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 the Interaction Assays
Take photos of each of your interaction plates. Make sure that the photos are sharp enough to evaluate later. Download them to your dropbox in Sakai. Email them to yourself and to each of your partners.
A sample completed assay is shown below.
Observe the colonies on your plate, comparing any differences in the appearance of the colony growth of each isolate, alone vs mixed. Look first at each control culture: the inoculum in each of the diagonal spots is a pure culture control as are the spots in the column on the far left. Compare each "spot" where two isolates are mixed to the control spots where each isolate is growing alone. Is either isolate growing better in combination than alone? If so, you have found a mutualistic (beneficial) interaction. For example, in the assay shown above, isolate #102 appears to have a positive effect on the growth of #101, classified as mutualism. Are there combinations that show a reduction in the amount of growth of either isolate compared to the growth of the control areas? If so, you have found an antagonistic (negative) interaction. An example in the assay plate shown above is between isolates #105 and #102. Number 105 seems to inhibit growth of #102, an example of an antagonistic interaction. Note that there are sometimes "edge" effects, differences in the appearance of the colony growth in the cultures along the perimeter of the plate as opposed to those growing in a more protected locations (such as the diagonal control colonies). Spend some time with these plates, carefully and fully evaluating all possible combinations of your soil community isolates for as many as possible examples of mutualism or antagonism. Record your results in your lab notebook with copies of the photos of your plates.
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?
Complete the Motility & NMN Tests & Analyze the Results
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?
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 Motility Tests for directions on performing confirmation tests.
TEST for MANNITOL as a useable carbon source
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?
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.
Test for reduction of NITRATE TO NITRITE
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. 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 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.
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 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.
Study for your Lab Practical.
In Lab 9 you will have a LAB PRACTICAL, an exam that tests your ability to function as a microbiologist in the lab. There will be two main categories of assessment: testing practical skills such as your ability to perform crucial techniques (aseptic transfer, streaking for isolation, performing a Gram stain, micropipetting, etc.) and testing for your understanding of the biological basis behind the work that you've done this semester. For example: we might give you a starch plate flooded with iodine and ask you which, if any, of the bacteria forming colonies on that plate are capable of digesting starch, what metabolic enzyme is produced by these bacteria that allow that digestion and/or what is the ingredient in the medium that allows this evaluation?
Wondering how to study? A lot of what we will evaluate you on, you already know how to do in your sleep, such as streaking for isolation using proper technique. You do not have to memorize the procedures in the wiki. You will have access to the directions that are printed in the wiki for performing techniques, such as Gram staining; however, you will not have access to how to read those tests or to the theoretical background information about those tests, such as the cell structural differences that the Gram stain uses as its differentiating principle. You should know that sort of information by studying the background information and protocols found in the Protocols section of the wiki for all the work that have you performed this semester. The protocols in the wiki that describe tests that you have not done are NOT included in the practical. However, even if you didn't do or get a positive test from those tests performed on your isolates, you should know how to evaluate the tests we all set up, such as the MNM soft agar deeps, eg. how to recognize a positive vs. a negative.
Don't forget that understanding of the molecular tools we used for identifying bacteria using their 16srRNA gene sequence. What we can learn from sequencing this gene is practical material. Your homework assignment on the theory behind that work should be a good study guide for that material as is all of the background information in the wiki in LABS 1-8 and in the general information page for our project in the main menu.
Continue to wind down and complete your work on your isolates. Start discarding your cultures.