Morphological Unknown

Solving an unknown is a lot like solving a mystery.  Think about the evidence logically and put the pieces together, and you should have little difficulty in determining the identity of your organism. Remember that the way you solve the unknown is as important-if not more important-than whether or not you correctly identify your organism.

Whatever you do, DO NOT perform all the available tests and then try to collate the data! 

That is the FASTEST way to get overwhelmed with extreme amounts of extraneous information that will do nothing but confuse you. Go through the process step-by-step, determine what tests will give you the most useful information, and perform only those tests.  Such an approach will save you lots of time and headache.

Also, the unknowns are not a huge chunk of your grade, so be sure to keep up with your lab work while working on them. The lab exercises you cover while working on your unknown will be on your lab practical, and your lab practicals count for much more of your lab grade than your unknown.

What does the title "morphological" unknown tell you?

Remember back to lab exercise 2-2, Cultural Characteristics.

What were you studying in that lab?

That's right!  Cultural morphology.

In this unknown, the cultural morphology of your organism will be one of your biggest sources of information.

After incubation, look at your plate.  Did you achieve separation of two distinct colonies? 

The first step is to streak your unknown broth for isolation on a TSA plate and incubate it for 24-48 hours. Be sure to vortex your broth before inoculating from it!

After incubation, look at your plate.  Are the colonies large? Punctiform?  What do the margins look like? The elevation? Is there any pigment?  Look at your notes from Exercise 2-2. Are there any bacteria you looked at that match the description of your unknown?

What should the next step be?

Perform a Gram stain on your organism. When Gram staining an unknown, the best method is to make three bacterial smears on the slide.  One should be a known Gram positive organism like Staphylococcus aureus.  The other should be a known gram negative organism like Escherichia coli .  In the middle, make a smear of your unknown organism. Then perform the Gram stain as usual.  If the known Gram positive and Gram negative organisms look like they're supposed to, then you can be sure your unknown organism stained correctly. If the known organisms are not the correct colors, you know there was a problem with your staining technique and that you need to perform the stain again on a new set of smears.

From this Gram stain slide, you should be able to determine the Gram specificity of your unknown and learn something about the cellular morphology. You may even be able to determine the cell arrangement of your organism. These will all be very helpful pieces of information in determining your unknown.

Now what?

Based on your results so far, if it seems like it would be a useful source of information, perform a motility test on your organism using the semisolid motility media with the TTC indicator.

The next step will depend on your results so far.  For some organisms, a capsule stain can give valuable information. Be sure to only perform the capsule stain on a sample of your organism that is 5 or more days old. Capsules don't form on “fresh” cells.

Would an acid-fast stain be helpful? An endospore stain?

Once you've streaked for isolation, examined the colonial morphology, performed a gram stain, performed a motility test (if helpful), and performed any other stains that are helpful to solving your unknown, you should be able to determine the identity of your unknown.  Fill out an unknown data sheet and turn it in to your instructor.

You're done!