What is Gram Staining? 

Named after Hans Christian Gram, a Danish bacteriologist, Gram stain is one of the most powerful staining techniques within microbiology. This technique was introduced in 1882 to identify pneumonia-causing organisms. The Gram staining technique uses crystal violet or methylene blue as primary staining colors to distinguish gram-positive from gram-negative organisms. Under a microscope, the gram-positive organisms appear purple-brown, retaining the primary color. Gram-negative organisms appear pink or red as they do not acquire the color of the primary stain. 

To Understand the Gram Staining Technique Better

An example of a bulletproof vest vs. thick wooden fence analogy can be taken to understand the technique. Though the bulletproof vest is pretty thin compared to the thick wooden fence, a bullet cannot penetrate the bulletproof vest. The person wearing the vest, much like the gram-negative organism, will stay unharmed. However, a bullet shot at the thick wooden fence is bound to go through the barrier and blast it. The wooden fence, like the gram-positive organism, will get a hole in its structure. 

The most widely used technique, the gram stain, is a differential and complex procedure. The domain bacteria are isolated as per their cell wall composition through steps including staining and decolorization. Gram-negative bacteria have cell walls that contain thin layers of peptidoglycan, which forms 10 percent of the cell wall. Gram-positive bacteria have thick layers of peptidoglycan, which forms 90 percent of the cell wall. 

Difference between Gram-Positive Bacteria and Gram-Negative Bacteria

When the staining process is initiated, all bacteria pick up the color of crystal violet dye. However, using solvent dissolves the lipid layer of gram-negative organisms. This dissolution leads to the loss of the primary gram stain from gram-negative cell walls. In gram-positive organisms, the solvent dehydrates the cell walls leading to the closure of pores. The closed pores prevent the stain from diffusing, and so the bacteria retain the stain. Decolorization length is critical as prolonged treatment with a decolorizing agent would remove all the stains from the cell walls of both kinds of bacteria. The last step of counterstaining stains the gram-negative bacteria pink, distinguishing them from the gram-positive bacteria. 

"Gram-positive and gram-negative bacteria"

How is Gram Staining Performed?

Swabs contain clinical specimens from the throat, nostrils, wound, rectum, and cervix. The most common specimens used are blood, sputum, ascitic fluid, cerebrospinal fluid, synovial fluid, urine, and pleural fluid. The collected specimen should always be stored in sterilized containers. The steps to use the Gram stain are complex and precise. 

  1. The slide is prepared with a smear of the collected specimen or bacterial culture. The smear is fixed with heat. 
  2. The smear is stained with crystal violet, the primary dye that stains all cells purple. 
  3. The slide is washed with distilled water for few seconds. 
  4. The smear is then treated with the mordant iodine. The mordant helps retain the primary stain by forming a crystal violet-iodine complex or a CV-I complex. 
  5. This step is a differential one. The slide is washed with 95% ethyl alcohol, post which some bacteria hold on to the crystal violet stain, while some bacteria lose the stain and appear colorless. 
  6. The bacteria that retain the violet color are gram-positive, while the others are gram-negative. 
  7. The slide is washed with distilled water and drained. Safranin is used as a basic dye for counterstaining. The slide is washed again with distilled water and blot dry with an absorbent. 
  8. The slide is observed under a microscope. 
  9. The gram-positive bacteria retain the purple color and remain unaffected by the process of counterstaining. However, the gram-negative bacteria are de-colored and stained pink or red by safranin. 
  10. The gram-negative bacteria have thick cell walls with a simple chemical structure. Therefore, the alcohol treatment does not disrupt the CV-I complex, and consequently, the cells retain the purple color. 
  11. Gram-negative bacteria have thin cell walls which have a complex, multilayered structure. The lipopolysaccharide layer allows the CV-I complex to leak, leading to discolored cells. These cells later pick up the pink dye during counterstaining. 

Limitations of the Gram Staining Procedure 

The Gram staining might indicate false results if: 

  • Antibiotics have been used before submitting a specimen. 
  • The culture is too old or too young. 
  • The smear is fixed before air-drying it. 
  • The smear of the specimen is too thick. 
  • A low concentration of crystal violet is used. 
  • Too much heat is applied during heat fixation. 
  • The slide is excessively washed amid steps. 
  • There is insufficient exposure to iodine. 
  • The decolorization step is prolonged. 
  • There is excessive counterstaining. 
  • Finally, a lack of experience to prepare and observe the slide may lead to errors in results. 


Gram-positive bacteria 

Staphylococcus, Clostridium, Streptococcus, Listeria, Corynebacterium, Bacillus are some examples of gram-positive bacteria. 

Gram-negative bacteria 

Escherichia coli, Shigella spp., Pseudomonas aeruginosa, Salmonella typhi, Neisseria gonorrhoeae, Yersinia pestis, Chlamydia trachomatis are some examples of gram-negative bacteria. 

Smear preparation 

Preparing a smear is to fix bacteria on the slide and ensure that the sample does not get lost during the staining process. Smear can be prepared from both solid medium or broth medium. 

Cell wall 

A cell wall is an outer structural layer surrounding the plasma membrane of certain cells such as bacteria, fungi, and plants. The cell wall can be flexible, tough, or rigid. It acts as a barrier and provides protection and structural support to the cell. 

Stains or dyes 

Stains contain salts that are made up of positive or negative ions. A colored ion is called a chromophore, and the uncolored ion is called a counterion. The stains in which the chromophore is a positive ion are called basic dyes. If the chromophore is a negative ion, it is classified as an acidic dye. 

Heat fixing a smear 

The slide with the bacterial smear is first air-dried. It is then placed in a micro incinerator, or the side of the slide without the smear is passed a few times through a Bunsen burner. The slide can be stained once it is heat fixed. 

Common Mistakes 

Gram stain is a complex procedure, and interpreting slides can be a tricky affair. If the tiny smear is clumped, then there would be difficulty reading it. Another common mistake is over or under decolorization. Decolorization should be done for the right length of time and closely monitored to achieve the right results. For example, thicker smears need a longer duration of decolorization time. Cultures should not be stored for too long before the evaluation process. Old cultures stored for a long time tend to lose the peptidoglycan cell walls leading to wrong readings. 

Context & Applications 

This topic is significant in the professional exams for both undergraduate and graduate courses                                                                                                    

  • Bachelors of Science in Microbiology 
  • Bachelors of Science in Biology 
  • Master of Science in Microbiology 

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