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The page below is a sample from the LabCE course Reading and Reporting Gram Stained Direct Smears. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Under-decolorized or Over-decolorized Smears

Although this smear is the proper thickness, it is not appropriate for examination because the host cells are stained blue instead of red, indicating that the smear was under-decolorized. In addition, small flecks of precipitated stain are present. Notice that the precipitated stain is irregular in shape, which helps differentiate the flecks from bacteria.

A slide is also not acceptable for examination if microorganisms that should be gram-positive appear pink. This may indicate that either the Gram's iodine was not applied or the slide was over-decolorized.

Staining gram-positive and gram-negative control slides along with the patient's smear would confirm that proper staining technique was used.

If it is impossible to prepare a new smear, the poorly stained smear may still be salvaged. Remove immersion oil from the smear using xylol. Use appropriate procedures and personal protective equipment when using xylol, since it is a hazardous chemical. If the smear is under-decolorized, repeat the decolorization and counterstain steps. If the smear is over-decolorized, the slide should be stained again.