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BISC209/F13: Lab2: Difference between revisions
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→Abundance Test 1: Finishing the Standard Plate Count
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=='''Abundance Test 1: Finishing the Standard Plate Count'''== | =='''Abundance Test 1: Finishing the Standard Plate Count'''== | ||
Last week you started a standard plate count of the culturable microbes in your soil sample on dilute nutrient agar. Today you will complete that plate count to get one kind of enumeration of the microorganisms in your soil community | Last week you started a standard plate count of the culturable microbes in your soil sample on dilute nutrient agar. Today you will complete that plate count to get one kind of enumeration of the microorganisms in your soil community. <BR><BR> | ||
'''Calculating the number of colony forming units (CFU) per gram of soil'''<BR> | '''Calculating the number of colony forming units (CFU) per gram of soil'''<BR> | ||
Examine the plates at each dilution and find one dilution with between 30 and 300 colonies. You will not assess plates with well over 300 colonies or under 30; they should be designated as "invalid" in your lab notebook. Count all the surface and subsurface colonies on the replicate valid plates (those with 30-300 colonies). The colonies can be more easily counted by using a Quebec Colony Counter which allows proper illumination, a grid overlay, and slight magnification of the plate surface. (There are two colony counters in the lab.) divide the total number of colonies counted by the amount of inoculum plated times the dilution factor of that plate to obtain the number of bacteria per gram of soil. <BR> | |||
number CFU/(dilution plated*dilution factor) = number of CFU/gram<BR> | number CFU/(dilution plated*dilution factor) = number of CFU/gram<BR> | ||
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150/(0.1ml inoculum*1X10<sup>-6</sup>dilution)= 150X10<sup>7</sup> which in scientific notation is written as 1.5X10<sup>9</sup> CFU/gram <BR> | 150/(0.1ml inoculum*1X10<sup>-6</sup>dilution)= 150X10<sup>7</sup> which in scientific notation is written as 1.5X10<sup>9</sup> CFU/gram <BR> | ||
'''Record the number of '''CFU/ gram of wet soil''' in your lab notebook and enter your data on the course spreadsheet on the instructor's computer at the front of the lab. Calculate the average and standard deviation for the plates from your sample site. | '''Record the number of '''CFU/ gram of wet soil''' in your lab notebook and enter your data on the course spreadsheet on the instructor's computer at the front of the lab. Calculate the average and standard deviation for all the plates from your sample site. Note that this number is the average colony forming units/gram WET soil for your sample site.''' | ||
'''Soil bacteria are usually not recorded as number of colony forming units (CFU) in 1 gram of WET soil but instead as per gram of DRY weight. ''' Therefore, you will need to figure out your counts as DRY weight. Please weigh each of the three 1 gram samples that you left last week for oven drying. Average the new dry weights. The weights should be considerably less than 1 gram. | '''Soil bacteria are usually not recorded as number of colony forming units (CFU) in 1 gram of WET soil but instead as per gram of DRY weight. ''' Therefore, you will need to figure out your counts as DRY weight. Please weigh each of the three 1 gram samples that you left last week for oven drying. Average the new dry weights. The weights should be considerably less than 1 gram.<BR> | ||
Determine the % change in soil weight by subtracting the average dry weight from 1 g wet weight divided by the wet weight, then times 100. <BR> | Determine the % change in soil weight by subtracting the average dry weight from 1 g wet weight divided by the wet weight, then times 100. <BR> | ||
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Do you expect the results of this enumeration of soil microbes in your community to be the same, lower, or higher than the results of our other enumeration test (the DAPI DNA stain)? Why?<BR><BR> | Do you expect the results of this enumeration of soil microbes in your community to be the same, lower, or higher than the results of our other enumeration test (the DAPI DNA stain)? Why?<BR><BR> | ||
Don't discard any plates yet. | |||
=='''Abundance Test 2: Enumeration of Community Soil Microorganisms by Direct Count of Microbial DNA Stained & Viewed by Fluorescence Microscopy'''== | =='''Abundance Test 2: Enumeration of Community Soil Microorganisms by Direct Count of Microbial DNA Stained & Viewed by Fluorescence Microscopy'''== | ||
