LAB 7: Characterization of culturable soil bacteria
Before we get to work on characterizing our isolates, be aware of where are in indentification of those bacteria by 16s rDNA sequencing.
We have sent out all the amplicons that you prepared last week for DNA sequencing of the 16s rRNA gene. We must also send an appropriate primer. What's the sequence of such an appropriate primer? What's going on in those automatic sequencing machines in Danvers? Make sure you understand how Sanger sequencing works. We should have the data back by LAB9 when we are schedule to meet in a computer lab and learn to analyze the data generated so we can find out, we hope, a lot about the diversity of the soil bacteria in our habitat of interest.
Culturable Bacteria Characterization continued
Continue following the protocols for Tests to determine the role of a soil isolate
Concentrate on completing and evaluating the tests you have in process on your isolates(use the appropriate protocols-refer to LABS 5 and 6; in the Protocols section: Enzymes, Motility, and Carbohydrate Fermentation sections, addition to the Roles protocols.
Continue with/add new differential ID tests such as an SPECIAL STAINS (see the Special Stains section in the Protocols) that seem appropriate. For example, all Gram positive bacilli or any bacteria that showed unstained area in the cells when Gram stained should be stained for endospores.
All bacteria that were positive or ambiguous for motility in SIMs medium should be looked at in a wet mount and if positive in the wet mount, you could try the Flagella stain. All the "swarmers" (those bacteria that made lawns all over the plate)should be looked at in a wet mount too.
Highly mucoid colonies should be tested for the presence of a capsule using the capsule stain protocol.
If your Gram stain results were ambiguous or not what you expected from the descriptions of the bacteria that grow on the Enrichment media you used, you should probably repeat those Gram stains. Getting the Gram reaction right is crucial to the discussion of where these bacteria belong phylogenically and functionally.
If there extra test medium is available you may repeat any other tests that were ambiguous or add any you think might be useful for identifying your organism. You can also make a list of tests that would allow you to identify your isolates to species but were not available.
By this point, you should be wrapping up the testing, beginning to discard your plates and check with the instructor about freezing glcerol stocks of some of the isolates.
Are you using the data sheet (located in the Data folder of the lab conference) or one of your own design? Organization of all this information is key. Be sure that you have added the unique code numbers for your isolates so we can correlate identity from sequencing with your functional, morphologic, and metabolic tests. If you are pretty sure you know the genus of an isolate from our characterization tests, please add that as well.
1. All culture plates that you are finished with should be discarded in the big orange autoclave bag near the sink next to the instructor table. Ask your instructor whether or not to save stock cultures and plates with organisms that are provided.
2. Culture plates, stocks, etc. that you are not finished with should be labeled on a piece of your your team color tape. Place the labeled cultures in your lab section's designated area in the incubator, the walk-in cold room, or at room temp. in a labeled rack. If you have a stack of plates, wrap a piece of your team color tape around the whole stack.
3. Remove tape from all liquid cultures in glass tubes. Then place the glass tubes with caps in racks by the sink near the instructor's table. Do not discard the contents of the tubes.
4. Glass slides or disposable glass tubes can be discarded in the glass disposal box.
5. Make sure all contaminated, plastic, disposable, serologic pipets and used contaminated micropipet tips are in the small orange autoclave bag sitting in the plastic container on your bench.
6. If you used the microscope, clean the lenses of the microscope with lens paper, being very careful NOT to get oil residue on any of the objectives other than the oil immersion 100x objective. Move the lowest power objective into the locked viewing position, turn off the light source, wind the power cord, and cover the microscope with its dust cover before replacing the microscope in the cabinet.
7. If you used it, rinse your staining tray and leave it upside down on paper towels next to your sink.
8. Turn off the gas and remove the tube from the nozzle. Place your bunsen burner and tube in your large drawer.
9. Place all your equipment (loop, striker, sharpie, etc) including your microfuge rack, your micropipets and your micropipet tips in your small or large drawer.
10. Move your notebook and lab manual so that you can disinfect your bench thoroughly.
11. Take off your lab coat and store it in the blue cabinet with your microscope.
12. Wash your hands.
Study for your Lab Practical
Begin to wind down and finalize the characterization of your culturable isolates.
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