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BISC209/F13: Lab3: Difference between revisions
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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> large (18 X 100), sterile tubes with caps that can hold a volume of 10 ml and not spill it when vortexed | <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>1ml sterile, disposable pipets and a blue pipet pump | <LI> 4 1ml sterile, disposable pipets and a blue pipet pump | ||
<LI> 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> | ||
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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 last dilution tube. | 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''' | ||
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<BR> | <BR> | ||
1. | 1. Use sterile serological pipets to make 12 mls of the appropriate dilution (the one that gave you between 30-300 colonies in your Lab 2 plate count). Our goal is to end up with about 10<sup>5</sup> cfu in each well, this is an estimate that does not account for those organisms that did not grow on NA, but might grow in this community assay. For example, if you counted roughly 100 colonies on your 10<sup>-4</sup> dilution plates on your dNA enumeration plates, you would need to make 12mls of a 10<sup>-4</sup> dilution). You will accomplish this by pipetting 10.8 mL of 10mM phosphate buffer into a large sterile tube and adding 1.2 mL from your 10<sup>-3</sup> soil extract serial dilution tube. You need 12 ml of diluted soil extract to inoculate all the wells of a BIOLOG™ECO plate (10 ml for the plate and 2 ml extra.<BR><BR> | ||
2. If you will be using a multichannel micropipet, pour this 12ml of diluted soil extract (10<sup>6</sup> cfu/ml) into a sterile reservoir (a petri dish or a v-shaped plastic reservoir). If the multichannel pipet is unavailable, forget pouring the soil extract dilution into a reservoir. You can use your P200 to dispense individual aliquots directly into each well of the | 2. If you will be using a multichannel micropipet, pour this 12ml of diluted soil extract (10<sup>6</sup> cfu/ml) into a sterile reservoir (a petri dish or a v-shaped plastic reservoir). If the multichannel pipet is unavailable, forget pouring the soil extract dilution into a reservoir. You can use your P200 to dispense individual aliquots directly into each well of the BioLog plate as described in the next step.<BR><BR> | ||
4. Be careful to preserve the BIOLOG Eco™ plate's and its cover's sterility (eg. don't place it face down on your bench). Remove the cover and transfer 100µl of the soil extract dilution into each each well of the 96 well BIOLOG plate. Check visually the consistency of the amount of diluted extract in the micropipet tips after you have drawn up your aliquots to determine that you have no bubbles and that the quantity to be dispensed is the same. If the pipet tips appear unevenly filled or you have bubbles, do not dispense the inoculum into the wells! Start over. If you are unfamiliar with the use of multichannel pipets, ask your instructor to observe your technique. <BR><BR> | 4. Be careful to preserve the BIOLOG Eco™ plate's and its cover's sterility (eg. don't place it face down on your bench). Remove the cover and transfer 100µl of the soil extract dilution into each each well of the 96 well BIOLOG plate. Check visually the consistency of the amount of diluted extract in the micropipet tips after you have drawn up your aliquots to determine that you have no bubbles and that the quantity to be dispensed is the same. If the pipet tips appear unevenly filled or you have bubbles, do not dispense the inoculum into the wells! Start over. If you are unfamiliar with the use of multichannel pipets, ask your instructor to observe your technique. <BR><BR> | ||
5. Add 50μL of phosphate buffered saline (PBS) to each well (to account for dehydration over time of measurement). Note that this dilution does not change the number of cfus per well (10<sup>5</sup> cfus), but it does change the overall concentration of your soil microbes in this assay.<BR><BR> | 5. Add 50μL of phosphate buffered saline (PBS) to each well (to account for dehydration over time of measurement). Note that this dilution does not change the number of cfus per well (10<sup>5</sup> cfus), but it does change the overall concentration of your soil microbes in this assay.<BR><BR> | ||
