BISC209/F13: Lab2: Difference between revisions

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BISC209/F13: Lab2 (view source)

Revision as of 14:28, 22 August 2013

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→‎Abundance Test 2: Enumeration of Community Soil Microorganisms by Direct Count of Microbial DNA Stained & Viewed by Fluorescence Microscopy
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=='''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'''==
You can directly count a random sample of microbes from the soil extract that you prepared last week and then extrapolate the number per gram of soil. To make the microbes easier to count, you stained the nucleic acids of your soil community microbes (not just the bacteria) with a fluorescent 4'-6-Diamidino-2-phenylindole (DAPI) DNA stain. All the microbes in a 1ml aliquot of the 1% soil extract that you prepared last week were transferred in a Poisson distribution to a small piece of filter paper. Your instructor photographed the discreet bright "spots" of fluorescent DNA in several different areas of the filter paper distribution using fluorescence microscropy.  You will each count the discreet "spots" (individual microorganisms  = one fluorescent genome/cell) from and perform the calculations described below to assess the total microbial concentration in this ''culture-independent'' enumeration of your soil community's microorganisms. <BR><BR>
You can directly count a random sample of microbes from the soil extract that you prepared last week and then extrapolate the number per gram of soil. To make the microbes easier to count, you stained the nucleic acids of your soil community microbes (not just the bacteria) with a fluorescent 4'-6-Diamidino-2-phenylindole (DAPI) DNA stain. All the microbes in a 1ml aliquot of the 1% soil extract that you prepared last week were transferred in a Poisson distribution to a small piece of filter paper. Your instructor photographed the discreet bright "spots" of fluorescent DNA in several different areas of the filter paper distribution using fluorescence microscropy.  You will each count the discreet "spots" (individual microorganisms  = one fluorescent genome/cell) from and perform the calculations described below to assess the total microbial concentration in this ''culture-independent'' enumeration of your soil community's microorganisms. <BR><BR>
'''DIRECT COUNT OF MICROBIAL CELLS USING DAPI DNA: PROTOCOL''' <BR>
'''NOTE: The staining and imaging were performed by your lab instructor last week according to the protocol described below (provided here for reference only). YOU WILL NOT PERFORM THIS WORK IN LAB TODAY!'''<BR>
DAPI aqueous solution Stock conc. is 1mg/ml :  4'-6-Diamidino-2-phenylindole (DAPI) is known to form fluorescent complexes with natural double-stranded DNA.<BR>
<BR>
#If the  samples will not be processed for a while:  You need a final concentration of 4% parformaldahyde solution that will act as a preservative for the microbial cells in your soil extract samples.  Ask your instructor about this step.
# Add 5μL of 1mg/ml DAPI stock (Thermo scientific prod. # 62248) stain to 1mL of a fresh 1% diluted soil extract so that the effective concentration of DAPI is 5 μg/mL (1μg/mL may work as well).
# Incubate at 4°C for 20 min in the dark.
#Set up a vacuum flask and filter apparatus (125 ml side-arm flask,  Borosilicate base and fritted glass filter support with rubber stopper).
#Carefully place a 0.2μM glass fiber filter (Isopore membrane GTTP02500) onto the fritted glass filter support.
#Add the borosilicate glass funnel onto the base and clamp the two sections together using an anodized aluminum spring clamp. 
# Rinse the filter once with 1 mL sterile deionized water using a vacuum pump to provide 178mm Hg of force.  Wait until all the water is removed.
# Turn off the vacuum, gently break the vacuum by loosening the rubber stopper, re-tighten, and carefully transfer the 1mL of DAPI stained extract (made in step 1) onto the 0.2μM glass fiber filter.
#Turn the vacuum on and wait until all the solution is filtered.
#Rinse with 1 ml sterile deionized water to remove any background DAPI. 
# Dissassemble the funnel, gently breaking the vacuum by loosening the rubber stopper.
#Use forceps to carefully remove the filter and place it on a microscope slide.
#Add  1 drop of Fluoro-Gel with Tris buffer (Electron Microscopy Sciences Cat #17985)and a coverslip.
#Use the Nikon Eclipse 80i fluorescent microcope in L318C to view and photograph the DNA at 1000x magnification. 
#Use the Nikon NIS-Elements Imaging software, to photograph a field of view in white light (this allows you to see relative size and shape of the organisms in the sample, remember some will be eukaryotes), save this image, then do the same using the UV light at 350nm excitation wavelength (filter #1) so you can visualize the fluorescently labled DNA. 
#Save all images to a 209-2012 file folder.
# Count the unique spots of blue fluorescence; each indicates a soil microbe's genetic material (chromosome or nucleus). It is assumed that the bacteria are arranged in a Poisson distribution. For the most accurate counts, 20 fields or 400 bacteria should be enumerated to determine the number of bacteria per ml. <BR><BR>


'''COUNTING & CALCULATIONS:'''<BR>
'''COUNTING & CALCULATIONS:'''<BR>
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The area of each field of view at 1000X using the Fluorescent scope is 10487 μmeters<sup>2</sup>.  The area is determined for the microscope used, in our case,  a NIKON 80i fluorescent microscope.  The diameter of the filterable section of the borosilicate apparatus is 17 mm (8500 μmeter radius). Therefore, the area is 2.28 X 10<sup>8</sup>μmeter<sup>2</sup>.  Multiply the number of microorganisms counted on the photomicrograph by a factor of 2.17X10<sup>4</sup>  (2.28 X 10<sup>8</sup>μm<sup>2</sup> divided by 1.0487 x 10<sup>4</sup>μmeters<sup>2</sup>) to determine the number of organisms found in 1mL of filtrate of extract. Then correct for the 1:100 dilution factor of filtrate which is the number of organisms in 1 gram of wet soil.  Convert this to the number of organisms in the community in 1 gram of '''dry weight''' soil. <BR><BR>
The area of each field of view at 1000X using the Fluorescent scope is 10487 μmeters<sup>2</sup>.  The area is determined for the microscope used, in our case,  a NIKON 80i fluorescent microscope.  The diameter of the filterable section of the borosilicate apparatus is 17 mm (8500 μmeter radius). Therefore, the area is 2.28 X 10<sup>8</sup>μmeter<sup>2</sup>.  Multiply the number of microorganisms counted on the photomicrograph by a factor of 2.17X10<sup>4</sup>  (2.28 X 10<sup>8</sup>μm<sup>2</sup> divided by 1.0487 x 10<sup>4</sup>μmeters<sup>2</sup>) to determine the number of organisms found in 1mL of filtrate of extract. Then correct for the 1:100 dilution factor of filtrate which is the number of organisms in 1 gram of wet soil.  Convert this to the number of organisms in the community in 1 gram of '''dry weight''' soil. <BR><BR>


Post your mean (average of the areas of the filter counted) number of microbes/gm of soil to the spread sheet on the instructor's computer and on the board (all calculations must also be in your lab notebook!) from both the culture-dependent and culture-independent enumerations. Report them as the estimated number of microorganisms in 1gm of wet weight soil and 1gm of dry weight soil. Record these numbers in your lab notebook in scientific notation and as numbers (with an amazing number of zeros). Consider the relative insignificance of one gram of anything and the enormity of the number of microbes that thrive in a gram of soil! How is it possible that each of the microbial members of such a soil community can find a niche, obtain all the nutrients needed to grow and reproduce, and contribute to overall health of the soil and to the community of microorganisms? <BR><BR>
After each person has collected their data, post the numbers to the class spread sheet and, of course enter all calculations in your lab notebook. Record these numbers in scientific notation (and as numbers with no decimals and with an amazing number of zeros). <BR>


Now that we have some sense of the abundance of microbes in a soil community we can move on to investigating the richness (diversity) and the co-operative and competitive behaviors that maintain it.  
Now that we have some sense of the abundance of microbes in a soil community we can move on to investigating the richness (diversity) and the co-operative and competitive behaviors that maintain it.  
Janet McDonough
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