Posted by: loungekitten | April 16, 2009

The Gastrointestinal Bacteria Series

So, we had a series of labs that covered gastrointestinal bacteria.  Yes we collect our own samples.  🙂  That’s all I’m going to say about that.

So, first we cultured a TSA plate with our own bacteria to isolate a colony.  Here is my plate, with a single isolated colony:

My GI bacteria sample

My GI bacteria sample

Plus we streaked a MacConkey agar plate with standard samples of intestinal bacteria so we could observe the results and work with the bacteria in the second lab session.  Here is  a picture of that plate:

GI bacteria standards

GI bacteria standards

Unfortunately, the surface of the agar was wet when the plate was streaked, so some of my bacteria samples migrated into areas they should not have.  For example, the Escherichia coli (E. coli) sample, on the upper left section of the plate migrated into the section below it.  The bacteria we streaked, starting from the bottom of the plate and working in a clockwise direction: 1.  Enterobacter aerogenes, 2.  Proteus vulgaris, 3. Escherichia coli, 4. Shigella flexneri, and 5. Salmonella typhimurium.  A positive result for growth on MacConkey agar is indicated by a change in color of the bacteria, and it indicates that the bacteria can ferment lactose.  It is a test that differentiates between pathogenic and nonpathogenic enteric bacteria.  The two species that were supposed to have a positive result were the Escherichia coli and the Enterobacter aerogenes.  You can definitely see that the E. coli is positive.  My Enterobacter aerogenes also changed color, but only slightly – the change isn’t even visible in the picture.

Continuing with the E. coli and the Enterobacter aerogenes, the bacteria were used to inoculate test tubes containing two types of mediums; MR-VP medium (methyl red-Voges Proskauer) and SIM medium (sulfide indole motility).  The MR-VP tubes are filled with a broth containing dextrose, and if the bacteria produces acid when breaking down glucose then adding 10 drops of methyl red pH indicator indicate if acid is present.  E. coli should be positive, and Enterobacter aerogenes should be negative.  Here is a picture of the inoculated tubes after incubation – they are a little turbid.

MR-VP tubes for E. Coli and Enterobacter

MR-VP tubes for E. Coli and Enterobacter

Here is a picture of the Enterobacter tube after adding methyl-red indicator:

Negative MR-VP result for Enterobacter

Negative MR-VP result for Enterobacter

Here is a picture of the positive MR-VP result for E. coli:

Positive MR-VP result for E. coli

Positive MR-VP result for E. coli

The SIM tubes are filled with agar containing peptonized iron.  The agar turns a black color if the bacteria has cysteine desulferase, which is an enzyme that breaks down cysteine and produces hydrogen sulfide, which then combines with the iron in the medium to form FeS, the visible back color in the agar.

Here are the SIM tubes inoculated with E. coli and Enterobacter:

SIM tubes inoculated with E. coli and Enterobacter

SIM tubes inoculated with E. coli and Enterobacter

E. coli is negative and the Enterobacter is positive.  For the purpose of this lab, this is merely an observation.  The test that really needs to be run is the indole test.  The indole test proves that the bacteria has the enzyme tryptophanase, which breaks down the amino acid tryptophan.  To run the test, Kovac’s reagent is added to the inoculated SIM tubes.  If indole (produced when tryptophan is broken down) is present in the tube, the reagent will combine with the indole and a red color will be present.  If there is no color change, there is no indole present.  Here is a picture of the indole test for Enterobacter:

Negative indole test for Enterobacter

Negative indole test for Enterobacter

Here is the picture showing the indole test result for E. coli:

Positive indole test for E. coli

Positive indole test for E. coli

Circling back to the other bacteria on the MacConkey agar plate, these three bacteria were incoculated into three different triple-sugar-iron tubes.  These tubes contain many reagents, including sugars to test for fermentation, pH indicator to detect the presence of acid, and Ferrous sulfate to detect the production of Hydrogen sulfide.  To read the test results, you need to observe the color on the top of the tube (the slant), the color at the bottom of the tube (the butt), and the presence of a black color to detect Hydrogen sulfide.

For Proteus vulgaris, the slant color should be yellow (acid), the butt color should be yellow (acid), and there should be a black color present.  Here is a picture of the Proteus vulgaris TSI tube:

TSI tube for Proteus vulgaris

TSI tube for Proteus vulgaris

It is yellow on top, and of course there is Hydrogen sulfide present in the tube.  The butt color is also black, though it should have been (and most likely was at some point) yellow.

For Salmonella typhimurium, the slant should be red (alkaline), the butt should be yellow, and some of the tube should have turned black.  Here is a picture of the TSI tube for Salmonella:

TSI tube for Salmonella

TSI tube for Salmonella

I got the correct results for Salmonella.  It’s difficult to see in this picture, but just above the black area there is a faint yellow color, which would represent the butt color.

For Shigella flexneri, there is not supposed to be a black color in the tube.  The slant should be red, and the butt should be yellow.  Here is the picture of the Salmonella flexneri:

TSI tube for Shigella flexneri

TSI tube for Shigella flexneri

Red on the slant (alkaline), black in the tube, and no visible yellow color.  The results are wrong.  It turns out, after talking with other people in the lab, those who had used the Shigella flexneri had the same incorrect results as me – thus we concluded that the original sample was most likely the incorrect bacteria.  The other people in the lab who used the Shigella sonnei obtained the correct results for the TSI test.

And finally, we inoculated our own bacteria in an apparatus called the Enterotube II.  Here is a picture of mine after inoculation – my tube was not successfully inoculated:

Enterotube II after incubation

Enterotube II after incubation

It looks basically the same as the one that I have from before inoculating the Enterotube II.

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Responses

  1. you collected your own samples !? omg !@#$^

    • Yes 😉

      I work in an Emergency Room. . .I’ve collected samples that were a lot more disgusting. . .

      At least I didn’t have C-Diff!!!

  2. Thank you very much for posting this. I’ve been looking all over the internet for these particular organisms and their test results. 🙂
    My class did the SIM tubes last week for Escherichia coli, S. epidermidis, P. aeruginosa, and Enterobacter aerogenes. Unfortunately, the cultures that we were given were contaminated so our results were wrong. 😦

    • You are welcome! I know how frustrating it is to get unexpected results – it really is so satisfying to get it “right.” I’ve actually found really good biochemical test result pictures on Flickr, so a search on that site might be worth it if you’re stuck.

  3. hi, thanks for info that u have shared. It’s really help me in finishing my lab report for mini project.


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