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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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'''Glycerol Yeast Extract Agar (GYEA)''': a selective medium to enrich for many of the spore forming, antibiotic producing bacteria in the ''Actinomycetes'', Bacillus, and other groups of Gram positive spore formers.<UL><LI> | '''Glycerol Yeast Extract Agar (GYEA)''': a selective medium to enrich for many of the spore forming, antibiotic producing bacteria in the ''Actinomycetes'', Bacillus, and other groups of Gram positive spore formers.<UL><LI> | ||
Your instructors will heat dessicate (oven bake) your 3 one gram soil samples collected and weighed in LAB1 and return them to you in LAB2. You will use this heat shocked, dry soil sample to make a new soil extract for this protocol, which is based on spore resistance to dessication. Drying and heating the sample has encouraged spore generating bacteria to form a state that will allow them to survive harsh environmental conditions while killing off many of the microbes that can't make spores or survive the heat or lack of moisture. Now we need to coax those spores back into their vegetative state. The medium uses cycloheximide to inhibit fungal growth since many fungi make spores, too. | Your instructors will heat dessicate (oven bake) your 3 one gram soil samples collected and weighed in LAB1 and return them to you in LAB2. You will use this heat shocked, dry soil sample to make a new soil extract for this protocol, which is based on spore resistance to dessication. Drying and heating the sample has encouraged spore generating bacteria to form a state that will allow them to survive harsh environmental conditions while killing off many of the microbes that can't make spores or survive the heat or lack of moisture. Now we need to coax those spores back into their vegetative state. The medium uses cycloheximide to inhibit fungal growth since many fungi make spores, too. | ||
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Invert, and incubate the plate at RT. <li> | 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.<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.<li> | ||
Isolation streak any interesting colonies (preferentially chose those that appear like "little volcanos") 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.) | |||
<LI> | |||
'''Identification:<BR>'''<LI> | |||
These colonies may be slow growing, so check your plates every few days for up to 2 weeks. <LI> <LI> | |||
Incubate.<li> | Incubate.<li> | ||
'''Identification:'''<BR> | '''Identification:'''<BR> | ||
Look for hard, white, ridged colonies (little "volcanoes")characteristic of ''Streptomyces'' | Look for hard, white, ridged colonies (little "volcanoes")characteristic of ''Streptomyces'' or "powdered sugar" colonies with an indentation of agar around the colony. | ||
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, make a bacterial smear slide and Gram stain it(see Protocols for procedures)begin to examine the cellular morphology, arrangement, and cell wall structure. 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. | |||
</li></UL></LI></UL> | </li></UL></LI></UL> | ||
