Purpose: Design protocol for metabarcoding of oil ciliates to explore ciliate diversity.

Procedure:

1. Add 8 ml of Ludox HS 40 to a 15 ml conical tube
2. Quickly add 2 ml of liquid from the soil samples in the jars into the Ludox using a p1000
3. Carefully layer 2 ml of distilled water to the top of the Ludox mixture; do not mix
4. Centrifuge in a swinging bucket rotor for 15 min at 4300 x g
5. Carefully remove the rube and use a pipette to remove the cell layer from underneath the water layer
6. Transfer to a clean microfuge tube and label with your group’s number
7. Transfer 3-10 ul drops to a concavity slide and count the cells under 100x magnification; do this three times and record results
8. Centrifuge the remainder of the contents in your microfuge tube at 12,000xg for 1 minute
9. Remove the supernatant using a p1000 and store the pellet in the freezer

Results:

This is how our Ludox tube looked prior to centrifuging it in the swinging bucket rotor:

After, when we counted how many cells were present in three drops of the cell layer under the microscope, I counted that there were 12, 16, and 21 in my samples. The cells were very small, translucent, and had a circle/oval shape.

Conclusion:

We successfully created a Ludox gradient and were able to remove and observe the cells from the cell layer.

Next Steps:

The next step would be to try extracting DNA from the cells that are in our sample in order to begin the rest of the metabarcoding process.