Among the many ways bacteria can be differentiated, Gram staining remains a prominent technique in microbiology. This process categorizes bacteria as Gram-negative or Gram-positive, giving us insight into the cellular structure!
To begin the Gram staining process, a bacterial sample must be heat-fixed to a glass slide.
Heat Fixing Procedure
Use a sterile inoculating loop to place a drop of distilled water onto a clean glass slide.
With the loop, take a small sample of bacteria and spread it in the water on the slide.
Allow the sample on the slide to air dry completely.
Give the slide 2-3 passes across the bunsen burner, a couple of inches above the flame.
Gram Staining Procedure:
Flood the heat-fixed sample slide with crystal violet dye and let sit for 1 minute.
Hold slide upright and rinse with distilled water, avoiding spraying directly onto sample.
Flood the slide with iodine mordant and let sit for 1 minute.
Repeat step 2 to rinse the iodine off of the slide.
While holding the slide at a tilt, allow drops of ethanol to flow over the sample until purple no longer runs (maximum 10 seconds).
Ensure the ethanol is rinsed from the slide, using step 2.
Flood the slide with safranin dye and let sit for 1 minute.
Repeat step 2 to rinse the slide for the final time.
Gently pat the slide between sheets of bibulous paper to dry.
Observe the slide under a microscope at oil lens magnification.
Gram (+) positive bacteria will appear purple under the microscope, and Gram (-) negative bacteria will appear pink. This is because Gram (+) bacteria have thicker walls with more peptidoglycan, whereas Gram (-) bacteria have thinner walls with more lipids which allow the stain to wash off.
The staining procedure involves first dyeing the bacteria purple, and applying a mordant to fix the stain to them. Then if the bacteria are Gram (-), they are decolorized of the purple dye. Finally, the pink dye is applied so that the Gram (-) bacteria will be visible.
References:
Cleveland Clinic. (2022, March 16). Gram Stain: What It Is, Purpose, Procedure & Results. Cleveland Clinic. https://my.clevelandclinic.org/health/diagnostics/22612-gram-stainGram Stain Protocol. (2024).
Cornell University College of Veterinary Medicine. https://www.vet.cornell.edu/animal-health-diagnostic-center/testing/testing-protocols-interpretations/gram-stain-protocol
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