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BISC209/F13: Lab3: Difference between revisions
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1. | 1. Pairs will each 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). You will accomplish this by pipetting 10.8 mL of 10mM phosphate buffer (PB) into a large sterile tube and adding 1.2 mL from your 10<sup>-3</sup> soil extract serial dilution tube. Our goal is to end up with about 10<sup>5</sup> cfu in each well of rhe BIOLOG™ECO, 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 enumeration plates then there are 1X10<sup>7</sup> cells/ml, so you would need to make a 10 fold dilution (1X10<sup>6</sup> cells/mL). When you apply 0.1 ml of the 1X10<sup>6</sup> cells/mL to your plate, there are 1X10<sup>5</sup> cells present. You need 12 ml (10 ml for the plate and 2 ml extra) of diluted soil extract for each BIOLOG™ECO plate in order to inoculate all the wells. <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> | ||
