Unknown 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}: Observe bacterial colony morphology. {Colonies are large, beige or cream-colored, with irregular borders. } 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. } 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.

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I associate this type of appearance with rod-shaped bacteria in general, whether gram-positive or gram-negative. ] Perform catalase test. {Catalase test is positive. } 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.Observe results of tests inoculated during the last session [N[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. ]egative 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 {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 {This slant looks like the result for Shigella. [A[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. ]he result of this test definitely rules out Proteus vulgaris and Salmonella [typ[typhimurium? ]ause 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 {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. } 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! } 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.

} 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[Yes, at this point I think I have E. coli. ]ubate and observe results at next lab session. [Ide[Identification is due at the end of the next lab session.

] plate with original sample is discarded. [Aft[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. ]ults of tests inoculated in the last lab session: 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 {Positive result – colonies grown on the medium would have turned red.This test also rules out Escherichia coli and Enterobacter aerogenes. } Negative Sulfide production {No Hydrogen sulfide, so it is definitely not Enterobacter aerogenes. This test also probably proves that the unknown is not Salmonella [typ[typhimurium? ]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 {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 {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 [fle[flexneri or sonnei]e 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.