BISC209/F13: Lab7: Difference between revisions

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BISC209/F13: Lab7 (view source)

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→‎LAB 7: Diversity, Co-operation and Competition Among Selected Examples of Bacteria in a Soil Community: Assessing Bacterial Interactions, Functional Roles in the Nitrogen Cycle, and Misc. Characteristics
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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 growth patterns agree?  If not, how might you explain unexpected findings?<BR><BR>
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 growth patterns agree?  If not, how might you explain unexpected findings?<BR><BR>


<font size="+1">'''Complete the Motility Assessment, Mannitol Utilization Test, Nitrate reduction test, & Analyze the Results'''</font size="+1"><BR>
<font size="+1">'''Complete the Motility Assessment & Analyze the Results'''</font size="+1"><BR>
'''Mannitol Nitrate Motility Medium'''<BR>
'''Soft Nutrient Agar Deep Medium'''<BR>
1% Casein Peptone, 0.75% Mannitol, 0.1% Potassium Nitrate, 0.004% Phenol Red, 0.35% Bacteriological Agar. pH 7.6 at 25°C <BR><BR>
Use Nutrient Agar recipe but reduce agar content to 0.35% Bacteriological Agar. pH 7.6 at 25°C and pour into sterile glass tubes<BR><BR>
<font size="+1">MOTILITY ASSESSMENT</font size="+1"><BR>
<font size="+1">MOTILITY ASSESSMENT</font size="+1"><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>
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/F13: 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/F13: Motility | Motility Tests]] for directions on performing confirmation tests.<BR><BR>
<font size="+1">TEST for MANNITOL as a useable carbon source</font size="+1"><BR>
What functional advantage would bacteria have if they are able to use mannitol as a carbon source? Would having only some soil community members possess this functional capacity be advantageous to the soil community as a whole? How so? Remember that all metabolic processes are "expensive" in terms of energy and raw materials used. Does this testing give us direct rather than theoretical evidence of a community where members have different metabolic capabilities that contribute to the success of the community? Did the assessment we did previously of community carbon source utilization patterns and diversity provide additional evidence for functional metabolic diversity? Do you understand why we did these tests as part of this investigation?<BR><BR>
The ability of an isolate to ferment mannitol as a carbon source can be assessed as a color change from red to yellow when the isolate is grown in NMN medium. The NMN medium has a pH indicator that recognizes the acidic byproducts of fermentation and show this as a color change. If this test is positive in an isolate that you originally selected on Azotobacter medium, does that mean that the isolate is more or less likely to be in the Azotobacter group of nitrogen fixing bacteria?
Note that motility and ability to use mannitol as a carbon source should be evaluated ''before'' you add the indicator reagent to the tubes to test for nitrate reduction to nitrite as described below. <BR><BR>
<font size="+1">Test for reduction of NITRATE TO NITRITE</font size="+1"><BR>
Develop the nitrate to nitrite test in the MNM 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 can't test for all possible roles each of your isolates 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. A helpful image of the Nitrogen Cycle and its players can be found at [http://www.vtaide.com/png/nitrogenCycle.htm | http://www.vtaide.com/png/nitrogenCycle.htm]<BR><BR>
'''Gries reagent''' consists of solutions:<BR>
'''Solution A'''<BR>
Sulfanilic Acid 0.8% (v/v) in Acetic Acid 5N<BR>
'''Solution B'''<BR>
Alpha-Naphthylamine (0.001% v/v)
in Acetic Acid 5N
<BR><BR>
'''Control Organisms:'''<BR>
{| border="1"
|+
! Organism !! ATCC !! Motility !! Mannitol as C source !! Nitrate to Nitrite
|-
! ''Escherichia coli''
| 25922
| +
| +
| +
|-
! ''Klebsiella pneumoniae''
| 13883
| -
| +
| +
|-
! ''Proteus mirabilis''
| 25933
| +
| -
| +
|-
! ''Acinobacter anitrartum''
| 17924
| -
| -
| -
|-
|}
<br>


==Continue Antibiotic Production test started last week==
==Continue Antibiotic Production test started last week==
Tucker Crum
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