Gram Stain Procedure in Microbiology

What Gram Staining Is and How to Do It

Gram positive bacteria stain violet, while Gram negative bacteria stain pink or red.
Gram positive bacteria stain violet, while Gram negative bacteria stain pink or red. De Agostini Picture Library / Getty Images

The Gram stain is a differential method of staining used to assign bacteria to one of two groups (gram-positive and gram-negative) based on the properties of their cell walls. It is also known as Gram staining or Gram's method. The procedure is named for the person who developed the technique, Danish bacteriologist Hans Christian Gram.

How the Gram Stain Works

The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria.

The Gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain.

  1. The primary stain (crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-positive and gram-negative cells have peptidoglycan in their cell walls, so initially all bacteria stain violet.
  2. Gram's iodine (iodine and potassium iodide) is applied as a mordant or fixative. Gram-positive cells form a crystal violet-iodine complex.
  3. Alcohol or acetone is used to decolorize the cells. Gram-negative bacteria have much less peptidoglycan in their cell walls, so this step essentially renders them colorless, while only some of the color is removed from gram-positive cells, which have more peptidoglycan (60-90% of the cell wall). The thick cell wall of gram-positive cells is dehydrated by the decolorizing step, causing them to shrink and trapping the stain-iodine complex inside.
  1. After the decolorizing step, a counterstain is applied (usually safranin, but sometimes fuchsine) to color the bacteria pink. Both gram-positive and gram-negative bacteria pick up the pink stain, but it is not visible over the darker purple of the gram-positive bacteria. If the staining procedure is performed correctly, gram-positive bacteria will be purple, while gram-negative bacteria will be pink.

    Purpose of the Gram Staining Technique

    The results of the Gram stain are viewed using light microscopy. Because the bacteria are colored, not only is their Gram stain group identified, but their shape, size, and clumping pattern may be observed. This makes the Gram stain a valuable diagnostic tool for a medical clinic or lab. While the stain may not definitely identify bacteria, often knowing whether they are gram-positive or gram-negative is sufficient for prescribing an effective antibiotic.

    Limitations of the Technique

    Some bacteria may be gram-variable or gram-indeterminate. However, even this information may be useful in narrowing down bacterial identity. The technique is most reliable when cultures are less than 24 hours old. While it can be used on broth cultures, it's best to centrifuge them first. The primary limitation of the technique is that it yields erroneous results if mistakes are made in the technique. Practice and skill are needed to produce a reliable result. Also, an infectious agent may not be bacterial. Eukaryotic pathogens stain gram-negative. However, most eukaryotic cells except fungi (including yeast) fail to stick to the slide during the process.

    Gram Staining Procedure

    Materials

    • Crystal violet (primary stain)
    • Gram's iodine (mordant, to fix crystal violet in the cell wall)
    • Ethanol or Acetone (decolorizer)
    • Safranin (secondary stain or counterstain)
    • Water in a squirt bottle or dropper bottle
    • Microscope slides
    • Compound microscope

    Note it's better to use distilled water than tap water, as pH differences in water sources may affect results.

    Steps

    1. Place a small drop of bacterial sample on a slide. Heat fix the bacteria to the slide by passing it through the flame of a Bunsen burner three times. Applying too much heat or for too long can melt the bacteria cell walls, distorting their shape and leading to an inaccurate result. If too little heat is applied, the bacteria will wash off the slide during staining.
    2. Use a dropper to apply the primary stain (crystal violet) to the slide and allow it to sit for 1 minute. Gently rinse the slide with water no longer than 5 seconds to remove excess stain. Rinsing too long can remove too much color, while not rinsing long enough may allow too much stain to remain on gram-negative cells.
    1. Use a dropper to apply Gram's iodine to the slide to fix the crystal violet to the cell wall. Let it sit for 1 minute.
    2. Rinse the slide with alcohol or acetone about 3 seconds, followed immediately with a gentle rinse using water. The gram-negative cells will lose color, while the gram-positive cells will remain violet or blue. However, if the decolorizer is left on too long, all cells will lose color!
    3. Apply the secondary stain, safranin, and allow it to sit for 1 minute. Gently rinse with water no longer than 5 seconds. The gram-negative cells should be stained red or pink, while the gram-positive cells will still appear purple or blue.
    4. View the slide using a compound microscope. A magnification of 500x to 1000x may be needed to distinguish cell shape and arrangement.

    Examples of Gram-Positive and Gram-Negative Pathogens

    Not all bacteria identified by the Gram stain are associate with diseases, but a few important examples include:

    • Gram-positive cocci (round) -Staphylcoccus aureus
    • Gram-negative cocci - Neisseria meningitidis
    • Gram-positive bacilli (rods) - Bacillus anthracis
    • Gram-negative bacilli - Escherichia coli