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| Use both Glycerol Yeast Extract Media and Arginine-Glycerol-Salt (AGS) medium (AGS)'''</font color="purple"><BR> | | Use both Glycerol Yeast Extract Media and Arginine-Glycerol-Salt (AGS) medium (AGS)'''</font color="purple"><BR> |
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| '''It is important that we isolate representatives of this diverse group of organisms so make sure both soil samples from your habitat are enriched for ''Actinomycetes'' using both of the media described, Glycerol Yeast and AGS.''' Most of the ''Actinomycetes'' show a uniquely recognizable filamentous and/or leathery colonial morphology [http://www.microbelibrary.org/ASMOnly/details.asp?id=2566&Lang=&ISkip=20] that will help you find them. Most of the genera in this group produce a hyphal type of growth and form spores during their complex lifecycle. We will use two different media to enrich or select for various groups of ''Actinomycetes''. Each soil sample should be tested with both protocols. You will start the Glycerol Yeast Extract protocol described first in LAB2 and the AGS medium in LAB3. Culture and characterize as many different appearing colonies both these media as you can manage. | | '''It is important that we isolate representatives of this diverse group of organisms so make sure both soil samples from your habitat are enriched for ''Actinomycetes'' using both of the media described, Glycerol Yeast and AGS.''' Most of the |
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| '''Glycerol Yeast Extract Agar''': a less selective medium than Modular AGS medium to enrich for many of the antibiotic producing bacteria in the ''Actinomycetes''.<UL><LI>
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| '''Enrichment:'''<BR>
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| Soak a sterile cotton swab in the SOIL EXTRACT (Made in LAB 2). <LI>
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| Swab section 1 of a labeled isolation streak plate of Glycerol Yeast Extract Agar solid medium as described in [[BISC209: Streaking for Isolation | Streaking for Isolation ]].<LI> Follow the steps for [[BISC209: Streaking for Isolation | Streaking for Isolation ]] using a flame sterilized inoculating loop.<LI>
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| Allow the sample in section 1 to absorb into the agar before you,<LI>
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| Invert, and incubate the plate at RT. <LI>
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| Check your plate for the presence of colonies that look like a drop of powdered sugar with an indentation of the agar around the colony.<LI>
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| These colonies may be slow growing, so check your plates every few days for up to 2 weeks. (Look for the appearance of these colonies on your general purpose media too.)<LI>
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| '''Isolation:'''<BR>
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| Repeat isolation streaking on the same isolation medium until you have pure colony isolates.<LI>
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| ''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.<LI>
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| '''Identification:<BR>'''
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| When you have pure isolates, begin to examine the cellular morphology, structure, and metabolism of your isolates as described in LAB 5.</LI></UL></LI></UL>
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| '''Modular Arginine-Glycerol-Salt Agar medium (AGS) medium for ''Streptomyces'' sp enrichment''' <BR>
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| Note that you will not be able to start this protocol until LAB3. Your instructors will heat dessicate (oven bake) your soil samples collected in LAB2 and return them to you in LAB3. You will use the 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 AGS medium uses cycloheximide to inhibit fungal growth since many fungi make spores, too. We hope to select for the ''Streptomyces'', a common and important genera of soil bacteria. This group is an important source of many antibiotics used to control human bacterial infections. Every soil isolate should be tested with this enrichment protocol.
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| '''Enrichment:'''<BR>
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| Your lab instructor will return (in LAB 3) the dried 1 g soil samples, that you weighed out in LAB 2.<li>
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| Record the dried weight from the 3 samples in your lab notebooks<UL><LI>
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| Combine the 3 dried soil samples and then re-weigh to get 1 g of dried soil. Add it to 100 ml of dilute nutrient broth in a flask containing a magnetic stirrer. <li>
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| Agitate on a stir plate for 30 minutes.<li>
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| Allow the large particles to settle to the bottom of the flask for 30 minutes.<li>
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| Soak a sterile cotton swab in the broth. <LI>
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| '''Isolation:'''<BR>
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| Swab section 1 of a labeled AGS or modular AGS plate using your best isolation streak technique<li>
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| Follow the steps for [[BISC209: Streaking for Isolation | Streaking for Isolation ]].<li>
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| Allow the inoculum in section 1 to absorb into the agar before you, <LI>
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| invert, and incubate the plate at RT. <li>
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| Check your plate for colonies ''daily''.<li>
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| Isolation streak several different looking "little volcano" colonies onto new AGS plates (one colony/plate) using your flame sterilized inoculating loop. <LI>
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| Incubate.<li>
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| '''Identification:'''<BR>
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| Look for hard, white, ridged colonies (little "volcanoes"). The texture is tough and the colony will not be easily transfered. Try prying a small piece off with a sterile toothpick. 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.)
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| Once you have pure isolates, begin to examine the cellular morphology, structure, and metabolism of your isolates as described in LAB 5.
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| </li></UL></LI></UL>
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| <font color="purple">'''Lactic acid bacteria: <BR>
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| Use could probably successfully use any enriched medium. We will use Nutrient Agar (not the dilute version). '''</font color="purple"><BR>
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| The products of sugar fermentation are often used in classical culturable microbiology as one step in the identification process. Among these sugar fermenting organisms there are some bacteria that produce only lactic acid as the product of sugar catabolism.
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| <UL><LI>We will not have to use selective or enrichment media that is specific for this group as they grow well on nutrient agar. Steak your soil extract onto nutient agar and it is likely that you will isolate some of these bacteria from this medium or from other media. <LI> The characterization of this group is based on colony morphology, cell morphology, biochemical screening tests, and information in The PROKARYOTES or Bergey's Manual.
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| Any organisms that tests positive in the OxFERM test may belong to this group and should also be tested on other available carbohydrate media for differentiation.
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| <UL><LI>
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| Select colonies that you isolate from your nutrient agar streak plate (or dilution plates). A combination of your Gram Stain data and OX-Fem test will indicate if these are aerotolerant microbes that ferment glucose to produce lactic acid under anaerobic conditions.
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| </LI></UL>
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| ==Enrichment media and Isolation protocols (Starting with SOIL)== | | ==Enrichment media and Isolation protocols (Starting with SOIL)== |
