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Jenna M. Lang added Isolation.md
about 10 years ago
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#Isolation
##Swab
Using a sterile cotton swab, swab the area you intend to sample for 10 to 15 seconds, as if you were trying to clean the area. Try to rotate the swab to ensure that all sides touch the surface.
##Plate
Gently (so as not to break the agar surface) streak the swab across the entire surface of an agar plate. Be sure to rotate the swab as you are doing so to ensure that all sides of the swab make contact with the plate. Incubate the plate at the desired temperature (usually 37C or room temperature) for 1-3 days.
##Dilution Streak (streaking for individual colonies) X2
After incubation, choose desired colonies (we typically attempt to maximize the diversity of colony morphologies) and dilution streak them onto individual plates. For a review of dilution streaking technique see for example:
http://www.umsl.edu/~microbes/streakplates.pdf
After growth to visible colonies, repeat the dilution streaking to help ensure purity of the culture. If you have access to a X microscope, visually inspect a smear of cells to confirm a single cell type. Some organisms will only grow in tight association with others, and a mixed culture will prove difficult to work with. If no microscope is available, just proceed with the overnight culture.
##Liquid Culture
After the second dilution streaking, a liquid culture is needed both for long-term storage and for DNA extraction. Pick a single colony from each dilution streak plate into 5mls of culture media and grow for 1-3 days until cloudy. Once the liquid culture is ready, prepare a 10% final concentration glycerol stock for long-term storage at -80C from 2mL of the sample.
