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BISC209/F13: Lab3: Difference between revisions
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1. Use sterile serological pipets to make 12 mls of the appropriate dilution ( | 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 BioLog plate as described in the next step.<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 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> | ||
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'''MEASURING MICROBIAL GROWTH''' | '''MEASURING MICROBIAL GROWTH''' | ||
The intensity of color change is monitored in each of the wells by taking spectrophotometer readings once a day at A<sub>590</sub> nm. You and your partners need to figure out a schedule to divide up the work of collecting these data | The intensity of color change is monitored in each of the wells by taking spectrophotometer readings once a day at A<sub>590</sub> nm. You and your partners need to figure out a schedule to divide up the work of collecting these data. Make sure that you take a photo of your plate against a white background on the final day of measurement. There are a few digital cameras available or you can use your own camera.<BR><BR> | ||
We will use the MolecularDevices SpectraMax 190 located in the lab and the SOFTmaxPRO6.3™ program. | We will use the MolecularDevices SpectraMax 190 located in the lab and the SOFTmaxPRO6.3™ program. | ||
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