Solving an unknown is much 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.
It is recommended–and may be required by your professor–to develop a dichotomous key or flowchart outlining the tests you will perform to identify each organism.
Go through the process step-by-step, determine what stains or tests will give you the most useful information, and perform <strong>only</strong> those stains or tests. Such an approach will save you LOTS of time and headache.
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.
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?
Perform a Gram stain on your organism. You can perform the Gram stain from your initial broth–you will be able to see cellular arrangement more accurately from the broth–or from an isolated colony on your plate.
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 should, 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.
The next steps 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? What about a motility test? Catalase?
With a proper dichotomous key, flowchart, or table, you should be able to determine your morphological unknown in as few as two class periods.