Mixed Unknown

Now you have your mixed unknown; where do you begin this time?

Remember from your morphological unknown that 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 does.

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!

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

If so, streak each organism out on its own TSA plate and then continue with your investigation. If not, try your streak for isolation again.

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.

Some instructors will have you streak more plates than just the TSA on the first day of your unknown. Some of these plates are selective media; some are differential media, and some are both.  Be sure to really take the time to figure out what information each of these plates gives you. Which organisms will grow on each plate? What, for instance, do pink colonies on a MAC plate mean? These results will give you a large chunk of data to use in beginning to solve your unknown.

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 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 the 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 fermentation tests may provide useful information. A catalase test, blood agar plate, or nitrate reduction test may help narrow down the possible organisms.  The IMViC tests might help. 

Your best bet is to look at what each test tells you and devise a flowchart to determine what path to take when determining your unknown.  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, and your mixed unknown will be a snap!