Purpose:

This week, the purpose of our experiment was to conduct the modified Ludox Protocol and practice our pipetting skills. We were able to learn new pipette techniques and helped in determining the accuracy of the micropipettes. We were also able to learn how to conduct an accurate and precise protocol to obtain a pure result.

Procedure:

Pipetting 

1. Obtain a p1000 pipette, pipette up 1000uL and place it on a weigh boat on a scale.
2. Repeat this process 10 times and see if the scare weighs 1 gram. If so, the pipette is calibrated and is going to give accurate measurements.
3. Obtain a serological pipette and pipette approximately 10mL.
4. Place these 10mL in a weigh boat and weigh it on a scale. It should weigh 1 gram.
5. Record these questions in the Questions that matter.

Modified Ludox Protocol

1. Collect 5 grams of fresh top soil, pour it through a screen, and add 10mL of H2O using a serological pipette. Do this in a film bottle.
2. Shake the bottle to mix the sediment. Do this until the soil is evenly distributed throughout the mixture.
3. Place the film bottle on the vortex and allow 5-10 minutes for the soil to mix.
4. After shaking, let the vial sit undisturbed for 1-2 minutes. Transfer 3.68mL of soil water to a clean glass tube and add 320uL of 25% Glutaraldehyde. Vortex briefly to mix well. The final concentration of Glutaraldehyde should be 2%.
5. Add 16mL of Ludox to a 50mL conical tube. Inject 4mL of fixed sample into the Ludox tube by placing the p1000 micropipette 2mL below the surface. Do this carefully as to not disturb the solution.
6. Carefully layer food coloring on top of the surface, about 2mL.
7. Weigh the total sample and be sure it is the same mass as your corresponding groups. If not, add food coloring to readjust.
8. Centrifuge 4300xg for 15 minutes in a swinging bucket rotor. This will level out your mixture so that each layer will become independent.
9. Remove the liquid from the cell layer, it will be a moderately thick layer in the middle/top of the mixture. Remove a total of 4mL and transfer to two labeled 2ml microfuge tubes. Use a p1000 for this process and remember that 1000uL is equal to 1mL. You may want to cut the tip of the pipette to prevent debris from clogging the tip.
10. Centrifuge the 2mL tubes at 3000xg for 5 minutes to pellet out the cells. The pellets should be at the very bottom of the vial.
11. Mark the tubes so you know the side was toward the outside of the centrifuge.
12. remove the super natant with a p1000 without disturbing the pellet. This can be done by turning the tube so that the pellet is toward the top and remove the liquid which is on the opposite side. This process needs to be done carefully as you do not want to extract the pellet by accident.
13. Add 100uL of PBS to each pellet and resuspend the pellet by pipetting it up and down in the solution. Combine the cell suspension pellets in 1 tube. The total volume in the one tube should be 200uL.
14. Place five 2uL drops on a slide and count the cells using the 40x lens. Record the total number of cells per uL and obtain a class average of cell numbers. It should be noted that most the cells will be in cyst form as it takes about 24 hours for them to come out.
15. If you are experiencing trouble locating any cells, at a 10uL stain of iodine to 10uL of cells.

Control Tubes

1. Add a known amount of stock ciliate to 5 grams of autoclaved soil. Dr. Adair added about 1000uL of Tetrahymena culture to the soil. There was roughly 2 million cells in the sample.
2. Threat the sample in the same manner as the experimental samples.
3. Calculate the efficiency of the method by using the formula [(Total number of cells added – Total number of cells recovered in extraction) / Total number of cells added] * 100.

Clean up

• The used serological pipettes should go in the waste box, not the trash can.
• Rinse the remaining soil into the bucket and wash the plastic containers you used. Be sure not to get any soil in the sink.
• Put the remaining Gluteraldehyde in the waste bottle by the sink. Rinse the tube with bleach and water to sanitize it.
• Place the Ludox tubes in the rack by the sink for later freezing.
• Spray windex on the counters and wipe to disinfect any possible substances.

Results:

• Sample Weight: 40.9 grams
• Number of cells per uL: We were unable to observe the sample as we ran out of time. This step will take place next week.
• Label: We labeled our conical tube (PIN) and our microcentrifuge tubes (PIN) as well.
• Mass of 1ml of water using the p1000 micropipette= 0.9 grams (Our pipette was inaccurate)
• Mass of 0.1ml of water using the p1000 micropipette= 0.1 grams
• Mass of 1ml of water using the serological pipette= 0.9 grams (this could be as a result of user error)
• Mass of 0.1ml of water using the serological pipette= 0.2 grams (this could also be as a result to user error, the serological pipettes are difficult to use with smaller quantities of liquid)

Conclusion:

We placed our 2 microcentrifuge tubes on the rack labeled as PIN. We hope to extract and count the cells next week. I hope to begin DNA extraction soon as that step of the process is soon. This lab taught us how to use a serological pipette as well as practicing the accuracy of our pipetting skills. We were also able to gain more experience regarding the Ludox protocol. These skills will be very valuable as we increasingly get more time in the lab. I am excited for the upcoming weeks and hope we can extract the DNA from these pellets.