Microbiology Lab 352
Unknown Plan: Isolation and Identification of Environmental Microbes
For your unknown project, you will research an environment and choose an
organism to isolate and identify (either at the genus or species level) that is
commonly found in that environment. You can choose to use the Winogradsky
column as your environment, or find another environment of your choice (i.e. pond
water, soil sample etc.). At the end of this handout are listed some general types of
organisms to help you choose. You should research several organisms in the
environment of your choice (Bergey’s manual is a good resource) to find out what
is interesting about these, how you can isolate a particular organism and what tests
you will need to perform for isolation. You must cite at least two peer-reviewed
primary research articles.
Since this laboratory has limited resources, at the end of this handout are
listed all of the different tests that are available. Some of them we will have gone
over in lab, while others we have not. If you need help understanding any of the
protocols, ask your TA. If there are any tests you want to use that are not listed, ask
your TA about them before the proposal is due, and depending on availability of
reagents, you may be able to use that test. In your unknown proposal, you should
list enough tests to figure out if you have the correct organism down to the genus
level (four or five tests should be enough).
Your unknown plan should be approximately 3 pages double-spaced and
should include the following:
1. A relevant title
2. An introduction describing one of the organisms you are going to isolate and
the environment that you will be isolating from.
3. A description of where in the Winogradsky column your organism will be
located. If you are choosing s different environment (not from the
Winogradsky) make sure to describe that environment and describe the
different types of organisms that live there. Samples of potential medical
concern will not be allowed in the lab. You do not need to describe how you
will sample from the Winogradsky column: Your TA will go over that in class.
4. Often, your intended microorganism is not initially isolated, so you should
have a good idea of some other organisms that are commonly found in your
environment so that if your results do not match your intended isolate, you
would have an idea of what your isolate is. This will require some
background research, potentially from peer reviewed literature.
5. A description of the media you plan to use to isolate your organism (using
selective media will increase (but not guarantee) the chances that you find
the organism you are looking for).
6. A description of all the tests you will be using and their expected outcomes.
You should briefly describe how the tests work, and should demonstrate that
you understand what they are testing, but you do not need to go into detail
about how they work/how they are conducted. Everybody’s first test
should be a Gram stain.
7. Works cited page: do not cite any websites ending with .org or .com don’t
site Wikipedia. The works cited page is in addition to the 3 pages required
for the proposal.
8. In addition to the written proposal, you should also provide a flow chart that
shows the order of tests that you intend to conduct, expected outcomes for
your organism and what the tests are actually testing.
9. In addition to your proposal, you’ll need to submit a ‘pre-proposal’ by the
end of the weekend to me that explains succinctly (in 2-3 sentences) which
organism you intend to isolate and from where.
The following is a list of media you may use for isolation, and the types of
organisms they tend to select for
Some organisms are difficult to isolate, you may want to discuss these with your TA.
1. R2A (general media for environmental samples)
2. Sulfur phototroph (selects for green and purple sulfur phototrophs)
3. Non-sulfur phototroph (selects for cyanobacteria and green and purple nonsulfur phototrophs)
4. MSA (Mannitol Salt Agar, selects for Gram+ and Stapylococcus spp.)
5. EMB or MacConkey (either selects for Gram-)
6. TSA (general media used in our lab, good for most spp.
7. Nitrogen Fixing (selects for organisms that fix N2)
8. Nitrate reduction (Nitrogen reducers)
9. Sulfate reducing media (selects for organisms that can reduce sulfur)
The following are tests that you can perform on your organism (if you have
any questions about these tests, ask your TA):
Microscopic Techniques:
1. Gram stain (required)
2. Wet mounts
3. Endospore stain
4. Capsule stain
5. Acid-fast stain
Culturing (in stock):
1. MSA agar
2. EMB agar
3. McConkey agar
4. DNAase agar (used to differentiate Staphylococcus species primarily; based
on ability to degrade DNA)
5. Thioglycollate
6. Carbohydrate media with Durham tubes
• Glucose
• Sucrose
• Lactose
• Mannitol
7. TSI slants
8. Starch Hydrolysis plates
9. IMViC (Indole, methyl red, voges-proskauer, indole, citrate tests) – used to
differentiate enteric bacteria
10. Urease (used to detect the ability to degrade urea)
11. Coagulase (tests the ability to cause fibrin clots in animal plasma)
12. Oxidase (tests for the presence of cytochrome c oxidase – an indicator of
aerobic metabolism)
13. Catalase (tests the ability to reduce oxygen to H2O2, a trait of aerobes, or
facultative aerobes)
14. Lysine and Ornithine decarboxylase (tests the ability to degrade these two
amino acids)
15. Phenylalanine deamination (tests the ability to degrade phenylalanine)
16. Blood agar hemolysis (used to test for the ability to degrade red blood cells -generally used to distinguish organisms of clinical concern, especially
Streptococcus species & Staphylococcus species)
17. Nitrate reduction
18. Sabouraud agar
19. Plate count agar
Culturing (let the TA know in advance you will be needing the following media):
1. Casein
2. Nitrogen free (selects for nitrogen fixers)
3. Non-sulfur (selects for non-sulfur phototrophs)
4. Sulfur-reducing (selects for sulfate reducing organisms)
5. Sulfur (selects for sulfur phototrophs)
6. R2A (minimal media used to select organisms with low nutrient
requirements)
The following is a list of groups of organisms commonly found in Winogradsky
columns:
1. Cyanobacteria
2. Green sulfur phototrophs
3. Purple sulfur phototrophs
4. Green non-sulfur phototrophs
5. Purple non-sulfur phototrophs
6. Clostridium
7. Beggiatoa
8. Desulfovibrio
9. Pseudomonas
10. E. coli
11. Nitrogen cycling organisms (nitrogen oxidizers, reducers, fixers)
12. Sulfur cycling organisms (sulfur oxidizers or reducers)
Know how your organisms will grow and incubate accordingly.
For example; anaerobic, phototrophic, temperature.