Gram stain – What is Gram-positive and Gram-negative bacteria?

The Gram stain is a very important step in the initial characterization and classification of bacteria. After all, this staining method allows bacteria to be viewed under an optical microscope, since without staining it is impossible to observe them or identify their structure.

Gram’s staining method received its name in honor of the Danish pathologist Hans Christian Joachim Gram who made the discovery in 1884 and, incidentally, to this day remains the most used in clinical analysis and microbiology laboratories. Through staining it is possible to identify and differentiate the two main groups of bacteria, whether Gram-positive or Gram-negative.


In short, the Gram staining procedure allows bacteria to retain color based on differences in the chemical and physical properties of the cell wall. In fact, the use of dyes allows to increase the contrast and highlight the bacterial structure.

Coloring involves 3 main steps:

  • Coloring with crystal violet (a water-soluble dye, purple);
  • Decolorization (using ethanol / acetone);
  • Counter staining (using Safranina dye, red).


At first, there are different degrees of permeability in the wall of Gram-positive and Gram-negative microorganisms.

Gram-positive bacteria retain the violet crystal due to the presence of a thick layer of peptidoglycan (polymer consisting of sugars and amino acids that originate a kind of mesh outside the bacteria’s cell membrane) in their cell walls, appearing in color purple.

Gram-negative bacteria, on the other hand, have a thinner peptidoglycan wall that does not retain the violet crystal during the discoloration process and is red in the final staining process.

Examples of Gram-positive bacteria

Bacillus, Nocardia, Clostridium, Propionibacterium, Actinomyces, Enterococcus, Cornyebacterium, Listria, Lactobacillus, Gardnerella, Mycoplasma, Staphylococcus, Streptomyces, Streptococcus.

Gram positive

Examples of Gram-negative bacteria

Escherichia, Helicobcater, Hemophilus, Neisseria, Klebsiella, Enterobacter, Chlamydia, Pseudomonas, Salmonella, Shigella.



  1. Cover the smear with methyl violet and then leave for approximately 15 seconds;
  2. Add an equal amount of water to the slide covered with methyl violet and then let it act for another 45 seconds;
  3. Drain the dye and wash in a stream of running water; Cover the slide with diluted lugol (1/20) and let it act for approximately 1 minute;
  4. Drain the lugol and wash in a stream of running water;
  5. Add ethyl alcohol (99.5º GL) on the slide; bleaching it, until no more dye is released;
  6. Wash in a stream of running water;
  7. Cover the slide with safranin and let it act for approximately 30 seconds;
  8. Wash in a stream of running water;
  9. Allow to air dry, or dry gently with the aid of a clean filter paper;
  10. Visualize under the microscope. Right after, read in immersion objective (100 X).

Gram staining method


In fact, the methodology will help in the identification of a bacterial infection, determining what type of bacteria is. In other words, the differentiation of Gram-negative and Gram-positive bacteria allows the doctor to determine an effective treatment for the patient, thus applying the appropriate therapy in each case.

Even though not all bacteria can be differentiated through Gram stain, the methodology certainly has great application in clinical diagnosis and biological research.


by Abdullah Sam
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