September 30

Lab 6: Staining

Braden Hanson

September 29, 2016

Date Performed: September 27, 2016

Procedures:  

  1. Obtain a compound microscope.
  2. Using the proper techniques, properly clean both the objective and and ocular lenses.
  3. Obtain an empty slide, and prep clean it properly.
  4. Make a wet mount using the provided tetrahymena at the front of the lab.
  5. Obtain a cover slip.
  6. Before placing the cover slip over the sample on the slide, gently rub vaseline on the corners of the coverslip so that it doesn’t crush the tetrahymena.
  7. Place the coverslip on the slide.
  8. Take approximately 20μL of Methyl green-pyronin and put it on the edge of the cover slip so that it will diffuse across the wet sample.
  9. Place the slide on the stage, and observe the stained ciliates.

Observations: 

I was able to take some pictures through the ocular lens and this is what I got from staining the slide. This was one of the less darker regions of the slide. Perhaps, 20μL was too much stain.

img_3332

Was not able to count the population of tetrahymena in my experimental design trials. Will do on Friday during open lab.

Conclusions: 

Staining is a relatively easy process. It is a few extra steps to add to making a wet slide. However, it should be noted that when staining the slide, the amount of stain should be extremely low. The amount I used made half of my slide to dark to even look at. Once a proper amount of stain is diffusing throughout the slide, it will be helpful to observe the cell and different parts of the cell as well.

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September 29

Lab 6: The Compound Microscope (II)

Jess Hastings

Date of Experiment: 9/27/2016

Experiment Title: The Compound Microscope (II)

Rationale: The purpose of this lab was two learn how to distinguish between Primary Sources and all other online literature, learn about and practice staining with methyl green-pyronin, and collect data on the progress of your experiment.

Procedure: 

Staining:

  1. Obtain slide and clean it with alcohol
  2. Put 1 small drop of Tetrahymena on the slide
  3. Put Vaseline on all 4 corners of the cover slip so that the ciliates aren’t smashed when you put the cover slip on top
  4. Place cover slip on top of drop containing Tetrahymena
  5. Place 1 drop of methyl green-pyronin on the side of the cover slip
  6. Look at slide under compound microscope

Observations:

When the drop of stain was placed on the side of the cover slip, it then diffused through the drop of water and into the Tetrahymena cells. Once the stain had diffused, when the slide was looked at under the compound microscope. I could clearly see both of the nuclei in the Tetrahymena. The stain also helped to outline the shape of each organism.

Conclusion:

Staining is a helpful technique that could be used in the lab. Because the stain enters into the cell of the organism you can see structures inside the cell clearly.

 

Experiment:

Rationale: The objective in this lab was to count the populations in each well of Tetrahymena after one week of being exposed to either natural, artificial, or no light.

Procedure:

  1. Obtain a watch glass
  2. Obtain a p10 micropipette
  3. Obtain Tetrahymena media
  4. Obtain 24-well plate from designated area (cabinet, window, or counter)
  5. Create a 1:10 dilution in the watch glass using 1 uL of liquid from Tetrahymena well 1 and 9 uL of Tetrahymena Media
  6. Under dissecting microscope, count the number of moving Tetrahymena and record
  7. Repeat Steps 5 & 6 for Wells 2-4 (including control)

Observations/Results:

Note: During set up, my 24-well plate was bumped. This caused liquid to spill from the plate, but we think it was just condensation that had built up under the lid, because all 3 wells with Tetrahymena look consistent.

Observations: Many of the Tetrahymena in my dilution were not moving, even after shaking the watch glass. Also, there were visible moving slower than both of the samples that had been kept in artificial light and darkness.

Calculations: After counting how many moving Tetrahymena there were in my 1:10 dilution, that number was multiplied by 1200 uL to get the number of Tetrahymena in the well. To get the number of Tetrahymena per mL the total number of Tetrahymena in the well was divided by 1.2.

Results for 24-well plate in Natural Light:

table1-for-post

Conclusion:

There seems to be a difference between the activity on my plate verse the other two plates. This could mean that natural light does effect the life and population growth of Tetrahymena. This could explain why ciliates usually live in soil where it is dark.

 

My plate was stored by the window in Dr. Adair’s lab labeled with my name.

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September 29

Running the Experiment – Lab 5

Timothy Tateossian – September 28

– Lab complete September 27

 

In this lab, there were a couple of things that got stained, at least I stained some organisms and other organisms unfortunately stained my wells.

We started the lab making wet mounts of Tetrahymena and stained them with methyl green-pyronin. It stained their nuclei a nice dark blue. While the macro nuclei were relatively easy to spot, I cannot remember specifically seeing any micronuclei.

 

Experiment:

I left the last lab session with my wells filled up and ready to go. However, it seems that some mold got into my wells and found the environment rather suitable. They took over and nothing was left but somehow my control well survived.

Thus, in the last 15 minutes of lab time I made the valiant effort to completely reset up my wells. I quickly mixed in the NaCl solution I needed and threw my wells together. Here is the breakdown of what I did:

Control Well

One well

100µl Tetrahymena

900µl of Peptone culture

Experimental Wells

Five wells

100µl Tetrahymena

100µl of 0.01 M of NaCl Solution

800µl of Peptone culture

 

Until I realized, I have 1 minute to count these things!

What I did was take out 1.0µl from the Tetrahymena culture and put it on a small petri dish. Then I estimated how many were in the drop. With that, I used proportions to estimate how many organisms were in the wells.

15 Tetrahymena / 1 µl = X Tetrahymena / 100 µl

Of course, this means there are 1500 Tetrahymena in each well.

 

Next week, I plan on counting each well to see if the salt has affected the number of Tetrahymena in each well. Looking forward to it!

 

Category: General, Timothy Tatéossian's Notebook | LEAVE A COMMENT
September 29

Experiment Start-up – Lab 4

Timothy Tateossian – September 28

– Lab complete September 20

 

In this lab, I examined all my previous ciliate wells from earlier weeks. And then, I decided to kill them all. I dumped them all out, sterilized with bleach solution, and now I have a clean, ready to use cell culture plate for the actual experiment. I got rid of everything so let me make one final comment on what was alive:

 

Well A: Signs of life still there! Out of all these weeks!!

Well B:  1 ciliate was reported to be alive (strangely, last week there was nothing…?)

Well C: Dead

Well D: Dead

Well E: Dead

Well T: Dead – well was completely dry/no liquid

1st Dil: Prolific movement

2nd Dil: Dead

3rd Dil: Dead

 

Anyways, they are all dead now!

 

We also practiced on a compound microscope, so we learned on a prepared slide and afterwards made our own wet mount. I made my wet mount with a Euglena, and on the highest power (1000x) one can really see the chloroplasts and apparently there was a red eyespot (???). I am colorblind so I am taking other people’s word for it^^.

Experiment Design:

We have finalized the molarity, as discussed below:

Braden: 0.1 M

Kristin: 0.5 M

Timothy: 0.01 M

Yanni: 1.0 M

We created a normal serial dilution (Start with 200µl and take out 20µl for the next while adding 180µl to keep the amount of solvent equal) and found that the concentration per well is 1500 organisms per 100µl where 100µl equals 1 well.

We decided that there would be 3 trial runs each with a one-well control and a one-well variable. The variable well for me will be only different in that it will have 0.01 M NaCl in it. I will measure growth by the number of cells in the well after 1 week and 2 weeks.

Category: General, Timothy Tatéossian's Notebook | LEAVE A COMMENT
September 28

Lab 6: Begin Experiments and Practice with Staining

Taylor Guynup

September 27, 2016

Objective: The objective of this lab was to begin our experiments, collect data, and to practice staining organisms, which is a skill we will use when researching the soil ciliates.

Staining:

Staining is used to darken specimen to make viewing them under a microscope easier. In this lab, staining is used to clarify the nuclei of the ciliates. The steps of staining an organism is shown below:

  1. Clean and dry slides
  2. Place a drop of the specimen on the slide
  3. Line the coverslip with vaseline
  4. Place the coverslip on the slide (one edge first then the other to avoid bubbles)
  5. Place a drop of stain on the slide next to the edge of the coverslip, the stain will diffuse throughout the media

Here is a link to an article on a type of staining called gram staining. This staining technique is used to determine gram negative bacteria from gram positive bacteria.

Experiment:

During this lab we conducted our experiment. Our experiment’s purpose was to discover the effect of voltages on Tetrahymena’s behavior after different amounts of time. We used a 9 volt battery and copper wire to deliver the voltage to the organisms. Each of us exposed the Tetrahymena to the voltage for a different amount of time specifically 1.5, 3, and 5 minutes. We are still collecting data and comparing and sharing our data. One thing that happened in all three trials was when we placed the wire in the media, there was a reaction (pictured below). Our Tetrahymena are stored in the drawers in the back of the classroom.

Reaction when placed in media

Reaction when placed in media

Conclusion: 

Staining organisms will be useful in the future labs to be able to more clearly see the organism and the organelles within the organism. Our experiments are still being conducted and we are still collecting data.

Category: General, Taylor Guynup's Noteook | LEAVE A COMMENT
September 28

Lab 5: Compound Microscope Practice and Experimental Design

Taylor Guynup

September 20, 2016

Objective: The objective of this lab was to to learn the differences between compound and stereoscope microscopes, to learn how to use a compound microscope, to learn how to make a wet mount, and to further design our experiments.

Compound vs. Stereoscope:

Stereoscope microscopes:

  • Have a working distance
  • 3D
  • have a zoom magnification
  • are low magnification

Compound microscopes:

  • low/no working distance
  • 2D
  • no zoom magnification
  • are high magnification

How to use a compound microscope: 

  1. Clean microscope with cleaning tissue and alcohol

    Source: http://www.sciencedirect.com/science/article/pii/S1438463904700887

    Source: http://www.sciencedirect.com/science/article/pii/S1438463904700887

  2. always start on lowest power
  3. use course adjustment knob to focus (ONLY USE ON LOWEST POWER)
  4. adjust clarity using the fine adjustment knob
  5. switch to high powers and use the fine adjustment knob to clarify image

How to prepare a wet slide: 

  1. Clean and dry slides
  2. Place one small drop (if drop is too big then the cover slip will slide around) on the slide
  3. Optional: place vaseline along the edges of the cover slip to prevent the crushing of your species
  4. Place one side of the cover slip on the slide and slowly lower, do not place the coverslip directly on the slide as this will create bubbles
  5. When finished with slide, rinse slides and put them away according to directions

Experimental Design: 

We decided to change parts of our original experiment. We will now all be using the same voltage with different time exposures. We will be using 9 volt batteries. Each person will expose the Tetrehymena to the voltage for different amounts of time (1.5 min, 3 min, and 5 min). We will be observing the behavior of the Tetrahymena before, during, and after the exposure to the voltage. The Tetrehymena will be in a 6,000 microliter media and there will be 600 microliters of the specimen.

http://www.sciencedirect.com/science/article/pii/S1438463904700887

Above is the link to an article that has a similar experimental design to ours.

 

Conclusion:

Compound microscopes will be useful in observing individual organisms, however we will be using the stereoscopes in our experiment because we want to see the organisms interacting as a whole. Our Tetrahymena are stored in petridishes in the drawers in the back of the classroom. Next class we will begin our experiments.

 

 

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September 27

Biology Lab: Week 5 Lab Write- Up

Biology Lab: Week 5 Lab Write- Up

Name: Kayla Wilson

Date: Tuesday, September 20, 2016

Lab Title: Lab 5: Experimental Design Execution

Experimental Procedures:

Experiment:

  • Our experiment will test how ciliates (specifically, Tetrahymena) survive and reproduce at various pH levels.
  • Materials: 3, 24- well plates, 5 pipettes, Tetrahymena sample, test tubes, pure lemon juice, household bleach, pure water, pH testing strips, and a dissecting microscope.
  • Hypothesis: Our collective hypothesis is that the Tetrahymena ciliates will thrive most dominantly in a neutral environment. The more neutral (closest to 7) the pH, the more likely the Tetrahymena are to survive.
  • Dependent variable: the number of living Tetrahymena.
  • Independent variable: the various pH levels.
  • Controls: the basic solution used for contrasting purposes, the non-varying amount of Tetrahymena sample placed in each well, and the fact that we have multiple wells per pH level.

 

Actual Experiment:

  • Clean old ciliates out of 24- well plate using bleach and water. Dry well.
  • Start by diluting a Tetrahymena sample. Place 450 um of Tetrahymena media and 50 um of Tetrahymena in well number one. Next, place 450 um of Tetrahymena media and 50 um of the sample from well number one, in well number two. One dilution is enough to get the sample to contain a countable number of ciliates.
  • Each member of our group has a different sample of Tetrahymena within their 24- sample well.
    • Sample one: acidic. First, add 20 um of the diluted Tetrahymena sample to each of the nine wells. Then add 450 um of additional Tetrahymena media to each of these nine wells, for the purpose of not letting the Tetrahymena sample dry out. Then, add lemon juice (because it has a pH of exactly two) to all nine of the wells. However, the amount of lemon juice in each of the wells will vary. The chart below indicates how much lemon juice to add per well:
1- 1 drop 4- 2 drops 7- 3 drops
2- 1 drop 5- 2 drops 8- 3 drops
3- 1 drop 6- 2 drops 9- 3 drops
  • Sample two: neutral. First, add 50 um of the diluted Tetrahymena sample to three wells. Then add 450 um to each of these three wells, for the purpose of not letting the Tetrahymena sample dry out. Then, add three drops of D.I. water (because it has a pH of exactly seven) to all three of the wells.
1- 1 drop 2- 1 drop 3- 1 drop
  • Sample three: basic. First, add 50 um of the diluted Tetrahymena sample to nine wells. Then add 450 um to each of these nine wells, for the purpose of not letting the Tetrahymena sample dry out. Then, add pure bleach (because it has a pH of exactly ten) to all nine of the wells. However, the amount of bleach in each of the wells will vary. The chart below indicates how much bleach to add per well:
1- 1 drop 4- 2 drops 7- 3 drops
2- 1 drop 5- 2 drops 8- 3 drops
3- 1 drop 6- 2 drops 9- 3 drops

Interpretations and Conclusions:

  • After our Tetrahymena dilution, each well contained approximately 20 Tetrahymena per 10 um.

Sample Storage:

My sample of ciliates is stored in a drawer within the classroom. It is labeled “Kayla Wilson” on the side in black sharpie.

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September 27

Lab 5: The Compound Microscope and Experiment Set-up

Therese Riesberg

Date of Lab: 09 November 2016

Objectives: Learn how to use a compound microscope to study ciliates, set up experiment

In this lab, we learned how to read and report magnification, as well as how to clean the microscope lens with isoprophenol, how to prepare slides, how to prepare a wet prep, and how to use a counting chamber.

 

Procedure:

Wet Prep

  1. clean the slide with water and paper towel
  2. put one small drop of ciliate culture on the slide
  3. carefully put a cover slip over the top- just enough to cover the drop of cell culture (the cover slip should not slide around)
  4. observe the slide under a compound microscope

Dilution / counting tetrahymena

  1. obtain 2 mL per group of fresh tetrahymena in culture tubes

    Image result for wet prep

    Compound Microscope Imagery Source:https://www.superteachertools.net/jeopardyx/answerkey.php?game=1407596466

  2. obtain 30 mL per tube of tetrahymena media in large media tubes. Use serological pipette so as to not contaminate.
  3. serially dilute the tetrahymena
  4. count the number of tetrahymena using counting chambers

Using a Compound Microscope

  1. work with your partner to read the microscope manual
  2. clean the microscope lens
  3. practice focusing on prepared slides
  4. make a wet prep and observe it under the microscope

Lab Set-Up

Our group was unable to set up the lab during this time because we did not have the necessary culture media prepared, as our experiment requires specific media containing different kinds of water to test in which tetrahymena survive in the highest quantities. During the lab time, however, we did set up our culture medium, which need to be autoclaved before they will be able to be used for the tetrahymena. The recipe we used is as follows:

  • 0.5 g proteose peptose
  • 0.5 g tryptone
  • 0.02 g K2HPO4
  • 100 mL water***

***we prepared two different sets of media- one contained bottled water, and the other contained mineral water.

 

Our prepared media are being autoclaved between the lab and the upcoming lab class (09/27/2016) and will be stored where Dr. Adair so chooses to store it after the autoclave process.

 

Conclusion:
Though we were not able to set up our experiment during this class, we were able to set up the media in which we would be testing the survival of our tetrahymena. It was interesting to see how it was made rather than just using pre-prepared materials. It makes the process seem that much more reproducible. Also, it was interesting to see how magnified things are under the compound microscope. It is a much greater magnification than when using dissecting microscopes, so it was a good difference to note.

 

In addition, I found an article on PubMed about keeping Tetrahymena alive. It describes different kinds of medium that can be used, including some that use skim milk and some that contain beans! I found this to be very applicable to the experiments the class as a whole is conducting. The link to said article is as follows: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608402/

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September 27

Lab 5: The Compound Microscope (I)

Name: Hope Willenborg

Date Lab Performed: Tuesday, September 20, 2016

Title of Lab: The Compound Microscope (I)

Procedure:
The purpose of the lab was to learn how to use the compound microscope and to set up our experiment. The first thing we did was clean the microscopes with lens paper. Then, we practiced using the compound microscope with prepared slides. We also made our own wet mount slide. In order to do that, we cleaned a glass slide and placed a drop of the sample in the middle of the slide. Then, we placed a cover slip over the drop of the sample. Once we could examine the sample under the microscope, we disposed of the sample and cleaned the slide. To do this, we simply tossed the plastic slip in the biohazard trash and washed the glass slide with soapy water.
The next part of the lab was to set up the experiment and perform a serial dilution to understand the original number of tetrahymena in a sample. We put 180 microliters of tetrahymena media and 20 microliters of tetrahymena in a well plate. We put another 180 microliters of tetrahymena media in a well plate with 20 microliters of the diluted solution to count up the tetrahymena.

Results:
We counted 2 tetrahymena in the final diluted solution, so that means there were only 20 tetrahymena in our original solution.
Because the lab did not house the equipment necessary to accomplish our original laboratory experiment, we had to alter it. We are still testing for pH, but instead of paramecium, we are now using tetrahymena. In order to do this, we will each set up a well plate with 15 wells filled with diluted tetrahymena. Then, we will add one drop of lemon juice to 5 of each of our wells, one drop of diluted bleach to 5 of each of our wells, and we will leave the remaining 5 wells as they are. We will then test the pH of the substances and count up the original amount of tetrahymena alive. We will then wait a week to continue to count up the number of alive tetrahymena. My hypothesis stays the same in the sense that when the pH changes outside the normal range of tetrahymena, the rate of survival will decrease.

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September 27

Lab #5: Compound Microscopes, and Experimental Lab

Ricardo Tellez

9/20/2016

Lab #5: Compound Microscopes, and Experimental Lab

Introduction:

This lab consisted on preparing the students on how to properly use a compound microscope, and the further development of our experimental Lab. Unfortunately my group wasn’t able to begin our experimental lab, so we came during open lab hours to initiate procedures with our experiment.

Compound Microscope

Procedure:

  1. Retrieve the designated compound microscope from the cabinet and place on the lab table.
  2. Carefully, using lens cleaning tissues clean the lenses of the microscope.
  3. Place a slide onto the slide placer of the microscope.
  4. Be able to adjust and zoom in to see specific parts of the slide using the coarse adjuster and fine adjuster.(DO NOT USE THE COARSE ADJUSTER ON ANY BUT THE LOWEST POWER)
  5. Use the different objective lenses to get more/less precise views of the slide.
  6. On a new slide place a drop of the solution  on the center.
  7. Cover the solution with cover slip.
  8. Observe through the microscope.
  9. Once finished, dispose of the wet cover by throwing it away in the bio hazard bin.
  10. Clean the wet slide using bleach and water, then store it.
  11. Carefully store the microscope in the cabinet.

Conclusion:

I was able to complete all the tasks successfully and now I know how to use a compound microscope, and I’m confident that I can do further experiment using this device.

Experimental Lab

Procedure:

  1. Dilution:
    • We diluted the population of Tetrahymena in order to have 300 specimens in one well. We will use this amount of Tetrahymena in each well in order to have many trials and to make sure our experiment is precise.
  2. Place the diluted Tetrahymena into the desired well.
    • We used only 12 wells (4 columns x 3 rows) to make our experiment.
  3. Seal the well plate with the modificated cap.
    • The cap is modificated because it is made so that each well will contain a light bulb.
  4. Turn the light bulbs on
    • We are testing 3 colors: red(A), green(C), and white(D).
    • The fourth color being tested is no light (B).
  5. Set the lightbulbs amps to 70.3 amps each, except for the Tetrahymena that will experience no light (B).

Conclusion

We have our lab set up and we will begin recording our observations when we go back to class on the 27th of September, 2016.

Category: Ricardo Tellez's Notebook | LEAVE A COMMENT