LAB 8 Finishing the project!
This is your last wet lab! Please discard all cultures as soon as you have recorded all your results and photographed your evidence. If you have positive antibiotic producers or AI producers, or cool interactions please show them to your lab instructor so they can be shared with the class and saved. Please do not leave any cultures in the lab while we are away for break. You will receive 5 "clean-up points" if we do not find any evidence of your cultures or tests. If we have to discard and clean-up later after you, you will not receive any of those 5 points.
Finding Evidence for Co-operation and Competition Among Cultured Members of a Soil Community
Complete Tests set up last week. Take photos for visual evidence of your findings.
Observe the colonies on your plate, comparing any differences in the appearance of the colony growth of each isolate, alone vs mixed. Note that the inoculum in each of the diagonal spots is a pure culture control as are the spots in the column on the far left. Note the "edge" effect, a difference in the appearance of the colony growth in the spots along the perimeter of the plate as opposed to those growing in a more protected locations (the diagonal control colonies).
Complete Antibiotic Production & Sensitivity Testing
Examine the plates and look for evidence of a zone of inhibition (no growth or reduced growth) of any of the "control" organisms in an area near the putative antibiotic producer's colonial growth. Evidence of antibiotic production should appear as a measurable zone of inhibition (section of a circle of no growth or reduced growth compared to the growth see on the control plate). The size of the zone of inhibition is indicative of the diffusion potential of the antibiotic and/or an indication of how sensitive the test organism is to the secreted inhibitor. Compare your results to other tested isolates in your lab section. Think about why an antibiotic might work differently on a Gram positive vs. a Gram negative organism or between two bacteria that are both Gram positive or Gram negative.
Take photos of any plates that show evidence of the presence of antibiotic producers in your soil community. If you found that your isolates did not appear to cause measurable inhibition of growth, does that mean that your isolate does not secrete any antimicrobial compounds? Explain?
Motility & MNM TESTS DEVELOPMENT AND ANALYSIS
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 look at your E. coli positive control to see an example of what radiating growth may look like. Hold your NA soft agar up to the light so that you can see the difference in the medium and the growth line. Get an uninoculated tube to compare color and turbidity. Motile organisms should exhibit detectable growth radiating from the stab inoculation line towards the periphery. Strictly aerobic organisms may show more growth radiating down from the surface than farther into the stab. Non motile organisms will exhibit growth only along the stab inoculation line. Consult with your instructor if you are having a hard time deciding on the motility results. Would it be useful for some soil community members to be motile? Why? Would having motile members in a soil community be useful to non-motile members, or would that mixture be disadvantageous to one or the other, or could it be both?
The NA soft agar deep is used exclusively for motility testing. The MNM tubes can be used for motility testing too but since that medium also tests for other metabolic capabilities, your NA soft agar should be your principle test for motility. If you have time, you can try to confirm that an isolate is motile by doing a flagella stain. See the Protocols section in the wiki on Motility Tests for directions on performing a flagella stain.
TEST for MANNITOL as a sole carbon source
What functional advantage would bacteria have if they are able to use mannitol as their sole 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?
The ability of an isolate to ferment mannitol as its sole carbon source can be assessed as a color change from red to yellow. The MNM 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?
The motility test and mannitol fermentation tests should be assessed in your MNM tubes before you add the indicator reagent to test for nitrate reduction to nitrite.
Test for reduction of NITRATE TO NITRITE
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. Nitrate-negative organisms are unable to reduce nitrates and they yield no color after adding the reagent. Nitrate-positive: The appearance of a pink or red coloration indicates that the nitrates have been reduced to nitrites. Be careful about interpreting negative reactions. It is possible that nitrogen cycling bacteria that reduce nitrate to nitrite may give a negative Gries test because the nitrite produced from reduction of nitrate has been further processed and now is absent by the time we do our testing. A positive test is meaningful but a negative test may not necessarily be evidence of incapability to reduce nitrate to nitrite and, thus, to be component bacteria in the nitrogen cycle.
Gries reagent consists of solutions:
Sulfanilic Acid 0.8% (v/v) in Acetic Acid 5N
Alpha-Naphthylamine (0.001% v/v)
in Acetic Acid 5N
| Organism || ATCC || Motility || Mannitol as sole C source || Nitrate to Nitrite
| Escherichia coli
| Klebsiella pneumoniae
| Proteus mirabilis
| Acinobacter anitrartum
Assessing Bacterial Morphology: morphological features visualized by special staining techniques
Today you will make smear slides (Protocols as Smear Slide Preparation Smear Slide Preparation)) to repeat Gram stains and to perform a few special stains to look for spores, capsules, or flagella ( Stains: Gram Stain).
Directions for the Schaeffer-Fulton Endospore stain and Capsule negative stain are found in the Protocols section of the wiki.
Stains : Simple, Gram, Endospore, Capsule. The confirmatory tests for motility are found in Protocols under MOTILITY.
All Gram positive bacilli or any bacteria that showed a spore shaped, unstained area in the cells when Gram stained should be stained for endospores. In addition, any Gram positive isolates growing from your dried soil extract on Glyerol Yeast Extract Agar (GYEA) medium should be stained for endospores. There is no need to stain Gram negative isolates for endospores. Would it surprise you to know that most of the spore forming bacteria are common soil organisms? Why would the capacity to form a highly protective, heat tolerant, dessication resistant, non-metabolic spore be useful to soil community microorganisms? Would this capacity give those members a competitive advantage to survive weather extremes? Would you expect a tropical greenhouse habitat to contain relatively fewer or more spore forming members than other habitats?
Confirmatory Tests for Motility
Directions for the Hanging Drop motility test and Flagella stain can be found in the Motility section of Protocols. All bacteria that were positive or ambiguous for motility in SIM medium should be looked at by Hanging Drop technique. Any "swarmers" (bacteria that spread all over the plate when cultured on solid medium)should be looked at by Hanging Drop, too. If the hanging drop test is positive and you have time after you have performed any other confirmation tests or special stains, you could try the Flagella stain, but don't worry if you don't have time for this stain. It is VERY difficult to see flagella even when they are coated with several layers of stain reagent that make the diameter larger. It is hard to get this stain to work well.
Detecting Capsules by Negative Stain
Highly mucoid (sticky and wet) colonies could be tested for the presence of a capsule using the capsule stain protocol if you have time. If you don't have time, don't worry. This test isn't particularly useful in acquiring evidence to fulfill the investigative goals of our project. The capsule stain protocol is found in the Special Stains section of PROTOCOLS.
If your Gram stain results were ambiguous or not what you expected from the growth patterns you observed on PEA and EMB media, you should probably repeat those Gram stains.
Make a table of the tests performed on the isolates from your soil community (your isolates and your teammates from the same sampling site) and write a results narrative for it. You should include in the table: Gram stain, description of the colony morphology, description of the individual bacteria and any characteristic arrangement (cocci, rods, with descriptors ie., large, small, bullet shaped, in chains, etc.), evidence of spores (either endospore stain positive or visualization of empty areas in vegetative cells on Gram stain), quorum sensing, antibiotic production, motility, indole, and H2S. Include the nitrogen cycling tests only if they worked. You may have to omit the nitrogen cycling product tests (as if you never did this testing---leave it out of M&M, too) if you had no change in any of these tests over the time period tested. Keep in mind that the medium we used tested positive for ammonia; therefore, if you didn't see a change in ammonia level over the testing period, you may omit any mention of the test for ammonia even if you were able to see change in nitrite or nitrate and can use that part of our testing. Leave out the interaction assay in this table and make a separate results figure of a photograph of the plate and discuss it in a separate paragraph in results.
The table part of the results narrative might start out, "In order to show the potential for functional co-operation and competition among members of a soil community, methylotrophic dinitrifiers, nitrogen cycling bacteria, and spore-forming bacteria were enriched and selected using media and protocols described in Materials and Methods. After isolation in pure culture these community members were tested for AI production in a quorum sensing assay using______. Each was tested for production of diffusable antibiotic compounds against known Gram positive and Gram negative bacteria. Production of endospores were visualized in Gram stain and by Shaeffer-Fulton endospore stain. SIM (sulfate, indole, motility) testing was performed on each isolate to detect other metabolic and physical capabilities. Evidence for a role in the nitrogen cycle was assessed using________. Table 1 shows the results of this testing." After this introduction, you can then analyze the findings shown in this table in order to then answer the our experimental question, "is there evidence for functional co-operation and competition among the bacteria isolated from your soil community?"
Preliminary Graphical abstract: See models in research reports found in recent issues of the journal Cell.
A description and examples of Graphical Abstracts can be found at http://www.elsevier.com/wps/find/authorsview.authors/graphicalabstracts . Pay particular attention to example #15 or those that are less molecular and more topically ecological. Because you will not create phylogenetic tree until after we analyze the sequencing data in LAB 9, you may leave a place holder for such an image (if you choose to use this type of data in your graphical abstract) and add it later. There is a folder in Resources in Sakai, called Images. Your instructor has uploaded images of the Wellesley Greenhouses including the Tropical room that you may use if you wish. NOTE that these images are available as an OPTION. It is not required to use them or even suggested that any be part of your graphical abstract!