Weird lab outcome

[starlathornhill]

For my path micro lab I had to swab certain places of my body (nose, ears, hands), plant them on MSA, chose a colony, streak isolate it on TSA, then gram stain it (in addition to performing a catalase and coagulase test).

When I did the gram stain of an isolated colony from my nose, I got gram negative staphylococci. I know the gram stain wasn't wrong because I redid it. I did a gram stain of another colony from the ear and it was gram positive, and the color was definitely purple, and definitely different, so it's not a problem with my interpretation.

How could I have gotten a gram negative colony? Since the samples were first grown on MSA I should have only isolated gram positive bacteria. Is it possible that the gram negative was surrounded by gram positives and was able to survive the night on the TSA, then was isolated during the streak plating and was able to grow?

I am thinking it is Neiserria sp. since they are common commensals on mucus membranes but I am unsure of how it would have survived being plated on MSA overnight!

[iRNAblogger]

So I have a couple guesses, but that is pretty weird.

 

1. MSA does inhibit the growth of gram - bacteria, but that doesn't mean that every single gram - bacteria in the world would be unable to grow in those conditions, there are probably some exceptions. You may have found an exception.

 

2. The bacteria might have an EC matrix that is heavy in lipids while also containing a thick peptidoglycan wall, which would allow it to survive on MSA, but then also prevent it from staining purple.

 

3. A simple contamination issue. You might have accidentally contaminated your TSA plate. If you were to re-do the experiment, I would streak onto an MSA plate for the growth step rather than TSA which is not selective.

[starlathornhill]

Hm, #2 might be it. I don't think its contamination because I definitely flamed the inoculating loop and the colony I chose was on the streak line.

Here's a picture of the two gram stains next to each other. Left (diplococci) is definitely a gram positive, and right (staph) is what I have identified as gram negative. It looks like my usual gram negative stain (it always comes out a really deep pink for me, not sure why). Is it possible I just over decolorized? Looking at the results of the other tests it looks to me like this should be S. aureus (coagulase positive) except that it stained pink.

[CharonY]

Well, some Gram positives have a thick outer layer, which sometimes inhibits Gram staining. I have not heard about a Staphylococcus sp. to exhibit this, however (more commonly found in actinobacteria). In addition, they often also do not counterstain well. There are of course also halophilic Gram negatives, as well as other Gram positives. Sarcina has some halophilic members, some do not stain well and some are part of the skin flora. But I do not know off the top of my head whether there is one to which all of it applies.