Some sources of error include:
Under- or over-decolorized smears
Thick smears are difficult to decolorize properly. In addition, the presence of precipitate (crystal violet stain crystals) is indicative of under-decolorization.
Overrinsing with decolorizer can cause over-decolorization. A slide is 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.
In this image...
- Although this smear (see image) 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.
Overheating
Overheating of smears (during a heat fixation step) may cause microorganisms or cells to lyse or become distorted.
Use control slides
Staining gram-positive and gram-negative control slides along with the patient's smear would confirm that the proper staining technique was used.
Note: If it is impossible to prepare a new smear, the poorly stained smear may still be salvaged. Remove immersion oil from the smear using xylene or a xylene substitute (a clearing agent, frequently used in the histology lab). Use appropriate procedures and personal protective equipment when using xylene, 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.
