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BISC209/F13: Lab2: Difference between revisions
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→Finishing the Standard Plate Count of Culturable Soil Microbial Community
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Today you will complete your first investigative goal in our semester long project: assessing abundance of a soil microbial community by enumerating its microbes by two methods, one culture dependent and one culture independent. Today you will also continue acquiring and practicing skills used by working microbiologists. You will complete your tests for abundance today. The results of the fluorescent DNA stain are available and your plate counts are ready to assess. In addition, you will continue to isolate a few bacteria of interest from your soil community in order to show evidence for richness (diversity) and potential for co-operative and competitive behaviors among community members. Next week we will begin testing the whole soil microbial community : a culture-dependent assessment of carbon source utilization diversity. | Today you will complete your first investigative goal in our semester long project: assessing abundance of a soil microbial community by enumerating its microbes by two methods, one culture dependent and one culture independent. Today you will also continue acquiring and practicing skills used by working microbiologists. You will complete your tests for abundance today. The results of the fluorescent DNA stain are available and your plate counts are ready to assess. In addition, you will continue to isolate a few bacteria of interest from your soil community in order to show evidence for richness (diversity) and potential for co-operative and competitive behaviors among community members. Next week we will begin testing the whole soil microbial community : a culture-dependent assessment of carbon source utilization diversity. | ||
==Finishing the Standard Plate Count | =='''Abundance: 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. Find the plate(s) among those of your serial dilution that contains 30-300 colonies. You will not assess plates with well over 300 colonies or under 30; they should be designated as "invalid" in your lab notebook and on the bottom of the plate. 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.) <BR><BR> | |||
'''Calculating the number of colony forming units (CFU) per gram of soil'''<BR> | |||
For each valid plate counted, 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> | |||
'''Calculating the number of colony forming units (CFU | |||
For each plate counted | |||
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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Use this % change to convert the CFU per gram wet soil to CFU per gram dry soil. For example: If your dry weight soil averages 0.75 grams, then (1g-0.75g)/1g * (100) = 25% change. The number of microbes should be 25% higher than the number calculated above/gram of wet weight. Calculate 25% (or whatever your conversion factor is) of your CFUs and add that number to the CFUs/ gram of wet weight soil. <BR><BR> | Use this % change to convert the CFU per gram wet soil to CFU per gram dry soil. For example: If your dry weight soil averages 0.75 grams, then (1g-0.75g)/1g * (100) = 25% change. The number of microbes should be 25% higher than the number calculated above/gram of wet weight. Calculate 25% (or whatever your conversion factor is) of your CFUs and add that number to the CFUs/ gram of wet weight soil. <BR><BR> | ||
'''Record the number of CFU/ gram DRY weight soil in your lab notebook''' and add this information to the chalk board. If you are unsure of the accuracy of your calculations of CFUs per gram of wet soil weight and per gram of dry weight, check with your instructor. You will need accurate counts | '''Record the number of CFU/ gram DRY weight soil in your lab notebook''' and add this information to the chalk board. If you are unsure of the accuracy of your calculations of CFUs per gram of wet soil weight and per gram of dry weight, check with your instructor. You will need accurate counts for some of our future testing.<BR> | ||
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Why did we perform so many dilutions when we set up our plate count in Lab 1?<BR> | Why did we perform so many dilutions when we set up our plate count in Lab 1?<BR> | ||
Why should you have only one dilution with 30-300 colonies?<BR> | Why should you have only one dilution with 30-300 colonies?<BR> | ||
Why did we make up to 4 replicates per site | Why did we make up to 4 replicates per site? | ||
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> | |||
Seal the edges | Seal the edges with parafilm of the valid dilute nutrient agar plate and store the plate in the cold room at the end of lab. Don't discard any other plates yet. | ||
== Enumeration of Community Soil Microorganisms by Direct Count of Microbial Genomes Stained & Viewed by Fluorescence Microscopy== | == Enumeration of Community Soil Microorganisms by Direct Count of Microbial Genomes Stained & Viewed by Fluorescence Microscopy== | ||
