Objective:

The goal of lab today is to test out our new ludox protocol that has been comprised of protocols that have been made be everyone in class. We hope to use this new protocol that has been created to further our experiment and help lead us to complete the metabarcoding process.

Purpose:

The purpose of today’s lab is to try another ludox protocol to see if this one will give us the results we desire. We hope that by changing our protocol once again we will get one step closer to being able to extract DNA from the ciliates and start the metabarcoding process and classify them.

Procedure:

1.) Collect 5 grams of fresh top soil (screened of debris) and add 10mL of water. Mix for 5-10 minutes by either shaking it up and down or using the vortex.

2.) After shaking, allow the vial to sit undisturbed for 1-2 minutes to let the soil settle.

3.) Transfer 3.68mL of soil water to a clean glass tube and add 368 micro-liters of 25% Gluteraldahyde and mix by vortexing or shaking briefly.

4.) Add 16mL of ludox to a 50mL conical tube. Inject 4mL of the fixed sample into the ludox solution.

5.) Carefully layer colored 2mL of water that has been dyed with one drop of food coloring on top of the ludox layer.

6.) Centrifuge at 4300 x g for 15 minutes in a swinging bucket rotor.

7.) Remove the liquid from the cell layer, about 1.5mL below the water-ludox interface. Make sure you move a total of 4mL. Transfer this to two labeled 2mL micro-centrifuge tubes.

8.) Centrifuge the 2mL tubes at 3000 x g for 5 minutes in order to completely pellet the cells.

9.) Remove the supernatant with a p1000 micro-pipette without disturbing the pellet and dispose of the liquid in your waste container.

10.) Add 100 micro-liters of the phosphate buffer saline (PBS) to each pellet and re-suspend them by flicking and pipetting up and down. Combine the cell suspension pellets together into one tube for a total of 200 micro-liters.

11.) Place five, 2 micro-liter drops on a concavity slide and count each cell present using the 40x lens. Record your number of cells per micro-liters, then obtain a class average of cell numbers.
*Most cells may be in cyst form.

-We only got to step 8 during lab today.

During lab:

-We also practiced out pipetting skills using serological pipets. We also measured to see if our pipetting skills were accurate by seeing if 1mL was equal to 1 gram, and 100 micro-liters is equal to one tenth of a gram.

Data/Observations:

During lab today, after the first centrifugation, the layers that had been created by the swinging bucket rotor were very undefined. The organic layer seemed to have mixed with the ludox which made it difficult to pipette out the organic layer. My groups conical tube weighed 40.5 grams originally but we had to add water to match the weight of the other group at our table. The weight of our conical tube was 41.0 grams before it was placed into the centrifuge. We didn’t have time to observe/ count our cells under the microscope because we ran out of time. We only got to step 8, we have been stopping at around the same spot in the past labs and have not been able to wash the pellet with any buffer yet.

Storage:

The conical tubes were collected by Dr. Adair to be properly disposed of and the 2mL micro-centrifuge tubes were left in the green racks at the end of the lab tables to be collected for centrifugation. We labeled our conical tube and 2mL micro-centrifuge tubes “Group #5 LSK”.

Future Goals:

In the future I hope that we eventually figure out a new ludox protocol that will allow us to gain the results we desire. I hope that the protocol that we come up with can be used by other researchers and future sections of this lab and help pave the way for future experiments. If we can create a more standardized protocol now, more experiments can be conducted in the future too further our knowledge of ciliates and the metabarcoding process.