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My main question is this: when you interpret gram stain results, does the majority color determine if its pos or neg?

We've been given unknowns and now have to determine what the organism is. I'm having trouble interpreting gram staining results. As far morphology they are all cocci and (sort of) in clusters but (this is the confusing part) they are both purple and pink. I understand that purple indicates gram+ and pink is gram-, but there should be one genus of organism not two. So shouldn't there be only purple or only pink, but not both? Should I be looking for the "majority" color and using that to decide whether I have pos or neg? That is, if its mostly purple, does that mean pos, ...but if so, then why are there still pink cocci on the slide?

I also understand why gram-pos appear purple, --that the crystal violet-iodine complex is retained due to thicker cell walls in gram-pos..... and thinner cell walls in gram-neg, so they are decolorized and appear the color of the counterstain.

Also I definitely did not see any rods in my slide, just cocci.

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The only reason that it shows both colors is due to imperfect manipulation while staining. You cannot count on the majority to determine the result of the stain.

In your specific case, rinsing or decoloration with EtoH was probably not done enough and safrainin wasn't completely eliminated.

The best way to be sure of the result would be to redo the coloring.

A good tip while staining is to have duplicates or triplicates.

Secondly, to answer what you said here:

I also understand why gram-pos appear purple, --that the crystal violet-iodine complex is retained due to thicker cell walls in gram-pos..... and thinner cell walls in gram-neg, so they are decolorized and appear the color of the counterstain.

Actually it is not only the thickness of the cell wall that will cause the bacteria to retain or not the crystal-violet. Gram positive bacteria have a plasma membrane then peptidoglycan on top while Gram negative bacteria have a plasma membrane, then peptidoglycan and another outer membrane on top. Crystal violet "sticks" primarily to the peptidoglycan.

Decoloring will remove the stain on the outer membrane of gram negative bacteria. You were right with the fact that Gram positive bacteria will have thicker peptidoglycan.

Gram + vs Gram - cell walls http://www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria

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  • $\begingroup$ I did 15 seconds of EtoH rinse but I'm hoping I can just do whole test again because it should be all purple , but its not gram neutral its definitely one microorganism $\endgroup$
    – user27969
    Nov 22, 2016 at 18:30

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