How to Get Better at Gram Staining

The gram stain is a critical first step in the identification of disease-causing bacteria. An excellent gram stain results in a microscope slide with a smear of bacterial cells clearly stained purple for gram-positive or red for gram-negative. The bacterial smear is thin enough to microscopically identify the shapes of individual cells. The slide is not contaminated with organisms other than your test sample. With practice, you can avoid the common gram stain errors that cause contaminated slides, slides without smears, smears that are too thick, slides with purple cells and red cells, and slides with cells stained the wrong color.

Things You'll Need

  • Inoculating loop
  • Bunsen burner
  • Flint striker
  • Bacterial sample
  • Microscope slide
  • Gram stain reagents
  • Squeeze bottle with water
  • Rinse tray
  • Light microscope
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Instructions

    • 1

      Take your time, paying attention to detail throughout the procedure.

    • 2

      Use sterile technique at all times --- from wiping your bench with 10 percent bleach before and after the staining procedure to sterilizing your inoculating loop before and after collecting your bacterial sample.

    • 3

      Make your bacterial smear by applying about a millimeter or two of bacterial sample to a drop of distilled water on a clean microscope slide. Swirl the sample to mix it with the water and then spread it to make a thin smear over the center and unlabeled portion of your slide.

    • 4

      Fix the smear to the slide by passing the microscope slide through the blue portion of the Bunsen burner flame at least three times, smear side up. Proper procedure is to gently move the slide side to side as you pull it through the flame, making sure the entire smear is being heated.

    • 5

      Flood the smear fully at each step, whether you're applying the stains, alcohol decolorizer or water rinse; insufficient coverage of stain and insufficient wash steps lead to slides with cells of both colors.

    • 6

      Rinse your slide completely during washing but don't over-rinse. Proper procedure involves holding the slide vertically while using a squeeze bottle to run a stream of water over the smear quickly and thoroughly, removing loose stain or decolorizer.

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