How to Perform a Gram Stain

Here are the five basic steps for performing a Gram stain:

1. Fix the sample to the slide
2. Stain the slide with the primary stain, crystal violet.
3. Treat the stain with iodine (a mordant).
4. Gently apply a decolorizer on the smear(s), then counterstain with safranin.
5. Gram-positive microorganisms such as Staphylococcus aureus appear bluish-purple. Gram-negative microorganisms such as Escherichia coli appear pinkish-red.

To learn more, check out the in-depth guide to Gram staining from MicrobeOnline. Read here.

Gram Staining Best Practices

Follow these best practices to perfect your Gram stain technique:

1. Don’t make your smear too heavy. Doing so may inhibit your ability to see the desired structures or organisms.
2. Always test your incoming stains and reagents. Products may not perform as expected if they were exposed to unfavorable conditions during shipping or storage.
3. Use a fresh culture. It should be less than 24 hours old. Gram stain reactions can be variable in older cultures.
4. Air dry the slide completely before fixing it.
5. Fix the specimen on the slide by using heat or methanol. If you are using  heat (for example, a flame), be careful not to overheat.
6. Don’t over-decolorize. An over-decolorized sample may lead to an incorrect result. Don’t squirt decolorizer directly on the smear. Instead, hold the slide at a slant and let the decolorizer flow over the slide. Stop decolorizing when the liquid flowing off the slide becomes clear. The decolorizing step should only take a few seconds. Rinse the decolorizer off with water.
7. Keep reagents closed between use.
8. Use the right microscope and lens required to view your sample. If you use the incorrect type of microscope or lens to view your stain, you may miss important details on the slide.
9. Take care of your microscope.
   • Clean your microscopes frequently with approved cleaners.
   • Only use immersion oil on the designated 100x objective lens. Immersion oil should only be used on dry microscopes.

Additional Resources

• LabCE Quality Control Smears Course: An online introductory course to Gram staining. Participants qualify for ASCLS P.A.C.E.-approved continuing education credits. Learn more.
• QC Microbiology Slides: Microbiologics offers a variety of control slides to help laboratories conduct training and proficiency testing. The Gram Stain Control Slide provides two air-dried and methanol-fixed droplets within two etched circles – one droplet of Gram-positive Staphylococcus aureus and one droplet of Gram-negative Escherichia coli. Learn more.

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