Grams Staining

Gram staining is a laboratory technique used to differentiate bacterial species into two groups based on the characteristics of their cell walls. The method was developed by Hans Christian Gram in 1884 and is widely used in microbiology to this day. The principle behind each steps are:

1. Primary Staining (Crystal Violet): The process begins with the application of crystal violet, a purple dye, to the bacterial smear. This stain is taken up by all cells.

2. Iodine Treatment (Gram’s Iodine): Iodine is then added to form a crystal violet-iodine complex within the cells. This step is crucial for the development of the reaction.

3. Decolorization: The smear is washed with a decolorizing agent (typically alcohol or acetone). This step differentiates bacteria into two groups based on their cell wall characteristics.

    a. Gram-Positive: Bacteria with thick peptidoglycan layers in their cell walls retain the crystal violet-iodine complex and remain purple.

    b. Gram-Negative: Bacteria with thinner peptidoglycan layers lose the crystal violet-iodine complex during decolorization. They are then counterstained with a red dye (safranin) to appear pink or red.

4. Counterstaining (Safranin): Gram-negative bacteria are counterstained with safranin, which imparts a red color to them. Gram-positive bacteria retain the purple color.

The key distinction in Gram staining is based on the structural differences in the cell walls of bacteria. Gram-positive bacteria have a thick layer of peptidoglycan, while Gram-negative bacteria have a thinner peptidoglycan layer surrounded by an outer membrane. This difference in cell wall structure affects the retention or loss of the crystal violet-iodine complex during the decolorization step. The Gram staining technique is valuable for categorizing bacteria and is an essential tool in microbiology for the initial identification of bacterial species. The information obtained from Gram staining helps guide further microbiological testing and treatment decisions.


  1. Bacterial Culture
  2. Microscope Slides
  3. Bunsen Burner or Alcohol Lam
  4. Inoculating Loop or Needle
  5. Crystal Violet Stain- 0.5-1.0%
  6. Gram’s Iodine Solution; 1% iodine or 2% KI
  7. Decolorizing Agent: ethanol (95%) or acetone.
  8. Safranin Stain- 0.5-1.0%
  9. Distilled Water in wash bottle
  10. Microscope:
  11. Immersion Oil (for Oil Immersion Objective):
  12. Bibulous Paper or Blotting Paper (soaking excess water)


  1. Take a clean grease free slide
  2. Pick a colony and emulsify with droplet of water and spread with loop to  make a thin smear or from the broth.
  3. Let the smear to dry
  4. Gently flood smear with crystal violet and let stand for 1 minute.
  5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
  6. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
  7. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide.
  8. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.
  9. Gently flood with safranin to counter-stain and let stand for 45 seconds.
  10. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
  11. Blot dry the slide with bibulous paper.
  12. View the smear using a light-microscope under oil-immersion.


Gram staining observations are based on the color and morphology of bacterial cells under a microscope after the staining process. Here are the typical observations for Gram staining:

1. Gram-Positive Bacteria:

Color: Retain the crystal violet-iodine complex and appear purple or violet.

Morphology:   The morphology of Gram-positive bacteria can vary and may include cocci (spherical cells) or bacilli (rod-shaped cells).

 Examples: Staphylococcus, Streptococcus, Bacillus, Clostridium.

2. Gram-Negative Bacteria:

 Color: Lose the crystal violet-iodine complex during decolorization and appear red or pink after counterstaining with safranin.

 Morphology: Gram-negative bacteria can have diverse morphologies, including cocci or bacilli.

 Examples: Escherichia coli, Salmonella, Pseudomonas, Neisseria.

3. Cell Arrangements:

   Observations may include the arrangement of cells, such as singles, pairs, chains, clusters, or other patterns.

4. Size and Shape:

   Gram staining allows for the observation of the size and shape of bacterial cells, providing information about their general characteristics.

It’s important to note that the concentrations mentioned here are general guidelines, and variations can occur based on laboratory practices or specific kit formulations. It is recommended to follow the manufacturer’s instructions or laboratory protocols for the specific reagents being used.

Additionally, the timing of each step, especially during the decolorization step, is critical. Over-decolorization can lead to false-negative results, while under-decolorization can result in false positives. Gram staining is a technique that may require some optimization based on the characteristics of the bacterial species being studied.

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