BISC209/F13: Lab8

Lab 8: Finish Tests on Cultured Isolates

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?

Structural Diversity in Cultured Isolates Con't

Complete the Motility Assessment & Analyze the Results
Soft Nutrient Agar Deep Medium
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

MOTILITY ASSESSMENT
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?

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 Motility Tests for directions on performing confirmation tests.

Assignment

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