Purpose:

• To create our control for the Ludox protocol and adjust protocol for clarity purposes

Materials:

• Ludox tube
• Fresh top soil
• Distilled water
• Plastic vials
• Serological pipette
• Glass tube
• Vortex centrifuge
• Glutaraldehyde
• P1000 micropipette
• Colored water
• Bulb pipette
• Swinging bucket rotor
• Compound microscope

Procedure:

1. Collect 5 g of fresh top soil (screened of debris) and add 10 mL of distilled water into plastic vial
2. Mix soil mixture for 5-10 minutes on vortex centrifuge
3. Allow vial to sit for 1-2 minutes so clay and sand may settle on the bottom
4. After mixture has settled, transfer 3.68 mL of soil water to clean glass tube and add 368 ul of 25% glutaraldehyde to the soil mixture by using a serological pipette and P1000 micropipette
5. Vortex this new mixture on the vortex centrifuge for about 1 minute. Note: glutaraldehyde is added to prevent disintegration of the cells in the Ludox
6. Add 16 mL of Ludox to a 50 mL conical tube.
7. Inject 4 mL of the fixed sample into the Ludox tube by placing the P1000 about 2 mL below the surface of the Ludox solution. Note: Be sure to inject carefully to avoid disturbing the solution components
8. Carefully layer 2 mL of colored water on top of Ludox layer by using a bulb pipette
9. Centrifuge the conical tube at 4300xg for 15 minutes in a swinging bucket rotor
10. Remove the liquid from the organic matter layer below the water:Ludox interface
11. Remove a total of 4 mL from this layer and transfer to two labeled 2 mL microfuge tubes by using a P1000 micropipette.
12. Centrifuge the 2 mL tubes at 3000xg for 5 minutes to completely pellet the cells in order to concentrate cells into a pellet without much damage
13. Remove the supernatant with a P1000 without disturbing the pellet and dispose of the liquid in a waste container. Note: Be careful not to accidentally extract the pellet when removing the liquid from the tube
14. Add 100 ul of PBS (a buffer) to each pellet and resuspend the pellet by flicking and pipetting up and down.
15. Combine the cell suspension pellets together into 1 tube for a total of 200 ul.
16. Place five 2 ul drops on a slide and count the cells using the 40X lens on the compound microscope. Record cells/ul then obtain a class average. Note: If cells are not visible, stain mixture with iodine

Results/Conclusion:

After counting the cells, we hoped to obtain a 100% efficiency, which we will calculate in the next lab. There were some difficulties in measuring out the filtered soil due to the large size of the screen relative to the tube it was going in. After centrifuging, our Ludox tube had a very distinct layer of organic matter, which is good. Extracting the liquid surrounding the pellet was also a difficult task as it was very small. For future, use a smaller tip on the micropipette.