BISC209/F13: Lab3: Difference between revisions

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BISC209/F13: Lab3 (view source)

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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) '''  
Using your P200 micropipet and sterile tips, dispense 100µl of one of the soil extract dilutions into the center of each of the pre-labeled cellulose congo red agar replicate plates. 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. The ashed, acid-washed cellulose and the Congo red in the medium will allow enumeration of cellulose-digesting bacteria in soil. 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 the pre-labeled cellulose congo red agar replicate plates. 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. The ashed, acid-washed cellulose and the Congo red in the medium will allow enumeration of cellulose-digesting bacteria in soil. 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. Count the colonies on the replicate plates that show a clear halo, indicating the cellulose in the medium was digested by cellulase secreted from the bacteria in the colony.  The red color of the medium provides increased contrast between the cellulose containing medium and the halo or clear zone that indicates that cellulase has diffused out of bacterial producers and degraded the cellulose in the immediate vicinity. Count the number of colonies on the plate that are able to digest cellulose (clear zone around the colony). To calculate the % of cellulolytic microbes/CFU in a gram of wet soil you will use the 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>
'''Cellulose Congo Red Agar: '''<BR>
'''Cellulose Congo Red Agar: '''<BR>
0.05% K2HPO4; 0.025% MgSo4; 0.188% ashed, acid washed cellulose powder; 0.02% Congo red, 1.0% Noble Agar, 0.2% gelatin, 10%(vol/vol) sterile soil extract(10.5% air-dried, sieved soil that's autoclaved twice, allowed to settle, and the supernatant filtered); final pH 7.0 - 8.0. <BR>
0.05% K2HPO4; 0.025% MgSo4; 0.188% ashed, acid washed cellulose powder; 0.02% Congo red, 1.0% Noble Agar, 0.2% gelatin, 10%(vol/vol) sterile soil extract(10.5% air-dried, sieved soil that's autoclaved twice, allowed to settle, and the supernatant filtered); final pH 7.0 - 8.0. <BR>
Reference: Hendricks, C.W., Doyle, J.D., Hugley, B.  (1995)  A new solid medium for enumerating cellulose-utilizing bacteria in soil.  Applied and Environmental Microbiology ''61'', 2016-2010.  <BR><BR>
Reference: Hendricks, C.W., Doyle, J.D., Hugley, B.  (1995)  A new solid medium for enumerating cellulose-utilizing bacteria in soil.  Applied and Environmental Microbiology ''61'', 2016-2010.  <BR><BR>


4. '''Phosphate Medium (Pidovskaya medium [PVK]) is used to determine the % phosphate solubilizing microbes (those producing phosphatases) in a soil community:''' <BR>
4.   '''Phosphate Medium (Pidovskaya medium [PVK]) is used to determine the % phosphate solubilizing microbes (those producing phosphatases) in a soil community:''' <BR>


Using your P200 micropipet and sterile tips, dispense 100µl of one of the soil extract dilutions into the center of the two pre-labeled Phosphate medium (PVK) replicate plates. 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 the two pre-labeled Phosphate medium (PVK) replicate plates. 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. Count the colonies that show a clear halo on each of the replicate plates, indicating the phosphate in the medium was solubilized and processed by enzymes secreted from the bacteria in the colony.  Count the number of colonies that are able to solubilize phosphate (clear zone around the colony). To calculate the % of culturable, phosphate solubilizing microbes /CFU in a gram of wet soil you will use the 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>
'''Pidovskaya Medium Modified (Nautiyal, 1999)''' <BR>
'''Pidovskaya Medium Modified (Nautiyal, 1999)''' <BR>
1% glucose; 0.5% Calcium Phosphate [Ca<sub>3</sub>(PO<sub>4</sub>)<sub>2</sub>; 1% MgCl<sub>2</sub>.6H<sub>2</sub>O; 0.25% Magnesium Sulfate (MgSO<sub>4</sub>.7H20);0.2% (NH4)2SO4; 0.25% KCL; 0.0025% BromoPhenol Blue; 1.5-2% agar <BR>
1% glucose; 0.5% Calcium Phosphate [Ca<sub>3</sub>(PO<sub>4</sub>)<sub>2</sub>; 1% MgCl<sub>2</sub>.6H<sub>2</sub>O; 0.25% Magnesium Sulfate (MgSO<sub>4</sub>.7H20);0.2% (NH4)2SO4; 0.25% KCL; 0.0025% BromoPhenol Blue; 1.5-2% agar <BR>
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