Gram Staining- Principle, Reagents, Procedure, Steps, Results


Gram staining is a simple and rapid method to differentiate bacteria into two major groups: Gram-positive and Gram-negative. This distinction is based on the chemical and physical properties of their cell walls, which affect how they react to a series of stains. Gram staining is one of the most important and widely used techniques in microbiology, as it helps in the identification and characterization of bacteria, especially in clinical settings.

Gram staining was developed by a Danish physician and bacteriologist named Hans Christian Gram in 1882. He devised this technique while he was studying lung tissue samples from patients who had died of pneumonia. He noticed that some bacteria retained a purple dye called crystal violet, while others were decolorized by an alcohol solution and took up a red dye called safranin. He published his findings in a paper titled "Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten" (On the isolated staining of schizomycetes in section and dry preparations).

Gram staining is based on the principle that bacteria have different types of cell walls that vary in their thickness, structure and composition. Gram-positive bacteria have a thick layer of peptidoglycan, a polymer of sugars and amino acids, that forms the main component of their cell wall. Peptidoglycan can retain the crystal violet-iodine complex that forms during the staining process, and resist the decolorization by alcohol. Therefore, Gram-positive bacteria appear purple or blue under the microscope.

Gram-negative bacteria have a thin layer of peptidoglycan that is surrounded by an outer membrane composed of lipids, proteins and polysaccharides. The outer membrane can be dissolved by alcohol, which also removes the crystal violet-iodine complex from the peptidoglycan layer. Therefore, Gram-negative bacteria are decolorized by alcohol and appear pink or red after counterstaining with safranin.

Gram staining involves four steps: primary staining with crystal violet, mordanting with iodine, decolorizing with alcohol or acetone, and counterstaining with safranin. The procedure takes about 10 minutes to complete and requires a microscope, glass slides, inoculating loops, stains, water and ethanol or acetone.

Gram staining is useful for several purposes:

  • It provides a preliminary identification of bacteria based on their morphology (shape) and arrangement (clusters, chains, etc.).
  • It helps to select appropriate antibiotics for treating bacterial infections, as Gram-positive and Gram-negative bacteria have different susceptibilities to different drugs.
  • It aids in the detection of mixed infections by revealing the presence of more than one type of bacteria in a sample.
  • It serves as a quality control measure for bacterial cultures by ensuring their purity and viability.

However, Gram staining also has some limitations:

  • It cannot stain all types of bacteria, such as acid-fast bacteria (e.g., Mycobacterium), spirochetes (e.g., Treponema), mycoplasmas (e.g., Mycoplasma) and some intracellular bacteria (e.g., Chlamydia).
  • It may give false results due to over-decolorization or under-decolorization, poor quality of stains or slides, age or condition of bacterial cultures, or presence of interfering substances (e.g., blood or mucus).
  • It does not provide a definitive identification of bacteria, as some bacteria may have similar Gram reactions but different characteristics (e.g., Staphylococcus and Streptococcus are both Gram-positive cocci but have different biochemical properties).
  • It may not reflect the actual state of bacteria in vivo (in the body), as some bacteria may change their cell wall structure or composition in response to environmental factors (e.g., temperature, pH or antibiotics).

Therefore, Gram staining should be complemented by other methods of bacterial identification and characterization, such as biochemical tests, serological tests, molecular tests or culture methods.