Kinda difficult question about Gram staining?

Question

You are given a culture of an organism which is known to be Gram positive. You stain it by Gram's method and upon examination it appears to be red. What are the possible explanations for this?

I'd really appreciate the help! :p

Answer 1

the most common error in gram staining is over decolorizing. this would be rinsing off too much of the crystal violet with your decolorizer. when you are rinsing with your decolorizer, only rinse until there is no more purple color washing off. then stop and rinse with water. you could make several slides of known organisms and practice your technique. the strength of the decolorizer varies with the brand name, and sometimes with different lot numbers of the same manufacturer.

Answer 2

The most important step in gram staining is the alcohol decolorization step. If you don't decolorize enough then your gram negative bacteria may appear purple; if you decolorize too much, like in your case, then you have gram + bacteria appear pink.
What are you using to decolorize? This is the order of decolorizing power:
acetone - fastest
acetone/alcohol mix - intermediate
alcohol alone- slowest

Answer 3

You overstained, or you didn't wash effectively enough. Another issue might be the concentration of the Dye was too high. Carbol Fuschien has nonspecific staining at high enough concentrations. Perhaps you might want to do a 1:2 dilution to see if that helps.

Answer 4

It is most likely that you washed the gram stain off, or perhaps you accidentally skipped a fixing step prior to the final stain and wash?