I repeated a culture twice in an entry level microbiology class for our final lab project of culturing a provided unknown organism. One of the required tests on our dichotomous key was to culture a gram positive cocci in mannitol salt agar. After repeating the experiment twice as instructed, and doing an isolation streak as instructed, the part of the agar with no growth was acidic while the part of the agar with growth was neutral or slightly basic. Both cultures were incubated for around 48 hours, and the first one then refrigerated for another 48 hours while the second one was examined directly after incubation. My professor told me to disregard the results and treat it as a negative in my report, but I'm perplexed as to how this result came about.

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    $\begingroup$ Yeah it seems kind of impossible on first thought. The sterile plates are red, yes? Do you know what result is expected with your organism? $\endgroup$ – canadianer Apr 21 '17 at 2:31
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    $\begingroup$ In my experience, many TAs are not that thoughtful. Are you able to monitor the growth at more time points over the two days? If the plates were red to begin with, the only explanation I can think of right now is that the organism fermented mannitol and produced acid, turning the entire plate yellow. Once the mannitol was exhausted, it started metabolizing peptones which produces base and reverts the colour of the agar surrounding the growth to red. $\endgroup$ – canadianer Apr 21 '17 at 2:52
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    $\begingroup$ I was going to say that, if the plate was yellow to begin with, someone might have used the wrong indicator when they were preparing the plates (eg methyl red instead of phenol red). $\endgroup$ – canadianer Apr 21 '17 at 2:53
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    $\begingroup$ The orange colour you see is intermediate between red and yellow. It's likely that the pH was near the transition point (pKa) of the indicator. $\endgroup$ – canadianer Apr 21 '17 at 2:54
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    $\begingroup$ I can't think of a chemical explanation unless someone left an uncapped bottle of hydrochloric acid in the incubator. The plates are pretty simple. $\endgroup$ – canadianer Apr 21 '17 at 3:44

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