Finishing the term project
After break the labs will be focused on data analysis and the lab practical. This is a good time to begin discarding all cultures unless you need them for some test you were unable to complete. Please do not leave your cultures in the lab while we are away for break. If you want to repeat or need to finish any test, there is still time for you to do this but please leave subcultures made today in a designated plate holder so that I can put them in the cold room for you before Friday.
If you have interesting data today, please show it to your lab instructor as she will want to share it with the class.
Finding Evidence for Co-operation and Competition Among Cultured Members of a Soil Community
Complete Quorum Sensing & Mutualistic and Antagonistic Interactions Tests set up last week
THis is the last wet-lab for this term.
Analyze your quorum sensing and interactions assays. Take photos for visual evidence of your findings.
If the isolate produces AI and it is sensed by the CV026 mutant, a purple pigment will be produced by the mutant. What does this tell you about your isolate? Is there any reason to test the Gram positive isolates? Why or why not?
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 the diagonal spots is actually a single (hopefully pure) isolate. 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).
Did anyone see a change in the levels of nitrate, nitrite, or ammonia? Be sure you record the results on the team data sheet and post it to your team's DATA folder in Resources in Sakai so your teammates can access it. Include a column or row on your data sheet to record observations (such as when you detect evidence of growth by seeing an increase in turbidity compared to the McFarland standard, any change in smell, color, or appearance of the cultures). Discuss in with your team members the implications of your data. If you have no positive data, how might you explain this?
Add a few drops of Kovaks reagent to one tube for each organism tested by your team. Is there any evidence that some of these organisms have the enzyme Tryptophanase?
Complete Antibiotic Production & Sensitivity Testing
Examine the plates and look for evidence of a zone of inhibition (no growth) of "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). 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.
Take photos of any plates that show evidence 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? Why or why not?
SIM TESTS DEVELOPMENT AND ANALYSIS
Motility detection is possible due to the semisolid nature (low concentration of agar) of the SIM medium. Growth radiating out from the central stab inoculation line indicates that the test organism is motile. The motility test should be assessed first. Motile organisms will exhibit growth radiating from the stab inoculation line. Non motile organisms will exhibit growth only along the stab inoculation line. 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?
TEST FOR TRYPTOPHANASE and SULFUR REDUCTION
What functional advantage would bacteria have if they are positive for tryptophanase or able to produce hydrogen sulfite? Would having some soil community members with these functional capacities obviate the need for other members to have the same enzymes? 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? Will direct evidence that community members have the capacity to perform disparate roles be useful in answering one or more of your main investigative questions? Did the assessment we did previously of other exoenzymes and their relative prevalence in your soil community provide direct evidence for functional metabolic diversity as well, better, or just differently than the evidence you obtained from the SIM tests? Do you understand why we are doing these tests as part of this investigation?
The ingredients in SIM (sulfide-indole-motility) medium enable detection of two such metabolic capabilities that some bacteria have and others lack: digestion of tryptophan by the enzyme tryptophanase to indole and/or sulfur reduction with the production of hydrogen sulfide. SIM medium contains nutrients, iron, and sodium thiosulfate.
The indole test is used for detecting tryptophanase. Casein peptone is rich in tryptophan, which is attacked by certain microorganisms resulting in the production of indole. Bacteria possessing the enzyme tryptophanase cleave tryptophan, producing three end products. One of these end products is indole, produced in aerobic conditions; another is skatole, produced in anaerobic conditions. Amyl alcohol in Kovacs reagent acts as a solvent for indole or skatole, which then reacts with p-dimethylaminobenzaldehyde to produce a red rosindole dye. Organisms which do not produce tryptophanase produce no color change in SIM medium when Kovacs is added while bacteria positive for tryptophanase produce a red color when Kovacs reagent is added.
PROTOCOL for DEVELOPING AN INDOLE TEST
To detect indole production due to the enzyme tryptophanase, add three or four drops of Kovacs’ reagent and observe the fluid for development of a ring of red color(positive reaction)at the top of the tube.
DETECTING A POSITIVE H2S test:
The hydrogen sulfide test relies on the use of sodium thiosulfate and ferrous ammonium sulfate as indicators of hydrogen sulfide production. H2S reacts with sodium thiosulfate to produce ferrous sulfide, a black precipitate.
An insoluble black precipitate is seen as a positive result.
Approximate Formula* Per Liter
Pancreatic Digest of Casein - 20.0 g
Peptic Digest of Animal Tissue - 6.1 g
Ferrous Ammonium Sulfate - 0.2 g
Sodium Thiosulfate - 0.2 g
Agar - 3.5 g
Kovac's reagent: (per liter)
Amyl Alcohol 750.0 ml
Hydrochloric acid 250.0 ml
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.
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!
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