Ludox Centrifugation, 1/25/2018

Purpose: Can we get cells out of the soil and if so, can this procedure apply to our protocol?

Materials: Ludox (wear gloves!), high power centrifuge, lower power centrifuge

Procedure:

1.Add 8ml Ludox HS 40 to a 15 ml conical tube.

2. Add 2 ml of liquid from the soil samples in the jars into the Ludox using a p1000.

3. Add 2 ml of distilled water (with red food coloring) to the top of the Ludox mixture.

4. Weigh test tubes (both at the lab table).

5.Centrifuge in a swinging bucket rotor for 15 min at 4300 x g.

6. Use a pipette to remove 3-5 ml layer of cells.

7. Place three drops of cells onto a concavity slide and observe under the microscope.

8.Transfer cells to a clean 15 ml comical tube.

9.Dilute with buffer to 10 ml and spin at 4300 x g for 10 minutes to wash and pellet the cells.

10.Remove the supernatant and store the pellet in the freezer.

Mistakes: Not adding the water carefully to the Ludox and accidentally mixing the water into the Ludox.

Results:

Our sample contained a couple of cells. I personally was not able to find a ciliate, but Camille and Katy each found one small ciliate (using a high magnification).This proved that our method for extracting cells was successful. Future steps would be to use this protocol when attempting to extract cells for our major experiment with classifying ciliates based on DNA sequencing. An unexpected finding would be the ciliate Dr. Adair showed us, which had formed a protective layer to remain dormant during the cool weeks. A source of error would be not being able to solely extract the cells from the test tube with the Ludox in it. There was no way to ensure that only cells were taken up by the micropipette. This may explain why some samples taken with the micropipette had ciliates, but others did not.

Storage:

Our sample is labeled CKL and has a smiley face drawn on the cap.