BISC209/F13: Lab3: Difference between revisions

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BISC209/F13: Lab3 (view source)

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2. '''Starch Medium (2.5%(wt/vol) soluble starch in Nutrient Agar) is used to determine the % of amalyase producing, starch digesting culturable microbes when compared to the total number counted in NA:'''<BR>
2. '''Starch Medium (2.5%(wt/vol) soluble starch in Nutrient Agar) is used to determine the % of amalyase producing, starch digesting culturable microbes when compared to the total number counted in NA:'''<BR>
Using your P200 micropipet and sterile tips, dispense 100µl of one of the soil extract dilutions into the center of each of two pre-labeled starch agar plate at the appropriate dilutions. Choose the dilution that is expected to result in 30-300 cfu/g soil. Use a sterile, disposable spreader to evenly distribute the extract aliquot. Repeat for a soil extract dilution that is 10 fold more and one 10 fold less dilute. Incubate all plates at RT until mature, visible colonies appear. Move the culture plates to the cold room before they become overgrown.<BR>
Using your P200 micropipet and sterile tips, dispense 100µl of one of the soil extract dilutions into the center of each of two pre-labeled starch agar plate at the appropriate dilutions. Choose the dilution that is expected to result in 30-300 cfu/g soil. Use a sterile, disposable spreader to evenly distribute the extract aliquot. Repeat for a soil extract dilution that is 10 fold more and one 10 fold less dilute. Incubate all plates at RT until mature, visible colonies appear. Move the culture plates to the cold room before they become overgrown.<BR>
'''ANALYSIS in LAB 4''':<BR>
Choose the dilution that shows 30-300 colonies. Flood the replicate plates with Grams Iodine. Let the plate sit for at least 10 minutes, longer is fine. The plate should turn dark blue as the iodine binds to the starch; Pour off the excess iodine (into paper towels in the biohazard bag on your bench) and wait until the remaining liquid is absorbed by the plate.  Count the colonies that show a clear halo, indicating the starch in the medium was digested by amylase secreted from the bacteria in the colony.  Count the number of colonies that are able to digest starch (clear zone around the colony). To calculate the % of culturable microbes able to digest starch/CFU in a gram of wet soil you will use the Average total number of colonies calculated from your NA plate count. Make sure that both counts were made on the same dilution. If not, you will have to do a conversion.<BR> 
Reference: Beishir, Lois.  1996. Microbiology in Practice 6th ed.  HarperCollins Publishers Inc.  New York.  Module 33: 301-306. 
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3. '''Cellulose Congo Red Agar Medium is used to determine the % of cellulolytic microbes (those producing cellulase and able to digest cellulose) '''  
3. '''Cellulose Congo Red Agar Medium is used to determine the % of cellulolytic microbes (those producing cellulase and able to digest cellulose) '''  
Tucker Crum
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