Posted by: loungekitten | April 29, 2009

The Microbe Identification Lab Report

Purpose:  To identify an unknown bacterial specimen using basic laboratory technique and biochemical tests.  The unknown bacteria will be one of the following:  Enterococcus faecalis, Staphylococcus saprophyticus, Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Salmonella [I assume typhimurium], or Shigella [either flexneri or sonnei, we used both in our lab during the semester].

Procedure {and observations}:

  1. Observe bacterial colony morphology.  {Colonies are large, beige or cream-colored, with irregular borders.}
  2. Prepare two slides for gram staining and viewing under a microscope.  {Either my gram-stain slides were bad or the microscopes I chose for viewing were not good.  No bacteria were found under the microscope.}
  3. Prepare another gram stain of the unknown bacteria.  {Gram-positive, coccus shaped bacteria were observed under the microscope.  Also noted, the slide may have been inadvertently contaminated during the gram stain process by another person in the laboratory.}  [I do think that the gram-stain result is surprising - the two gram-positive cocci species we've been given form medium-sized, round, uniformly colored colonies on TSA plates.  When I say the borders of the bacterial colonies are irregular, I mean that both the shape is irregular and the consistency of the borders is not like the center of the colony.  I associate this type of appearance with rod-shaped bacteria in general, whether gram-positive or gram-negative.]
  4. Perform catalase test.  {Catalase test is positive.}
  5. Inoculate test tubes prepared with the following mediums – Triple Sugar Iron agar slant (TSI slant), Bile Esculin Agar slant (BEA tube), a methyl-red Voges-Proskauer tube (MR-VP tube) and a Urease tube.  Incubate the inoculated tubes, to be read at the following lab session.
  6. Observe results of tests inoculated during the last session [Note:  I used the wrong setting on my digital camera to take these pictures, so they are all blurry!  Also, thinking about the results from the last session, I am thinking that my bacteria is Staphylococcus saprophyticus - gram-positive cocci, positive catalase.  I did my research, and at this point I am expecting a positive urease result and a negative BEA result.]
    Negative result for urease

    Negative result for urease

    {The broth in this tube would have been pink if the urease test was positive.  So, that leaves Enterococcus faecalis, which would have a positive BEA result.}

    Negative BEA result

    Negative BEA result

    {This is a negative result for the BEA test.  So it’s not Enterococcus faecalis.  And I am out of gram-positive cocci species from my list of possible bacteria.  I need to start over from the gram-staining step.  But first I note the results from the other two tests.}

    Alk/A result for TSI slant

    Alk/A result for TSI slant

    {This slant looks like the result for Shigella.  [A helpful labmate saw my tube and said, "I know what you have. . .your tube looks like a rainbow!"  I shook my head, telling her I knew what she was thinking, but no, it wasn't that.  When I said that, I was still confused by all these conflicting results!  I really thought the TSI slant result was just rubbish at that point.]  The result of this test definitely rules out Proteus vulgaris and Salmonella [typhimurium?] because there is no Hydrogen sulfide in the tube, which would be indicated by the presence of a black color in the medium.}

    Positive MR-VP result

    Positive MR-VP result

    {10 drops of methyl red reagent were added to this tube, then gently mixed.  Red or pink color indicates a positive test.  There is only one bacteria on the list for which we saw a positive result in our regular lab sessions for MR-VP – Escherichia coli.}

  7. Prepare gram stain slide of bacteria.  {This time, gram-negative rods are observed.  Was what I saw last time contamination from skin bacteria, or am I seeing what I want to see?  The majority of the bacteria on this slide were pink – gram-negative.  I’m going with that!}
  8. Perform catalase test again to confirm catalase positive result.  {The catalase test can be tricky, though it is a simple test to perform.  False-positive test results are common.  I am redoing this test because most of the remaining bacteria species are catalase-negative — except for Escherichia coli, which I am sure is catalase positive.  Some of the others I am unsure of, and will need to research later.  A repeat test confirms that the unknown is catalase-positive.}
  9. Inoculate the following mediums with the unknown bacteria:  Sulfide Indole Motility tube (SIM tube),  a second MR-VP tube, a lactose broth tube (with Durham tube), and a MacConkey agar plate. [Yes, at this point I think I have E. coli.]  Incubate and observe results at next lab session.  [Identification is due at the end of the next lab session.]  TSA plate with original sample is discarded.
  10. [After thinking about it and researching at home, I am sure I have Shigella (flexneri or sonnei).  In fact I am sad I did all the extra work - I should have known.  By the way, I did a second MR-VP tube because, as we discovered in an earlier lab, there may have been a concentration problem with our methyl-red reagent that would give a false-positive result.  I plan to add less reagent to the tube in this session.]  Results of tests inoculated in the last lab session:
    Negative lactose fermentation test

    Negative lactose fermentation test

    {A positive test would have a color change to yellow, and the Durham tube would probably have a gas bubble in it.  This result rules out Escherichia coli and Enterobacter aerogenes.}

    Negative result for MacConkey agar

    Negative result for MacConkey agar

    {Positive result – colonies grown on the medium would have turned red.  This test also rules out Escherichia coli and Enterobacter aerogenes.}

    Negative Sulfide production

    Negative Sulfide production

    {No Hydrogen sulfide, so it is definitely not Enterobacter aerogenes.  This test also probably proves that the unknown is not Salmonella [typhimurium?] or Proteus vulgaris, but based upon experience in this lab throughout the semester I am not sure if this test is diagnostic for these species.}

    Negative indole test

    Negative indole test

    {This is the SIM tube shown above with 5 drops of Kovac’s reagent added to it.  If this test had been positive, there would have been a red or pink ring visible on top of the medium.  There is no color, so it is negative.  Interesting, E. coli would have been positive.  Shigella, apparently, is negative.}

    Positive result for MR-VP

    Positive result for MR-VP

    {I only added 5 drops methyl-red reagent, and did not mix it.  The tube, according to my reading, is not supposed to be mixed.  I already knew it was positive, though.  Just confirming!}

Result

The unknown sample is Shigella [flexneri or sonnei]. The TSI slant result is characteristic of Shigella species.  It is also catalase positive;  all but one strain is catalase positive (and the one that is catalase negative would have to be handled in a BSL-3 facility, not our regular lab).  Shigella is also methyl-red positive.

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Responses

  1. can i noe tat
    y whn we perform catalase test
    we mz we do lastly?
    i realize tat it will produce O2
    bt thn duno hw to prove tat
    y the O2 produce wil affect the colonies…
    thx^^

  2. I am also working on my unknown in my Microbiology class. So far, I have performed a Gram stain-Gram positive cocci. I also subcultured my unknown, twice. Today, I made 2 streak plates, one on EMB agar and the other on CNA, expecting my bacteria to grow on CNA, but not on EMB. I will, hopefully, be able to prove that Wednesday. I’m keeping my fingers crossed. Our next step is to make a list of all the Gram positive bacteria that we have worked with this semester and then figure out which tests we have worked with this semester to further prove my uknown as Gram positive. Any suggestions as to what tests I could perform to prove it is Gram positive? I would really appreciate it! Thanks!

    • I know that this is probably too late for your lab assignment, but it sounds as though you will get your answer based upon the streak plates you innoculated. We do not use these streak plates in our labs, but from what I read you will get your answer. If you need to determine your bacterium’s species, there are other tests and media you could use to differentiate. Some of the tests are not specific for a bacterial species, just another piece of evidence to prove which bacterium you have. Some media are really selective – Mannitol Salt Agar is selective for coagulase-positive Staphylococci, but you wouldn’t innoculate that plate unless you were fairly certain you had a Staphylococcus species.

      So, it would be difficult for me to give you a list of tests that would indicate a Gram-positive bacteria, in part because I don’t know what your objective is and also because labs tend to favor the use of certain media for experiments. Similarly, it would be difficult to give you a list of Gram-positive bacteria species because professors order cultures based upon the lab manuals they use. I get a ton of hits on this blog for Proteus mirabilis, but we didn’t work with that species in our lab, we worked with Proteus vulgaris!


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