Posted by: loungekitten | April 26, 2009

Microbiology Lab Practical

We have just about finished our lab practical for Microbiology.  A lab practical is a final in the lab, and for this class, the professor handed us a TSA plate with an unknown organism so we can identify it.  We know what the seven possible organisms are – and that’s it.  No recommendations, no guidance, and definitely not open lab notebooks or manuals.  We simply remember what we do each day, and we can go home and plan our next lab, getting closer to the answer.  For the first few days, we can inoculate various mediums and perform gram stains to help us identify our bacteria.  The final day is the day we go in observe final results, clean up, and give our answer.

The first day was a frustrating day for me.  I started by preparing two slides for gram staining.  I could not view these two slides with the first microscope I used, nor the second.  I prepared a third gram stain and while I was preparing it, someone contaminated it (a little soap from their hand-washing splashed onto the slide) but I continued with it because I figured that the contamination would wash off of the slide.  When I got back to my microscope. I tried to see the slide, but I couldn’t.  My professor saw that I was frustrated and adjusted the microscope for me.  But by the time I was able to look at it, it was already out of focus.  What I saw when I finally got the microscope back into focus were coccus-shaped, gram-positive organisms in pairs and chains.  So then I perform a catalase test, and it’s catalase positive.  Hmm, of the two gram-positive coccus-shaped species we’ve been given, only one is catalase positive – Staphylococcus saprophyticus. But just in case I’m off, I inoculate some other tubes for other biochemical assays – a BEA tube, a TSI tube, an MR-VP tube, and a urea tube.

Well, it’s a good thing I did the other tubes.  I did some research and saw that my urea tube would have a positive result if my bacteria really was Staphylococcus saprophyticus.  But the result I got was negative.  The BEA test was also negative, so it wasn’t the other gram-positive bacteria, Enterococcus faecalis.  At this point, I’m sort of in a panic, because something is clearly amiss.

My TSI results look something like this, except not as fancy:

TSI agar result

TSI agar result

In my head I know what it looks most like – the result for Shigella, but I’m confused by the direct conflict with what I saw on the gram stain.  Plus I know for sure that my bacteria is catalase positive, and I was not sure that Shigella is catalase positive. (All but one species are catalase positive, and our professor would not try to trick us by using the catalase negative one.)  Also, the result from my methyl-red test is positive, so my mind immediately turns towards E. coli as my unknown.

In any case, it is clear at this point that my bacteria is one of the gram negative species, and I need to do another gram-stain to confirm that in my mind.  So I prepare a fourth slide for gram staining, and I also prepare another slide for a second catalase test, just to confirm the catalase positive result.  (Although catalase tests are simple tests, it can easily produce a misleading result).  Yep, it’s definitely catalase positive – E. coli is catalase positive.  And the gram stain is definitely a gram-negative, rod-shaped organism, upon the examination of this slide.  I’m almost ready to put on paper that I’ve got E. coli, but I decide to do some other tests to confirm.  I inoculate a MacConkey agar plate, a lactose broth tube, a SIM tube and another MR-VP tube, since it’s  entirely possible that I added too much reagent for the methyl red test, so I wanted to run that test again.  Then we had to throw our plates away.

I left the lab jubilant and thinking I knew which bacteria I had.  Then I remembered the TSI tube looked like this when we ran the test in the lab:

TSI result for E. coli

TSI result for E. coli

And my TSI tube definitely did not look like that.  When I got home I researched for E. coli TSI test results, and I found that this result is characteristic of E. coli.

I have Shigella.  The TSI result is what is to be expected with Shigella.  It is catalase positive.  It is urease negative.  It has a positive methyl red test result.

For the record:  Shigella is known to be difficult to differentiate from E. coli. Some species even have a similar TSI result that is similar to that of E. coli.  Some biologists do not think that E. coli and Shigella are separate species.  And the reason I did not immediately think Shigella when I saw the TSI slant result?  Well, here is a photo of my Shigella slant from a previous lab session:

Incorrect result for TSI slant for Shigella

Incorrect result for TSI slant for Shigella

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