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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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Each pair will prepare a series of dilutions to use in the community physiology profiling analyses that we will set up today. | Each pair will prepare a series of dilutions to use in the community physiology profiling analyses that we will set up today. | ||
Gather the following materials:<BR><ul> | Gather the following materials:<BR><ul> | ||
<LI> | <LI> 4 large (18 X 100), sterile tubes with caps that can hold a volume of 10 ml and not spill it when vortexed | ||
<LI> | <LI> 4 1ml sterile, disposable pipets and a blue pipet pump | ||
<LI> 1 10ml sterile, disposable pipet and a green pipet pump | <LI> 1 10ml sterile, disposable pipet and a green pipet pump | ||
<LI> P1000 micropipet and sterile tips | <LI> P1000 micropipet and sterile tips | ||
<LI> Bottle of sterile water </ul><BR><BR> | <LI> Bottle of sterile water </ul><BR><BR> | ||
1. Label | 1. Label four large sterile tubes 10<sup>-3</sup>, 10<sup>-4</sup>, 10<sup>-5</sup>, and 10<sup>-6</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 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 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 10<sup>- | 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 10<sup>-6</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''' | ||
