Remember from your morphological unknown that 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.
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. You can find hints here on Microbugz as to whether a test will be useful for your organism.
With a proper dichotomous key or flowchart, you should be able to solve both your unknowns in three to four lab sessions.
Now you have your mixed unknown; where do you begin this time?
What does the title “mixed” unknown tell you?
That’s right! You have more than one organism in your tube.
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! You will probably have other plates available to streak on your first session. Possibilities include CNA, PEA, blood agar, MAC, and EMB. Find out what will be available and be sure to ask yourself what each plate will do and what information it will give you. Is the plate selective or differential? What types of organism will grow on that plate? These results will give you a large chunk of data to use in beginning to solve your unknown.
After incubation, look at your plate(s). Did you achieve separation of two distinct colonies on your TSA plate(s)?
If so, streak each organism for isolation on its own TSA plate and then continue with your investigation. If not, try your streak for isolation again.
NOTE:
When you streak a separate plate for each isolated organism, be sure to label each plate properly! You may choose to call one organism pick A and the other pick B. Whatever you decide to call them, be sure to label everything you do and keep your organisms separate! If you get confused about which organism you used to inoculate a test, you’ll have to do the test all over again, and you’ll lose time. Stay as organized as you can, and you’ll make your life much easier.
What should the next step be?
Perform a Gram stain on your organism. Just like last time, when Gram staining an unknown, the best method is to make three bacterial smears on the same slide. One should be a known Gram positive organism like <i>Staphylococcus aureus</i>. The other should be a known Gram negative organism like <i>Escherichia coli</i>. 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 the Gram stain slide, you should be able to determine the Gram specificity of your unknown. Once you know the Gram specificity of your organisms–there should be one Gram negative and one Gram positive–you can begin performing your biochemical tests.
Now what?
Now, follow your dichotomous key based on the information you have.
Remember: only perform the tests you need to perform!
Standard tests for Gram negative organisms begin with lactose fermentation. You will most likely have streaked a MAC plate already. Can you determine lactose fermentation from that?
Another set of standard tests involves the IMViC panel.
Urea and ornithine decarboxylase broths are also often used for identification of Gram negative organisms. What else–if anything–might you need to use? Might you need gelatinase? Nitrate broth?
What are some tests that will help you identify your Gram positive organism? One good place to begin is a catalase test. It will help you decide whether you should perform tests to identify streptococci or staphylococci/<i>Bacillus</i>. Will bacitracin be helpful for identifying streptococci? What about novobiocin; what is it useful for? You use coagulase (if it is available in your lab) to differentiate which organisms? MSA? Do you need to perform a salt tolerance test? Bile esculin?
Again, whatever you do, be sure you know why you’re performing a certain test and what information it will give you. Randomly inoculating tests without knowing what information they will give you is a recipe for disaster.
Be sure to keep good, detailed, organized notes as you go, and think about each step logically. If you do that, your mixed unknown will be a snap! If you don’t, you will be lost, confused, and frustrated. If you’re organized in your approach, you mixed unknown should be a fun puzzle. Enjoy!