BISC209/S12: Lab7 - Revision history

Tucker Crum: /* Complete the Motility & MNM Tests & Analyze the Results */

2012-03-14T17:47:01Z

Complete the Motility & MNM Tests & Analyze the Results

←Older revision		Revision as of 17:47, 14 March 2012
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	<BR>		<BR>
	'''MOTILITY'''<BR>		'''MOTILITY'''<BR>
-	Look for radiating growth around the stab line of inoculation of each isolate in each of your soft agar deeps. Motility detection is possible due to the semisolid nature (low concentration of agar) of these soft agar deeps. '''Growth radiating out from the central stab inoculation line indicates that the test organism is motile.''' First hold an ''E. coli'' positive control tube up to the light to see an example of radiating growth. Growth appears cloudier than the medium. Compare your positive control to an uninoculated tube and to a negative control culture of a non-motile organism ~~that your instructor has prepared~~. Non-motile bacteria exhibit growth in a tighter, defined line limited to where the organism was inoculated. In contrast, motile organisms exhibit detectable growth radiating away from the stab inoculation line towards the periphery. Strictly aerobic organisms may show more growth radiating down from the surface of the medium compared to the growth deep in the tube. Consult with your instructor if you are having a hard time deciding whether or not your isolates are motile. Why might it be useful for some soil community members to be motile? <BR><BR>	+	Look for radiating growth around the stab line of inoculation of each isolate in each of your soft agar deeps. Motility detection is possible due to the semisolid nature (low concentration of agar) of these soft agar deeps. '''Growth radiating out from the central stab inoculation line indicates that the test organism is motile.''' First hold an ''E. coli'' positive control tube up to the light to see an example of radiating growth. Growth appears cloudier than the medium. Compare your positive control to an uninoculated tube and to a negative control culture of a non-motile organism. Non-motile bacteria exhibit growth in a tighter, defined line limited to where the organism was inoculated. In contrast, motile organisms exhibit detectable growth radiating away from the stab inoculation line towards the periphery. Strictly aerobic organisms may show more growth radiating down from the surface of the medium compared to the growth deep in the tube. Consult with your instructor if you are having a hard time deciding whether or not your isolates are motile. Why might it be useful for some soil community members to be motile? <BR><BR>

	If you have time, you can try to confirm a positive preliminary motility test by doing a hanging drop motility wet mount or a flagella stain. See the Protocols section in the wiki on [[BISC209/S12: Motility \| Motility Tests]] for directions on performing confirmation tests.<BR><BR>		If you have time, you can try to confirm a positive preliminary motility test by doing a hanging drop motility wet mount or a flagella stain. See the Protocols section in the wiki on [[BISC209/S12: Motility \| Motility Tests]] for directions on performing confirmation tests.<BR><BR>

Tucker Crum: /* Complete the Motility & MNM Tests & Analyze the Results */

2012-03-14T17:20:12Z

Complete the Motility & MNM Tests & Analyze the Results

←Older revision		Revision as of 17:20, 14 March 2012
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	Look for radiating growth around the stab line of inoculation of each isolate in each of your soft agar deeps. Motility detection is possible due to the semisolid nature (low concentration of agar) of these soft agar deeps. '''Growth radiating out from the central stab inoculation line indicates that the test organism is motile.''' First hold an ''E. coli'' positive control tube up to the light to see an example of radiating growth. Growth appears cloudier than the medium. Compare your positive control to an uninoculated tube and to a negative control culture of a non-motile organism that your instructor has prepared. Non-motile bacteria exhibit growth in a tighter, defined line limited to where the organism was inoculated. In contrast, motile organisms exhibit detectable growth radiating away from the stab inoculation line towards the periphery. Strictly aerobic organisms may show more growth radiating down from the surface of the medium compared to the growth deep in the tube. Consult with your instructor if you are having a hard time deciding whether or not your isolates are motile. Why might it be useful for some soil community members to be motile? <BR><BR>		Look for radiating growth around the stab line of inoculation of each isolate in each of your soft agar deeps. Motility detection is possible due to the semisolid nature (low concentration of agar) of these soft agar deeps. '''Growth radiating out from the central stab inoculation line indicates that the test organism is motile.''' First hold an ''E. coli'' positive control tube up to the light to see an example of radiating growth. Growth appears cloudier than the medium. Compare your positive control to an uninoculated tube and to a negative control culture of a non-motile organism that your instructor has prepared. Non-motile bacteria exhibit growth in a tighter, defined line limited to where the organism was inoculated. In contrast, motile organisms exhibit detectable growth radiating away from the stab inoculation line towards the periphery. Strictly aerobic organisms may show more growth radiating down from the surface of the medium compared to the growth deep in the tube. Consult with your instructor if you are having a hard time deciding whether or not your isolates are motile. Why might it be useful for some soil community members to be motile? <BR><BR>

-	If you have time, you can try to confirm a positive preliminary ~~motilility~~ test by doing a hanging drop motility wet mount or a flagella stain. See the Protocols section in the wiki on [[BISC209/S12: Motility \| Motility Tests]] for directions on performing confirmation tests.<BR><BR>	+	If you have time, you can try to confirm a positive preliminary motility test by doing a hanging drop motility wet mount or a flagella stain. See the Protocols section in the wiki on [[BISC209/S12: Motility \| Motility Tests]] for directions on performing confirmation tests.<BR><BR>

	'''TEST for MANNITOL as a useable carbon source '''<BR>		'''TEST for MANNITOL as a useable carbon source '''<BR>

Tucker Crum: /* Complete the Motility & NMN Tests & Analyze the Results */

2012-03-14T17:19:48Z

Complete the Motility & NMN Tests & Analyze the Results

←Older revision		Revision as of 17:19, 14 March 2012
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	Did each of your isolates grow on PEA or EMB? What does that result mean about the isolate's cell wall composition? Do your Gram stain findings and PEA and EMB data agree? If not, what ideas can you generate to explain discrepancies?		Did each of your isolates grow on PEA or EMB? What does that result mean about the isolate's cell wall composition? Do your Gram stain findings and PEA and EMB data agree? If not, what ideas can you generate to explain discrepancies?

-	==Complete the Motility & ~~NMN~~ Tests & Analyze the Results==	+	==Complete the Motility & MNM Tests & Analyze the Results==
	'''Mannitol Nitrate Motility Medium'''<BR>		'''Mannitol Nitrate Motility Medium'''<BR>

Janet McDonough: /* Antagonistic and Mutualistic Interactions */

2012-03-13T18:05:50Z

Antagonistic and Mutualistic Interactions

←Older revision		Revision as of 18:05, 13 March 2012
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	[[Image:Interactions_slide4.jpg]] <BR>		[[Image:Interactions_slide4.jpg]] <BR>

-	Beginning with Isolate #2, inoculate a second 50 μl of each of your isolates into the column wells B2, B3, etc. (indicated by the green color).	+	Beginning with Isolate #2, inoculate a second 50 μl of each of your isolates into the column wells B1, C1, etc. (indicated by the green color).
	<BR>		<BR>
	Add 100 μL of nutrient broth to each of the wells containing your isolates (row wells A1-A8 and column wells B1-H1)		Add 100 μL of nutrient broth to each of the wells containing your isolates (row wells A1-A8 and column wells B1-H1)

Tucker Crum: /* Complete the Motility & NMN Tests & Analyze the Results */

2012-03-12T19:17:39Z

Complete the Motility & NMN Tests & Analyze the Results

←Older revision		Revision as of 19:17, 12 March 2012
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	'''Test for reduction of NITRATE TO NITRITE'''<BR>		'''Test for reduction of NITRATE TO NITRITE'''<BR>
-	Develop the nitrate to nitrite test in the NMN tube by adding Gries reagent (2 drops of solution A, and then 2 drops of the solution B) to the surface of the medium. Nitrite-positive: The appearance of a pink or red coloration indicates that the nitrates in the medium have been reduced to nitrites. Be careful about interpreting negative reactions as evidence that the organism does not contribute to the nitrogen cycle. We already know that some of these bacteria perform at least one specific role in this crucial cycle. How? Hint: Think about the selective media you used to enrich for nitrogen fixers and ammonium users. Those media provided highly limited nitrogen sources. ~~However,~~ for your isolates that weren't selected on Azotobacter medium or Simmons citrate~~, we don~~'t ~~know what~~ roles they might contribute to this cycle. It is possible for bacteria that reduce nitrate to nitrite to give a negative Gries test because the nitrite produced from reduction of nitrate has been further processed and is gone by the time you do your testing. A positive test is meaningful but a negative test may not necessarily be evidence of incapability to reduce nitrate. No color change: Either the organism was unable to reduce the nitrate in the medium to nitrite or the nitrite was reduced to ammonia.<BR><BR>	+	Develop the nitrate to nitrite test in the NMN tube by adding Gries reagent (2 drops of solution A, and then 2 drops of the solution B) to the surface of the medium. Nitrite-positive: The appearance of a pink or red coloration indicates that the nitrates in the medium have been reduced to nitrites. Be careful about interpreting negative reactions as evidence that the organism does not contribute to the nitrogen cycle. We already know that some of these bacteria perform at least one specific role in this crucial cycle. How? Hint: Think about the selective media you used to enrich for nitrogen fixers and ammonium users. Those media provided highly limited nitrogen sources. We have less information about nitrogen cycle contribution for your isolates that weren't selected on Azotobacter medium or Simmons citrate. We aren't testing for all possible roles they might contribute to this cycle. The Gries reagent test on those bacteria grown in MNM may give us evidence of one possible role they play, however, it is possible for bacteria that reduce nitrate to nitrite to give a negative Gries test because the nitrite produced from reduction of nitrate has been further processed and is gone by the time you do your testing. A positive test is meaningful but a negative test may not necessarily be evidence of incapability to reduce nitrate. No color change: Either the organism was unable to reduce the nitrate in the medium to nitrite or the nitrite was reduced to ammonia.<BR><BR>
	'''Gries reagent''' consists of solutions:<BR>		'''Gries reagent''' consists of solutions:<BR>
	'''Solution A'''<BR>		'''Solution A'''<BR>

Tucker Crum: /* Complete the Motility & NMN Tests & Analyze the Results */

2012-03-12T19:14:24Z

Complete the Motility & NMN Tests & Analyze the Results

←Older revision		Revision as of 19:14, 12 March 2012
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	'''Test for reduction of NITRATE TO NITRITE'''<BR>		'''Test for reduction of NITRATE TO NITRITE'''<BR>
-	Develop the nitrate to nitrite test in the NMN tube by adding Gries reagent (2 drops of solution A, and then 2 drops of the solution B) to the surface of the medium. Nitrite-positive: The appearance of a pink or red coloration indicates that the nitrates in the medium have been reduced to nitrites. Be careful about interpreting negative reactions as evidence that the organism does not contribute to the nitrogen cycle. It is possible for bacteria that reduce nitrate to nitrite to give a negative Gries test because the nitrite produced from reduction of nitrate has been further processed and is gone by the time you do your testing. A positive test is meaningful but a negative test may not necessarily be evidence of incapability to reduce nitrate ~~to nitrite~~. No color change: Either the organism was unable to reduce the nitrate in the medium to ~~Nitrite~~ or the ~~Nitrite~~ was reduced to ammonia.<BR><BR>	+	Develop the nitrate to nitrite test in the NMN tube by adding Gries reagent (2 drops of solution A, and then 2 drops of the solution B) to the surface of the medium. Nitrite-positive: The appearance of a pink or red coloration indicates that the nitrates in the medium have been reduced to nitrites. Be careful about interpreting negative reactions as evidence that the organism does not contribute to the nitrogen cycle. We already know that some of these bacteria perform at least one specific role in this crucial cycle. How? Hint: Think about the selective media you used to enrich for nitrogen fixers and ammonium users. Those media provided highly limited nitrogen sources. However, for your isolates that weren't selected on Azotobacter medium or Simmons citrate, we don't know what roles they might contribute to this cycle. It is possible for bacteria that reduce nitrate to nitrite to give a negative Gries test because the nitrite produced from reduction of nitrate has been further processed and is gone by the time you do your testing. A positive test is meaningful but a negative test may not necessarily be evidence of incapability to reduce nitrate. No color change: Either the organism was unable to reduce the nitrate in the medium to nitrite or the nitrite was reduced to ammonia.<BR><BR>
	'''Gries reagent''' consists of solutions:<BR>		'''Gries reagent''' consists of solutions:<BR>
	'''Solution A'''<BR>		'''Solution A'''<BR>

Janet McDonough: /* Confirmation of Gram stain results by Selective/Differential Media: */

2012-03-12T19:09:17Z

Confirmation of Gram stain results by Selective/Differential Media:

←Older revision		Revision as of 19:09, 12 March 2012
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	==Confirmation of Gram stain results by Selective/Differential Media:==		==Confirmation of Gram stain results by Selective/Differential Media:==
-	Did each of your isolates grow on PEA or EMB? What does that result mean about the isolate's cell wall composition? Do your Gram stain findings and PEA and EMB data agree? If not what ideas can you generate to explain discrepancies?	+	Did each of your isolates grow on PEA or EMB? What does that result mean about the isolate's cell wall composition? Do your Gram stain findings and PEA and EMB data agree? If not, what ideas can you generate to explain discrepancies?

	==Complete the Motility & NMN Tests & Analyze the Results==		==Complete the Motility & NMN Tests & Analyze the Results==

Janet McDonough: /* Confirmation of Gram stain results by Selective/Differential Media: */

2012-03-12T19:09:01Z

Confirmation of Gram stain results by Selective/Differential Media:

←Older revision		Revision as of 19:09, 12 March 2012
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	==Confirmation of Gram stain results by Selective/Differential Media:==		==Confirmation of Gram stain results by Selective/Differential Media:==
-	Did each of your isolates grow on PEA or EMB? What does that result mean about the isolate's cell wall composition? Do your Gram stain findings and PEA and EMB data agree?	+	Did each of your isolates grow on PEA or EMB? What does that result mean about the isolate's cell wall composition? Do your Gram stain findings and PEA and EMB data agree? If not what ideas can you generate to explain discrepancies?

	==Complete the Motility & NMN Tests & Analyze the Results==		==Complete the Motility & NMN Tests & Analyze the Results==

Tucker Crum: /* Antagonistic and Mutualistic Interactions */

2012-03-12T19:03:20Z

Antagonistic and Mutualistic Interactions

←Older revision		Revision as of 19:03, 12 March 2012
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	If the frogger is not available, use an 8 channel multichannel pipet set to 5µl and remove 5μL of culture from each well of your culture dish and deposit all of it onto an area of the NA square agar NUNC plate that is in the same location as in the 96 well culture dish. Again, be sure the tips are on tightly before loading the pipet. Repeat this procedure, with new tips, for each ROW of 8 wells until you have completed depositing the full array in the same orientation as the 48 wells.<br><BR>		If the frogger is not available, use an 8 channel multichannel pipet set to 5µl and remove 5μL of culture from each well of your culture dish and deposit all of it onto an area of the NA square agar NUNC plate that is in the same location as in the 96 well culture dish. Again, be sure the tips are on tightly before loading the pipet. Repeat this procedure, with new tips, for each ROW of 8 wells until you have completed depositing the full array in the same orientation as the 48 wells.<br><BR>
-
-	7. While the first plate is drying, you will follow the complete instructions twice more using your 4 isolates and the 4 isolates of each of your remaining partners. You will end with a total of 3 NUNC plates. You can discard the 96 well plates, they were only needed to mix the cultures.
-
-	8. Wait for your inoculated spots to dry, seal or cover the NUNC square tray, and incubate at Room Temp for a week. The 96 well plate was used only to mix the cultures so you can discard this in the appropriate manner. You will check on your assay and note any differences in the appearance of the colony growth of each isolate, alone vs mixed next lab. <br>
-
-


		+	7. Wait for your inoculated spots to dry, seal or cover the NUNC square tray, and incubate at Room Temp for a week. The 96 well plate was used only to mix the cultures so you can discard this in the appropriate manner. You will check on your assay and note any differences in the appearance of the colony growth of each isolate, alone vs mixed next lab. <br>

	==CLEAN UP==		==CLEAN UP==

Janet McDonough: /* Complete the Motility & NMN Tests & Analyze the Results */

2012-03-12T18:48:07Z

Complete the Motility & NMN Tests & Analyze the Results

←Older revision		Revision as of 18:48, 12 March 2012
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	'''Test for reduction of NITRATE TO NITRITE'''<BR>		'''Test for reduction of NITRATE TO NITRITE'''<BR>
-	Develop the nitrate to nitrite test in the NMN tube by adding Gries reagent (2 drops of solution A, and then 2 drops of the solution B) to the surface of the medium. ~~Nitrate-negative organisms are unable to reduce nitrates and they yield no color after adding the reagent.~~ Nitrite-positive: The appearance of a pink or red coloration indicates that the nitrates have been reduced to nitrites. Be careful about interpreting negative reactions as evidence that the organism does not contribute to the nitrogen cycle. It is possible for bacteria that reduce nitrate to nitrite to give a negative Gries test because the nitrite produced from reduction of nitrate has been further processed and is gone by the time you do your testing. A positive test is meaningful but a negative test may not necessarily be evidence of incapability to reduce nitrate to nitrite. No color change: Either the organism was unable to reduce the nitrate in the medium to Nitrite or the Nitrite was reduced to ammonia.<BR><BR>	+	Develop the nitrate to nitrite test in the NMN tube by adding Gries reagent (2 drops of solution A, and then 2 drops of the solution B) to the surface of the medium. Nitrite-positive: The appearance of a pink or red coloration indicates that the nitrates in the medium have been reduced to nitrites. Be careful about interpreting negative reactions as evidence that the organism does not contribute to the nitrogen cycle. It is possible for bacteria that reduce nitrate to nitrite to give a negative Gries test because the nitrite produced from reduction of nitrate has been further processed and is gone by the time you do your testing. A positive test is meaningful but a negative test may not necessarily be evidence of incapability to reduce nitrate to nitrite. No color change: Either the organism was unable to reduce the nitrate in the medium to Nitrite or the Nitrite was reduced to ammonia.<BR><BR>
	'''Gries reagent''' consists of solutions:<BR>		'''Gries reagent''' consists of solutions:<BR>
	'''Solution A'''<BR>		'''Solution A'''<BR>