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BISC209: Enrichment: Difference between revisions
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→Enrichment media to isolate specific soil bacterial community members based on functional roles(Starting with either SOIL or SOIL EXTRACT)
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Agitate on a stir plate for 30 minutes.<li> | Agitate on a stir plate for 30 minutes.<li> | ||
Allow the large particles to settle to the bottom of the flask for 30 minutes.<li> | Allow the large particles to settle to the bottom of the flask for 30 minutes.<li> | ||
Soak a sterile cotton swab in the broth. < | Soak a sterile cotton swab in the broth. <BR><BR> | ||
'''Isolation:'''<BR> | '''Isolation:'''<BR> | ||
Swab section 1 of a labeled plate of glycerol yeast medium (GYM) using your best isolation streak technique<br> | Swab section 1 of a labeled plate of glycerol yeast medium (GYM) using your best isolation streak technique<br> | ||
Allow the inoculum in section 1 to absorb into the agar before you, < | Allow the inoculum in section 1 to absorb into the agar before you, <li> | ||
Follow the steps for [[BISC209/S11: Streaking for Isolation | Streaking for Isolation ]].< | Follow the steps for [[BISC209/S11: Streaking for Isolation | Streaking for Isolation ]].<li> | ||
Invert, and incubate the plate at RT. < | Invert, and incubate the plate at RT. <li> | ||
Check your plate for colonies ''daily''. When well-isolated candidate colonies appear, use the tip of a sterile toothpick to pick up a small but visible amount of growth, being careful not to touch anything but the tip of the colony.< | Check your plate for colonies ''daily''. When well-isolated candidate colonies appear, use the tip of a sterile toothpick to pick up a small but visible amount of growth, being careful not to touch anything but the tip of the colony.<li> | ||
Isolation streak any interesting colonies (preferentially chose those that appear like "little volcanos" or "powdered sugar") onto new glycerol yeast plates (one colony/plate) using your flame sterilized inoculating loop after you have applied the growth from the toothpick to zone one of your streak plate. < | Isolation streak any interesting colonies (preferentially chose those that appear like "little volcanos" or "powdered sugar") onto new glycerol yeast plates (one colony/plate) using your flame sterilized inoculating loop after you have applied the growth from the toothpick to zone one of your streak plate. <li> | ||
''Actinomycetes'' and ''Streptomycetes'' are often tough leathery colonies, so transfer of these colonies is sometimes difficult. The powdery area may indicate spore formation: take a sample from this area, if possible. In any case, try to "break off" a piece of the colony with your sterile loop or with a sterile toothpick and transfer that piece of a colony to zone one of the new medium and then use your loop for streaking out the other zones. The tiny spores on the surface of the colony are likely to transfer to the next plate or tube when you work with it. (That's a good thing this time.) | ''Actinomycetes'' and ''Streptomycetes'' are often tough leathery colonies, so transfer of these colonies is sometimes difficult. The powdery area may indicate spore formation: take a sample from this area, if possible. In any case, try to "break off" a piece of the colony with your sterile loop or with a sterile toothpick and transfer that piece of a colony to zone one of the new medium and then use your loop for streaking out the other zones. The tiny spores on the surface of the colony are likely to transfer to the next plate or tube when you work with it. (That's a good thing this time.) | ||
<BR><BR> | <BR><BR> | ||
'''Identification:<BR>''' | '''Identification:<BR>''' | ||
These colonies may be slow growing, so check your plates every few days for up to 2 weeks. < | These colonies may be slow growing, so check your plates every few days for up to 2 weeks. <li> | ||
Incubate. | Incubate. | ||
< | <li> | ||
Look for hard, white, ridged colonies (little "volcanoes")characteristic of ''Streptomyces'' or "powdered sugar" colonies with an indentation of agar around the colony.< | Look for hard, white, ridged colonies (little "volcanoes")characteristic of ''Streptomyces'' or "powdered sugar" colonies with an indentation of agar around the colony.<li> | ||
Once you have relatively pure isolates without contaminating fungal growth, make a bacterial smear slide and Gram stain it(see Protocols for procedures)begin to examine the cellular morphology, arrangement, and cell wall structure of these bacteria. Look carefully for clear areas in the vegetative cells indicative of endospores. You will do an endospore stain in a later lab on any isolates that grow from this enrichment, but look carefully for this preliminary indication of endospores. | Once you have relatively pure isolates without contaminating fungal growth, make a bacterial smear slide and Gram stain it(see Protocols for procedures)begin to examine the cellular morphology, arrangement, and cell wall structure of these bacteria. Look carefully for clear areas in the vegetative cells indicative of endospores. You will do an endospore stain in a later lab on any isolates that grow from this enrichment, but look carefully for this preliminary indication of endospores. | ||
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