| MadSci Network: Microbiology |
Greetings Jee,
You have asked a question that trouble many students performing the Gram Stain.
Old, damaged, or antibiotic-treated bacteria that are normally Gram-positive may appear Gram-variable or even Gram-negative, presumably because their cell walls are more permeable to the decolorizing agent. The opposite does not hold true -- Gram-negative bacteria do not become Gram-positive in appearance with age or damage. When repeated Gram stains suggest the presence of organisms (often yeasts) that do not fit the clinical picture, consider the possibility that the Gram-stain reagents have become contaminated.
Most errors in the interpretation of Gram-stained smears are due to errors in the preparation of the slide, such as making the smear too thick, excessive heat-fixing, and under- or overdecolorization.
The examiner may consider the smear to be adequately decolorized if Gram-positive (purple) bacteria are found next to a properly decolorized (red-counterstained) polymorphonuclear leukocyte. If the smear happens to contain only Gram-negative (red) bacteria, this guide will be lacking, and other proof is needed that the slide has not been overdecolorized. By looking at one of the thicker parts of the smear, one can often find purplish cells that have been marginally decolorized. Finding red bacteria nearby will then confirm that the organisms are Gram-negative and not simply over decolorized.
An excessively thick smear is easily recognized because it resists decolorization and microscopically shows dark masses of structures that cannot be individually distinguished. A convenient way to check staining technique, if problems are encountered, is to stain a sample of one's own mouth flora, which contains both Gram-positive an Gram-negative bacteria as well as epithelial cells.
Ordinarily, a new smear should be prepared if the original one is technically inadequate. However, if this is impossible, a badly stained smear can occasionally be salvaged by removing the immersion oil with xylol and then restaining, as follows: An underdecolorized slide is given further decolorization followed by counter staining an overdecolorized preparation is again put through the entire Gram-stain sequence.
I hope this is sufficient information and I want to thank you for taking the time to send in a question to MadSci Network.
June Wingert
MadSci Admin
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