Date: April 27, 2017

Objective: 

• Further our understanding of the how to classify Ciliates by using DNA Extraction

Procedure: Metadata Collection

1. Collected soil sample from a muddy patch in front of my dorm
2. Record information about soil on Metadata sheet. Information includes: color, date, time of day, location, last rainfall, etc.
3. Take the weight of a weigh boat without your soil in it, then 5g of your soil into the weigh boat and weigh them. Record this information
   • Weigh soil boat system again after 1 week to find percent of water composition
4. Place 5mL of your soil into a falcon tube and then place 5mL of water into the same tube
   • After 1 week take the soil composition of your falcon tube
   • Use the water in your tube to find the pH
5. Take some of your soil and it into a non-flooded dish. Place enough water to where there is a little bit of runoff but do not flood the dish
   1. If you find any ciliates culture them in Ceraphyll

DNA Extraction

1. Transfer 400 uL of culture ciliates into a centrifuge tube
2. Centrifuge tube for 5 minutes at 6000g
3. Add 200 uL of Chelex into the tube and vortex for a minute
4. Incubate at 56 degrees Celsius for 8 minutes
5. Place tube in a 100 degree Celsius water bath for 8 minutes
6. Centrifuge at 16000g for 3 minutes
7. Place the supernatant into another centrifuge tube and label
8. Determine the purity of the sample using a nano drop spectrometer

Gel Electrophoresis 

1. Add100 ml of 1XTAE and 1.5 g of argose to a 250 mL flask and microwave.
2. Assemble tray by placing the comb into the tray with the two bumpers on each side.
3. Fill the tray with gel about halfway up the combs.
4. Thermocycling procedure:
   • Denaturation at 95 degrees C for 30 seconds
   • 35 cycles:
     1. Denaturation at 95 degrees C for 30 seconds
     2. Annealing at 56 degrees for 20 seconds
     3. Elongation at 72 degrees C for 2:30 seconds
   • Extension at 72 degrees C for 5 minutes
5. Add 5 ul of the ladder to the product using a micropipet in the gel tray.
6. Place the lid on the tray after loading the DNA samples into their respected loading zones, and turn it on at 100 volts for 30 minutes
7. Place the tray under UV light in order to see results.

Picture of Gel Electrophoresis: Due to a complication in the Gel Electrophoresis process our DNA was largely corrupted and any data that could’ve been collected from the process was ruled inconclusive 

Ideas For Future Experiments:

• Try culturing ciliates in something other than ceraphyll
• Instead of waiting a week to check on our cultured ciliates, we can check on them after a day or two.
• Have backup DNA just in case something happens

Date: February 23, 2017

Goals:

• Analyze our groups collective data
• Present our data in graph form in class

Procedure: 

1. Present our data for the class, explaining how we analyzed our data and why we chose which graph method we used for presenting our data.
2. Open excel and create data points so that we could use to learn how to effectively find things such as: standard deviation, mean, median, mode, how to create a histogram, and how to perform an F-test and a T-test.

Observations: 

• Answering questions on the setup of our graph helped to clarify any questions we had about whether or not we were representing the data correctly. We also learned that it is better if our counts our converted to per 1 mL instead of per 2 micro-liters.

Conclusion: 

• The skills we learned using excel will help us to better analyze our data and it will also help us have a better understanding of the meaning behind the numbers we are given; how they would relate to the Tetrahymena.

Future Ideas: 

• Any questions about how to properly execute what we learned are answered on Canvas.

Date: February 16, 2017 – February 26, 2017

Goals:

• Determine the number of Tetrahymena after 24 hours and after 1 week

Procedure After 1 Week:

• Take 2 micro-liters of your control, place on a concavity slide and view under the compound scope
• If too many Tetrahymena to count, take 5 micro-liters of your control and place it into 45 micro-liters of PPT media in a petri dish
• Take 2 micro-liters of this dilution and place it on a concavity slide and place it under the compound scope to count the cells
• Dilute your solution until there is a small enough number of cells in your 2 micro-liters to count
• Do this procedure twice for each of your wells
• Also do the same procedure for your treated cells

Procedure After 24 Hours:

• My cells were place in a Glutaraldehyde solution that preserved them until I could count them. When working with glutaraldehyde you must use gloves and note an extra calculation step that must be done at the end.
• Take 2 micro-liters of your control, place on a concavity slide and view under the compound scope
• If too many Tetrahymena to count, take 5 micro-liters of your control and place it into 45 micro-liters of PPT media in a petri dish
• Take 2 micro-liters of this dilution and place it on a concavity slide and place it under the compound scope to count the cells
• Dilute your solution until there is a small enough number of cells in your 2 micro-liters to count
• Do this procedure twice for each of your wells
• Also do the same procedure for your treated cells

Observations: Counts after 1 Week 

Control:

• A6 – 10^-2 dilution – counted 2 Tetrahymena
• B5 – 10^-2 dilution – counted 1 Tetrahymena
• B6 – 10^-2 dilution – counted 2 Tetrahymena

Treatment:

• C4 – 10^-1 dilution – counted 2 Tetrahymena
• C5 – 10^-2 dilution – counted 8 Tetrahymena
• C6 – 10^-2 dilution – counted 4 Tetrahymena

Observations: Counts After 24 Hours 

Control:

• B2 – 10^-3 dilution – counted 1 Tetrahymena
• B3 – 10^-3 dilution – counted 7 Tetrahymena
• B4 – 10^-2 dilution – counted 3 Tetrahymena

Treatment:

• C2 – 10^-1 dilution – counted 6 Tetrahymena
• C3 – 10^-2 dilution – counted 2 Tetrahymena
• D1 – 10^-1 dilution – counted 7 Tetrahymena

Conclusion: 

• My data seemed to conclude that there were more Tetrahymena in the treated well-plates after 24 hours and 1 week. The next step would be to analyze my data and form a better conclusion about the impact of Glyphosate on the Tetrahymena.

Date: February 9, 2017

Goals: 

• Practice counting cells using C-chips
• Set up our experiment
• Practice serial dilutions

Procedure Counting with C-chip:

1. Use 20p Micro-pipetter to inject 20 micro-liters of stock solution into each side of the c-chip
2. Look at the C-chip under the compound scope record the number of cells in and side A and B in your notebook. When counting only count the 4 outer corners and the middle cell. Only count the cells that are touching the top, left hand sides, and the middle. Do not count the ones that are touching the bottom and right sides.
3. Average your counts from side A then from side B, and calculate the amount of cells in 1 mL of solution. Equation: count avg. / 2×10^-4 mL = x cells / 1 mL. Only use 2×10^-4 mL if you have a .2mm C-chip.  

Procedure for Experiment: 

1. Designate 12 clear well-plates for you Tetrahymena
2. Using the 1000p Micro-pipetter, place 900 micro-liters of Tetrahymena into all 12 well-plates
3. Designate 6 well-plates to be your control and, using the 100p micro-pipetter, place 100 micro-liters of PPT growth media in along with the 900 micro-liters of Tetrahymena.
4. Designate the remaining 6 well-plates as your treatment and place 10 micro-liters of 0.5% of Glyphosate in along with the 900 micro-liters of Tetrahymena.
5. Record how cilia act in new environment
6. Use the 10p micro-pipetter to take 10 micro-liters of one control sample and place it on a concavity slide under the compound scope.
7. Do the same for your treated sample

Observations: 

• Side A: 13 combined cells / 5 = 2.6 as the average cell count
• Side B: 8 combined cells / 5 = 1.6 as the average cell count
• Calculated stock concentration in 1 mL as outlined in procedure for counting with a C-chip
• Hour 0: The cells in the control – some cells had gotten stuck together by a thread looking substance
• Hour 0: The cells in the Treatment – the cells seemed to be moving very rapidly

Conclusion: 

• The Tetrahymena only showed a minimal amount of change

Future Ideas: 

• Practice calculating and diluting so that the experiment doesn’t take so long

Date: February 2, 2017

Goals of Lab:

• Practice diluting substances
• Practice counting Tetrahymena in a diluted substance
• Practice calculating how many cells are in 1 mL of the original stock solution

Procedure for Practice:

Equipment – 2 Micro-fusing tubes, 1 Micro-fusing tube with dye and water, 200p and 20p Micro-pipetter

1. Put 180 microliters of water into both micro-fusing tubes using the 200p Micro-pipetter
2. Take 20 microliters of dye, using the 20p micro-pipetter, and place into your D1 solution (original solution labelled D1)
3. Make a 20 microliter dilution from your D1 one solution and put it into your D2 solution
4. Take a 20 microliters from your D2 solution and transfer it to your D3 solution.

Procedure: 

1. The 500 microliters of Tetrahymena will be your “stock” that you will use to make a 1:10 dilution into your micro-fusing tubes.
2. Place a 10 microliter drop of each solution on a well plate and count the number of Tetrahymena. Starting from your stock solution all the way until your 4th dilution.
3. Record the number of cells you see in the stock solution and in each dilution
4. After your last dilution use a calculation to determine the number of cells in 1mL of stock solution

Observations: 

• Stock Solution: N/A (too many to count)
• 10^-1 dilution: 15 cells
• 10^-2 dilution: 10 cells
• 10^-3 dilution: 3 cells
• 10^-4 dilution: 0 cells

Conclusion: 

• Making serial dilutions for a substance with too many cells is a very useful tool in determining the amount of cells are in the original solution.

Future Experiment Ideas:

• Take notes of all of your calculations
• Use vortex to spread around the Tetrahymena so that they all aren’t congregated in one area

Date: January 26, 2017

Goals:

• The goals of this lab were: to get a better understanding of how to use the Dissecting and Compound microscopes
• Learn how to use and the purpose of different pipettes
• Introduce us to the Tetrahymena

Background:

• Before the lab was started, we learned the different parts of the Compound microscope we were using. The skills we learned last week in the ciliates lab helped us prepare for actually viewing the Tetrahymena through the Compound microscope.

Procedure:

• Observe any alive ciliates in your 24-well plate under the Dissecting Microscope
• Choose the well plate a good amount of alive ciliate ( for me it was well plate A3 – Blepharisma)
• Using the plastic pipette, transfer some ciliates to a Watch Glass and observe under the Dissecting Microscope
• Then, using the same pipette, transfer a smaller sample of ciliates to a concavity slide and observe under the Compound Microscope (Only 10x and 40x, do not view on 100x objective)

Introduce the Tetrahymena

• Using the Micropipette, draw a 400 microliter solution and place it into a new well plate. (I placed mine into B1)
• Then take 100 microliters of Tetrahymena and place them in the same well plate
• Transfer 50 microliters of Tetrahymena from the well plate into a Watch Glass and record what you see under the Dissecting microscope
• Transfer 20 microliters of Tetrahymena from the Watch Glass onto a Concavity Slide and observe what you see under the Compound Microscope

Observations:

Tetrahymena

• Very small
• Oval like shape
• Quick Movements
• There was a lot of Tetrahymena in just 20 microliters of culture

New Equipment Introduced:

• Serological Pipette: used for measuring large quantities of liquids
• Micropipetters: used for measuring much smaller quantities of liquids
• Compound Microscope: Can achieve a much higher magnification than the Dissecting microscope
• Watch Glasses
• Concavity Slides

Conclusion:

• The compound microscopes are really helpful for viewing small things and organisms up close
• Different types of pipetters can give you a more precise amount of liquid depending on how much you need

Ideas for Future Experiment:

• Be careful with the many parts used in a lab
• Avoid Contamination
• Keep track of the purposes of the equipment you’re using
• With a many step lab like this, make sure to keep detailed notes of your process
• Work with your group members if you are having a hard time viewing anything through your microscopes

Date: January 19, 2017

Learning Objectives:

• Understand the idea behind constructivist learning
• Use the dissecting microscope correctly
• Observe and characterize ciliates using dissecting microscope

Procedure: 

• Clean desks with 10% bleach solution in spray bottles
• Obtain a clean 24-well plate
• Use plastic pipette to place each unknown into a separate well. Write down which unknown you placed into which well. Fill wells so that the solution covers the bottom of the well. To avoid decontamination, do not mix the pipettes!
• Observe each unknown under the dissecting microscope and fill out the table below. Make sure you add plenty of detail to your description.
• Make detailed description of each ciliate, and provide tentative identification along with reasoning.
• Tape observations into your lab notebook for instructor review.
• Discuss “Questions that Matter” with your group, write your answers, and turn in to your TA.

Observations: 

Sample B (Well A1):

• Shape: Long elongated body, worm-like shape
• Size: Large in length, but skinny
• Movement: Snake like movements
• Location in Media: Towards the edge of the media
• Other Characteristic: Head of ciliate swivels from side to side before moving
• Conclusion: Spirostomum

Sample A (Well A2):

• Shape: Short with tapered head (cigar shape)
• Size: Fairly small
• Movement: Quick spiral-like movements
• Location in Media: Kind of everywhere
• Other Characteristics: Body kind of undulates when it moves
• Conclusion: Paramecium

Sample C (Well A3):

• Shape: Cone-like shape, aperture at the head that looks like a mouth
• Size: Slightly bigger than sample A and thicker
• Movement: Slow up and down movements, kind of looks like its cruising
• Location in Media: More towards the edge of the media
• Other Characteristics: Dark red in color
• Conclusion: Blepharisma

Sample D (Well A4):

• Shape: Kind of rectangular in shape, with a dark C shape inside
• Size: Very Small
• Movement: Jerky movements
• Location in Media: Closer to edge but ventures out into middle
• Other Characteristics: C shape is the nucleus
• Conclusion: Euplotes

Sample E (Well A5):

• Shape: Thin tail, with large bell shaped head. Could also be described as trumpet looking.
• Size: Pretty large
• Movement: Up and down movements, likes to twirl. Snapped its tail in like a turtle, new position made it look completely circular.
• Location in Media: More in the center of the media
• Other Characteristics: Kind of green with black coloration along the sides, they’re funny little dudes
• Conclusion: Stentor

Final Notes: 

• Make sure to have the dissecting microscope adjusted well enough so that you can see out of both eyepieces. If you feel you didn’t get enough information during the class period make use of the extra lab meetings offered on Fridays.

Future Suggestions: 

• Either takes lots of pictures of you work or frequently sketch what you observe, this makes it easier to remember what progress you have made with your observations.