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BISC209/F13: Lab3: Difference between revisions
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→Make another Soil Extract and Serial Dilution
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<LI> Bottle of sterile water </ul><BR><BR> | <LI> Bottle of sterile water </ul><BR><BR> | ||
1. Label four large 18 mm sterile tubes 10<sup>-3</sup>, 10<sup>-4</sup>, 10<sup>-5</sup>, | 1. Label four large 18 mm sterile tubes 10<sup>-3</sup>, 10<sup>-4</sup>, 10<sup>-5</sup>, etc. until you reach the proper dilution series for your soil extract.</sup>.<BR> | ||
2. Pipet 9 ml of sterile water into each tube using a 10 ml sterile, disposable serologic pipet. (You may use the same sterile 10 ml pipet for all of the tubes if asepsis is maintained.) <BR> | 2. Pipet 9 ml of sterile water into each tube using a 10 ml sterile, disposable serologic pipet. (You may use the same sterile 10 ml pipet for all of the tubes if asepsis is maintained.) <BR> | ||
3. Using a different sterile, disposable 1ml serologic pipet for each transfer, transfer 1ml of the 1% soil extract (1:100 dilution made of 1gram of soil) to the next dilution tube labeled 10<sup>-3</sup>; mix well by vortexing.<BR> | 3. Using a different sterile, disposable 1ml serologic pipet for each transfer, transfer 1ml of the 1% soil extract (1:100 dilution made of 1gram of soil) to the next dilution tube labeled 10<sup>-3</sup>; mix well by vortexing.<BR> | ||
4. Using a new pipet, transfer 1ml of the 10<sup>-3</sup> dilution to the tube labeled 10<sup>-4</sup>. Mix well. (Mixing 1ml of the 10<sup>-3</sup> dilution with 9ml of sterile water makes a 10<sup>-4</sup> dilution. )<BR> | 4. Using a new pipet, transfer 1ml of the 10<sup>-3</sup> dilution to the tube labeled 10<sup>-4</sup>. Mix well. (Mixing 1ml of the 10<sup>-3</sup> dilution with 9ml of sterile water makes a 10<sup>-4</sup> dilution. )<BR> | ||
5. Continue to transfer 1ml aliquots (after mixing well) from each dilution to the next dilution tube of water until you have carried the dilution to the | 5. Continue to transfer 1ml aliquots (after mixing well) from each dilution to the next dilution tube of water until you have carried the dilution to the last dilution tube. </sup>. | ||
6. You now have diluted soil to use in all of the community level profiling analyses, described in the following protocols. The appropriate dilution for each assay depends upon the CFUs/gm (ml)that you calculated in your original Lab 1 soil sample. Check your previous calculations for CFUs/gram of soil WET WT (use the wet weight not dry weight calculation) and have that information available to use today. Divide up the work for each of the community assays with your partners. Before you leave lab today you must make sure that you and your partners have a workable plan to collect data and check your cultures as you perform: <BR><ul> | 6. You now have diluted soil to use in all of the community level profiling analyses, described in the following protocols. The appropriate dilution for each assay depends upon the CFUs/gm (ml)that you calculated in your original Lab 1 soil sample. Check your previous calculations for CFUs/gram of soil WET WT (use the wet weight not dry weight calculation) and have that information available to use today. Divide up the work for each of the community assays with your partners. Before you leave lab today you must make sure that you and your partners have a workable plan to collect data and check your cultures as you perform: <BR><ul> | ||
<li>'''Prevalence of Exoenzyme producers able to digest Starch, Cellulose, and/or solubilize Phosphate''' | <li>'''Prevalence of Exoenzyme producers able to digest Starch, Cellulose, and/or solubilize Phosphate''' | ||
