Purpose:

The purpose of this lab was to practice pipetting with both micropipettes and serological pipettes. We wanted to make sure that we were accurate with out measurements and that it would not be a problem when it comes to do doing the correct protocol. We also modified the protocol to have it be more accurate and hopefully work in giving us the cell layer we want.

Procedure

• Pipette and weigh amounts of water using micropipettes
  • 1mL – 1g
  • 0.1mL – 0.1g
  • 0.01mL – 0.01g
• Pipette and weight amounts using the serological pipette
  • 1mL – 1g
  • 0.01mL – 1g

New Protocol:

1. Collect 5g of soil in a container, use the screen to help in not having any debris and add 10mL of water into the container
2. Put the lid on and mix for 5-10 minutes, shaking and using the vortex
3. Let the mixture sit for 5 minutes in order to let the soil settle to the bottom
4. Transfer 3.68mL of the soil water on the top into a glass tube then add 320ul of 25% Glutaraldehyde. Shake or mix for 1-2 minutes
5. Obtain a 50mL conical tube that is filled with 16mL of Ludox. Then add 4mL of the soil water in the glass tube to the ludox about 2mL below the surface
6. Slowly add 2mL of red water on the top of the Ludox tube, make sure the weight of your tube if the same as the other group at your table
7. Centrifuge the tube for 15 minutes on 4300xg
8. Carefully take out the layer of cells right below the water layer and transfer into a new microfuge tube
9. Centrifuge the tubes for 5 minutes at 3000xg
10. Begin to remove the water on top of the tube, avoiding taking out the pellet at the bottom
11. Add 100ul of the buffer PBS to each pellet, combine the two tubes together for a total of 200ml
12. Place 5 2ul drops on a slide and look at them under a 40x lens
13. Total number of cells counted/10ul =cells/ul

Data:

Did not have time to put drops on a slide and get cell counts

• Ludox tube weight: 40.3g
• Weighed out exactly 5g of our soil

We did have some issues with our soil sample and the soil water seeming to have too much soil in it. Our soil did not exactly all settle to the bottom in the beginning when we were obtaining our soil water. We believe this is because we used such fine soil.

We have our small microfuge tubes stored that are labeled with SAM as well as our ludox tube that has SAM and 40.3g on the top.

Conclusion:

It was good to practice our pipetting skills before we did the protocol to make sure that our pipetting was not the reason why we had so issues with the past protocols. Based on our observations this protocol should be a lot better than last time and hopefully helped in avoiding the problems we had in the past. We were able to extract the cell layer but did not have time to isolate out pellet and then be able to look at the cells under the microscope.