Purpose: 

Begin the first stages of designing a protocol for the metabarcoding of soil ciliates to explore the ciliate biodiversity

Procedure:

1. Add 8 mL Ludox HS 40 to a 15 mL conical tube.
2. Quickly add (inject) 2 mL of liquid from the soil samples in the jars into the Ludox using a p1000.
3. Carefully add (layer) 2 mL of distilled water to the top of the Ludox mixture, not mixing it together.
4. Centrifuge in a swinging bucket rotor for 15 minutes at 4300 g.
5. Carefully remove the tube and use a pipette to remove the cell layer.
6. Transfer to a clean microfuge tube.
7. Transfer 3-10 microliter drops to a concavity slide and count the cells using 100 magnification.
8. Centrifuge the microfuge tubes for 1 minute.
9. Pipette the liquid out of the microfuge tube, carefully not touching the pellet.
10. Freeze the pellet in order to preserve the sample.

Data Collection:

• our sample, before extraction,with the Ludox and distilled water weighed 21.4 grams
• 
• Above is our sample before centrifugation.
• 
• Sorry I don’t know how to turn the photo, but above is our sample after centrifugation.
• Our sample did not have a distinct layer of organisms, so we had to pipette a larger amount of the sample to observe.
• We ended up extracting about 3 mL of the sample.
• Our sample had a good amount of debris
• We looked at 3 different drops of the sample and found about 3 or 4 ciliates

Conclusion:

The lab was successful, which is great because it means that our protocol is adequate as of right now. It was a little challenging trying to identify whether some of the organisms were ciliates, protists, or debris. But with help from my TAs, I was able to distinguish each of the organisms. The net step of the protocol is to completely isolate the ciliates and denaturation.