2. Place micropipette 2 mL deep into the ludox solution and inject the soil sample.
  3. Place 2 mL of the artificially dyed red distilled water to the top of the mixture.
  4. With the group at your table, weigh each test tube to make sure that they weigh the same.
  5. Centrifuge the mixture in a swinging bucket rotor for 15 minutes. This shouold be at 4300 x g.
  6. Use a micropipette to remove the cell layer from the Ludox solution, this should be directly under the distilled water.
  7. Place the cell layer or band into smaller centrifuge tubes in equal mass/volume.
  8. Place 3 1 uL drops of the cell layer on a concavity slide and place it under a microscope at x100 magnification.
  9. Count the ciliates in the drop to get an estimate of the number of ciliates in your concentration.
  10. Label all the small tubes (LAK 21, star on lid), spin the tube in the centrifuge at 12000 x g for 1 minute.
  11. Remove the excess Ludox from the test tube by micro-pipetting the excess solution away. Dispose of waste properly.

Results: After the centrifugation in the spinning buckets, my partners and I noticed a lot of grass in our sample. To get the grass out we pipetted it out and disposed of it. We didn’t have much luck under the microscope as we only seen a few ciliates. The ciliates we did see were dead due to the high pH of the Ludox. Unfortunately, we didn’t have time to stain our drops with iodine to see them better. Because everyone is working with the same sample and other groups were able to see ciliates better than our group, we aren’t losing hope because we know that the ciliates are there.

The picture above shows our conical test tube after we completed step 3 from our procedure.

The picture above shows our conical test tube after we completed step 5 from our procedure. In the picture, you can see that the spinning buckets centrifugation separated our solutions into multiple phases using density.

Conclusion: Todays lab was pretty fun. Seeing all the advanced equipment in the lab was awesome and really sparked an interest for research for me. Overall, I think we can say that this protocol for isolating the ciliates from our soil sample was a success and will be able to be used in our official experiment. Today’s lab was important in that it’s going to serve as a building block for the rest of the semester as we expand on the protocol  for our experiment. I can’t wait for next lab!

Category: Angelica Torres | LEAVE A COMMENT
January 26

January 25, 2018 – Lab 3: Ciliate Extraction Procedure

Purpose:

The purpose of today’s lab is to practice the cell extraction procedure to see if it would actually work in our metabarcoding experiment.

Procedure:

  1. Add 2 mL of dirt sample to 8 mL of Ludox solution in flacon tube
  2. Add 2 mL of red-dyed water to the top of the Ludox solution in the same falcon tube
  3. Make sure weight is the same as the group across from you so the solution will go through the centrifuge properly
  4. Use a swinging bucket rotor centrifuge for 15 minutes at 4300 x g
  5. Remove from centrifuge and carefully extract cell layer and put in microtube
  6. Take a small sample to observe under a microscope for ciliates and try and estimate cells/mL
  7. Put remaining samples in a microfuge to isolate DNA pellet
  8. Remove from microfuge after it finishes and extract the water
  9. Freeze the remaining pellet

Observations:

Conclusion:

In conclusion, the procedure worked! We successfully isolated ciliates using the Ludox solution. We extracted the cell layer as best we could, and we have DNA being preserved in the freezer. Our next stop is using the polymerase chain reaction.

Category: Steven Sanders | LEAVE A COMMENT
January 26

Lab 3: Ludox Centrifugation 01/25/18

Purpose:

The purpose of this lab was to isolate ciliates as well as learn some more basic laboratory techniques. We were given a Bermuda grass sample and had to extract the ciliates. After we had extracted the ciliates we observed them under a microscope. This method, Ludox Centrifugation, would work very well in the process of extracting ciliates DNA. This lab really helped me understand the process of metabarcoding and how many intricate details are involved.

Procedure:

  • Add 8mL of Ludox HS 40 to a 15mL conical tube.
  • Quickly add 2mL of liquid from the soil samples in the Ludox using a p1000. It is very helpful to cut the tip off to prevent dirt blockage.
  • Carefully add 2mL of distilled water to the top of the Ludox mixture. Be careful this liquid does not mix with the sample of Ludox.
  • Centrifuge in a swinging bucket rotor for 15 minutes at 4300 x g. It is crucial to have the same weight as our other group to avoid misbalancing in the centrifuge.
  • Carefully remove the tube and use a pipette to remove the cell layer. It should be visible under the distilled water layer.
  • Transfer the cell later to a clean microfuge tube. Be sure to only put 1mL of sample into each microfuge tube.
  • Transfer 3-10uL drops to a concavity slide and count the cells using 100x magnification. Record any observations in the lab notebook.

Part 2:

  • After viewing and making observations, label the microfuge tubes.
  • Spin the cells in the microfure at 12000xg for 1 minute.
  • Remove the supernatant using a p1000 without taking out the bottom pellet, the ciliates.
  • Store the pellet in the freezer.

Results:

We obtained 6 microfuges worth of sample which was a large number. However, I was unable to find any in my sample. This was largely due to the fact my microscope was not working properly. My partner, Preston, was able to find several ciliates in his sample. We had about 6mL of sample which was much larger than most groups. We are unaware of anything we might have done to cause this. There was quite a bit of debris in our sample but after using the microfuge, all the debris scattered and we were left with a pure pellet at the bottom. A possible source of error could have been the fact we maybe extracted too much debris which made our sample harder to purify. We completed the cell isolation and now the next step is the DNA extraction then PCR/ Gel Electrophoresis and so on. This is a picture from out sample after the centrifugation but before we put the ciliates in the microfuge tubes. As you can see we have the distilled water on top, ciliates/debris in the middle, and the ludox on the bottom. It is all based on density.

Conclusion:

Over the course of this experiment our group was able to learn some basic laboratory skills. We were also able to learn more about metabarcoding and how to isolate the ciliates in a sample. These skills will be very beneficial to me in the future and I am glad I am learning them now. Currently, our pellets are being stored in a freezer so they will be able to be used next week.

Category: Ian Johnson | LEAVE A COMMENT
January 26

Cell Isolation 1/25/18

<Purpose>

Understand the process of cell isolatation and conduct an experiment using the Ludox isopycnic centrifugation.

Overall. we are performing the process of cell isolation -> DNA extractions -> PCR/Gel Electrophoresis Validation -> Metabarcoding -> Analysis of Sequencing data.

Today, the main purpose was to isolate the cells.

<Procedure>

  1. Add 8mL of Ludox HS 40 to a 15 mL conical tube
  2. cut the tip of the micropipette
  3. Quickly add 2mL of liquid from the soil samples in the jars into the Ludox using p1000 (use p1000 then add two times/ avoid grass and sticks)
  4. Carefully add 2mL of colored distilled water to the top of the Ludox mixture
  5. Weigh the tube
  6. Compare the weight with another group
  7. The group with less weight should add appropriate amount of water to make the weight equal.
  8. Centrifuge in a swinging bucket rotor for 15 mins at 4300 x g
  9. Carefully remove the tube and use a pipette to remove 3-5ml layer of cells and place in two different microcentrifuge tube
  10. Using a micropipette, place 5 ul of the liquid on a concavity slide and observe the cells
  11. Record what you observe
  12. Centrifuge the two tubes and observe the pellets
  13. Using a pipette remove the liquid above the pellets

<Result>

The Ludox mixture after centrifuging showed layer of different segments. It displayed sediments at the bottom, ludox and water columns and distilled water column. Between the ludox and water column there were faint particles floating around. This was extracted and observed. When looked into the microscope, there were some dust particles and round ones that suggested to be a cell. However, it was unable to clearly see what the cells were and even if they were cells. An attempt to take a photo was made, but the photo did not display clearly what was observed.

<Conclusion>

The experiment itself was very interesting. There was an opportunity to observe a different laboratory containing the swinging bucket rotor, which further brought interest in the lab. Although not clearly observed, the result suggested there maybe cells present in the sample collected. The next step to this process is DNA extraction and PCR. PCR would be used to amplify the DNA and hopefully result enough to go through the process of metabarcoding in the end.

<Final Sample>

The final tube was labeled JJN 22 and was submitted to the LA for storage. Ludox would be sent to a different lab to be process and discarded appropriately.

Category: Jae Lee | LEAVE A COMMENT
January 26

Lab 3 Extraction Protocols

Lab 3 Ciliate Isolation and DNA Extraction Protocols 1/26/2018

Purpose

The purpose of  lab was to start the process of DNA extraction by using Ludox to separated cells out from the Bermuda grass sample.

Procedure

  1. Inject 2 mL of the dirt sample into 8 mL of the Ludox solution.
  2. Layer 2 mL of the dyed water on top of the Lodux dirt mixture.
  3. Weigh your sample and make sure it’s within a tenth of a gram of the other sample. (our’s was not, so we added 300 microliters of water)
  4. Centrifuge the sample for 15 minutes at x4300g
  5. After removing the tube from the centrifuge, pipette out the layer of separated cells and place in a new 2 mL tube. ( We were able to pipette out about 1 mL)
  6. Add drops of the separated cells onto a concavity slide to observe. (We located a few cells within our sample)
  7. Place the tube of cells into the microfuge and spin to pellet the cells
  8. Prepare the cells for the freezer by removing the supernatant.
  9. Store the cells in the freezer.

Conclusion

Separating the cells with the Ludox worked incredibly well. It was fascinating to see the change from the dirt, to the separated cell layer, to the pellets. Before this lab, I was confused about how we would even start trying to extract DNA, so this lab was useful because it demonstrated those few beginning steps. It was advantageous to see how a experiment like this is built through trial and error. I think this will be a lot easier to mimic now that we know what it looks like.

Category: Lindsay Haag | LEAVE A COMMENT
January 26

Lab #3-Ludox Gradient Density Centrifugation (1/25/18)

Purpose:

The purpose of this experiment was to use centrifugation in order to try to isolate ciliates from the soil sample.

 

Procedure:

  1. Use a p1000 micropipette to pipette 2mL of the designated soil sample to a conical tube. (Note: the conical tube already contains 8mL of Ludox in it.)
  2. Pipette 2mL of dyed water into the conical tube.
  3. Weigh your group’s conical tube and your partner group’s conical tube. One group may need to add more water to their tube in order to make the two masses equivalent.
  4. Place the conical tubes in a centrifuge for 15 minutes at x4300 g.
  5. Pipette the soil/organism layer into the microcentrifuge tubes. (Note: ours filled up 4 microcentrifuge tubes.)
  6. Extract a 10uL sample from each microcentrifuge tube, place it on a concavity slide, examine it under a compound microscope, and try to identify/count ciliates if there are any.
  7. Centrifuge the microcentrifuge tubes for 10 minutes.
  8. Remove the the liquid from the tube and store the pellet in the freezer.

 

Results:

The mass of the conical tube and Ludox/water/soil sample was 21.4g. After centrifuging the tube for 15 minutes, it was evident that our particular sample had a large layer of organisms. There were not any ciliates identified, although it is possible that taking a second examination could yield better results. Ankan noted that there was an amoeba in one of the tubes, but that is the extent of identification we reached.

Here is an image of the microcentrifuge tube. The liquid was in the process of being extracted so that the pellet could be isolated and placed in the freezer.

 

Conclusion:

This lab was very informative, fun, and straightforward. I thought it was cool to see the microbiology lab and all of the different technology that they use. It was also interesting learning about the Ludox centrifugation technique in the last lab and then getting to actually apply it in this lab.

Category: Lexi Caudill | LEAVE A COMMENT
January 26

Lab 3: Ludox Gradient Centrifuge Protocol 1/25/2018

Purpose:

The purpose of lab eight was to complete the Ludox gradient centrifugation protocol that we were introduced to by Dr. Adair in the previous lab. She introduced to us all the steps, however this week we actually carried out the steps. This lab allowed us to see sides of the BSB that we had not previously seen before. After completely the protocol, we began to set up the experiment for next week. We prepared the cells for DNA extraction with the use of a microfuge at a force of 12000 x gravity. After it was completed, it left behind a pellet that will be examined next week.

Procedure

  • Add 8mL of Ludox HS 40 to a 15mL conical tube
  • Take 2mL of liquid from the soil sample provided and quickly inject it into the Luxox about 3 centimeters below the surface.
  • Add 2mL of distilled water with red food coloring to the top of the Ludox making sure not to mix the water and Ludox
  • Weigh your conical tube and make sure the masses are identical to the one it will be paired with in the group next to you
  • Label the conical tube
  • Locate a swinging bucket rotor and centrifuge the conical tube for about 15 minutes at a force of 4300 x gravity
  • Remove the tube and use a pipette to remove the visible cell layer
  • Place the removed cell layer solution into a clean microfuge tube
  • Remove 3 drops of around 3-10microliters and place them on a concavity slide
  • View each drop under the microscope at a magnification of 100x Record observations/count any ciliates
  • Label centrifuge tubes
  • Spin the cells in the microfuge at a force of 12,000 x gravity for one minute
  • Remove the liquid from the tube carefully  make sure to leave the pellet at the bottom
  • Place centrifuge tubes on the rack and put them away
  • Clean up your group’s work station

Data and Observations Data and Observations

Our group found 3 ciliates and 1 cyst throughout all 9 drops  The ciliates did not move while under observation due to alkaline Ludox solution  The Ludox from the drops had cracks in them as they dried up

Conclusion

After completing the Ludox gradient centrifugation protocol, the group was able to locate three ciliates and one cysts. It was extremely difficult to determine what was a ciliate and what was not a ciliate, because the ciliates were not moving to begin with. The process was not that time consuming, but it was also just one of the first steps in order to obtain the cell pellet for future study for DNA extraction. In the future we will be examine the cell bellet we obtained at the end of this lab from the microcentrigue.

Category: Austin Scott, Uncategorized | LEAVE A COMMENT
January 26

Lab 3: Ciliate Isolation and DNA Extraction Protocols 01/25/2018

Introduction

After our presentations last week, we were able to finalize some of the methods we wanted to use. These included the use of eDNA, 18S primers, cell separation using the Ludox, and DNA amplification using DNA barcoding versus metabarcoding. In order to confirm the validity of these protocols we will be exploring them in lab. Our work flow for the overall experiment is as follows: ciliate isolation -> DNA extraction -> PCR/Gel electrophories -> metabarcoding -> analysis.

Purpose

The purpose of today’s lab was to test the Ludox protocol, explore the lab and determine our workflow for the next few weeks. After the power point of with an overview of what we will be doing, we began to dive in.

Data/Observations

Part 1

  1. Add 8ml of Ludox HS 40 into a 15 ml conical tube
  2. Quickly inject 2 ml of liquid from the soil samples in the jars in to the Ludox
    1. Make sure to inject below the Ludox, not on top
  3. Carefully add 2 ml of the colored water to the top of the Ludox solution
  4. Centrifuge in the swinging bucket for 15 mins at 4300 x g
    1. Make sure you and the other group at your table have the same mass (21.3g)
  5. Remove the tube and carefully remove the cell layer – under the water layer
  6. Transfer the cell layer into a microfuge tube
  7. Transfer 3-10 microliters onto a concavity slide and observe for ciliates

Part 2

  1. Label the microfuge tubes
  2. Spin cells in microfuge at 12,000 x g for 1 minute
  3. Remove the excess water with a pipette
    1. Leaving the pellet of cells in the tube
  4. Store pellet in the freezer

It was difficult finding cells at first but, after multiple drops we found a few.

Conclusion

At the end of today’s lab we were, for the most part, were successful in extracting the cell layer and observing them under the microscope. The Ludox and density separation was successful in dividing based on density. Seeing the lab was really cool and informative.

Category: Sydney Gauss, Uncategorized | LEAVE A COMMENT