BISC209/F13: Lab8

Finding Evidence for Co-operation and Competition Among Cultured Members of a Soil Community

Complete the Interaction Assays
If you haven't been provided with a digital image, please take a photo 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.

If you have positive antibiotic producers or evidence for interesting interactions, please show them to your lab instructor so they can be shared with the class.

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 isolates do not secrete any antimicrobial compounds? Explain?

Assessing Isolates for Physical Characteristics by Special Stains

Today you will make smear slides (Protocol found at Smear Slide Preparation) and then perform a few special stains to look for endospores and capsules. You may also want to repeat some of your Gram stains if you got ambiguous results on a previous attempt or if your EMB and PEA growth patterns conflicted with an earlier staining result.

Special Stains:
Directions for the Gram Stain, Schaeffer-Fulton Endospore stain and Capsule negative stain are found in the Protocols section of the wiki at Stains.

Detecting Endospores
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. 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?

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. A capsule is a secreted, protective, adhesive, polysacchride envelope layer outside the other parts of the cell envelope. Some, but not all bacteria produce a capsule. It tends to be a virulence factor in bacteria that are human pathogens because it makes it more difficult for the immune system to recognize the bacterium with a capsule as a foreign invader.

Repeating Gram Stains
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. If there are PEA and EMB plates available, consider reinoculating your latest cultures onto these media. Perhaps your earlier cultures were not pure? If not, what effect might this have on your genomic data? What other implications might there be?

Assignment

Graded Assignment:
Directions for this assignment can be found at: Assignment 8