BISC209/F13: Lab3: Difference between revisions

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BISC209/F13: Lab3 (view source)

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1. Use sterile serological pipets to make 12 mls of the appropriate dilution.  Our goal is to end up with about 10<sup>5</sup> cfu in each well; therefore, you need a working solution that has 10<sup>6</sup>CFUs/ml, because 0.1ml contains 10<sup>5</sup>CFUs. This is an estimate that does not account for those organisms that did not grow on NA, but might grow in this community assay.  You need 12 ml (10 ml for the plate and 2 ml extra) of this working solution for each BIOLOG™ECO plate in order to inoculate all the wells. You will accomplish this by pipetting 10.8 mL of 10mM phosphate buffer into a large sterile tube and adding 1.2 mL from the serial dilution that gave you 10<sup>7</sup>CFUs/ ml. You can find that information in the Enumeration DATA sheet in the Data folder. <BR><BR>
1. Use sterile serological pipets to make 12 mls of a 10-3 dilution of your soil extract.  You will need 12 ml (10 ml for the plate and 2 ml extra) of this working solution for each BIOLOG™ECO plate in order to inoculate all the wells. Our goal is to end up with about 10<sup>5</sup> cfu in each well. Pipet 10.8 mL of 10mM phosphate buffer into a large sterile tube and add 1.2 mL from the serial dilution that gave you 10<sup>3</sup>CFUs/ ml. <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 100μL individual aliquots directly into each well of the BioLog plate as described in the next step.<BR><BR>
2. If you will be using a multichannel micropipet to inoculate your BIolog plate, pour this 12ml of diluted soil extract 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 100μL individual aliquots directly into each well of the BioLog plate as described in the next step.<BR><BR>
3. 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 or use a multichannel pipet with new tips set to 100μL.  If you are using the multichannel pipet,  check visually the consistency of the amount of diluted extract in each of 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! Expel the fluid back into the reservoir and start over. If you are unfamiliar with the use of multichannel pipets, ask your instructor to observe your technique. <BR><BR>
3. 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 or use a multichannel pipet with new tips set to 100μL.  If you are using the multichannel pipet,  check visually the consistency of the amount of diluted extract in each of 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! Expel the fluid back into the reservoir and start over. If you are unfamiliar with the use of multichannel pipets, ask your instructor to observe your technique. <BR><BR>
4. 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>
4. 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, but it does change the overall concentration of your soil microbes in this assay.<BR><BR>
5. Replace the cover of the plate and label one '''side''' of the cover. DO NOT LABEL ON the top or bottom to avoid interference in the light passage during spectrophotometric readings. Use a piece of your team color tape and include your initials, lab section, date, and soil sample code.<BR><BR>
5. Replace the cover of the plate and label one '''side''' of the cover. DO NOT LABEL ON the top or bottom to avoid interference in the light passage during spectrophotometric readings. Use a piece of your team color tape and include your initials, lab section, date, and soil sample code.<BR><BR>
6. Take a time 0 reading at A<sub>590nm</sub> using the SpectraMax 190 plate reader as described in the next section of these instructions.<BR><BR>   
6. Take a time 0 reading at A<sub>590nm</sub> using the SpectraMax 190 plate reader as described in the next section of these instructions.<BR><BR>   
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