Microbiology Basics

Gram (-/+) staining

Post by Hazel Silistre, Alumna of Institut Pasteur
Inspired by her international name, Hazel has left Turkey in 2008 to get her university education in Germany and has lived in multiple countries ever since. Her interest to understand life by deciphering bacterial mechanisms led her to pursue a PhD in microbiology. After finishing her PhD at the University of Nottingham, she has left the U.K. and continued her research at Institut Pasteur in Paris. The creative aura of Paris has given her courage to leave the laboratory and follow her passion for writing full-time. She is committed to communicating science to the public and aspires to be a science journalist.

Let’s say that you got a bacterial infection and when you stopped by your doctor’s office to discuss the results of the urine or the blood test, he happened to tell you the name of the bacterium. Would you be Googling it when you get back home? If you do, the first thing you will come across is a Gram -/+ distinction. “……………. is a gram-negative / gram-positive bacterium”. What does this mean?

Gram-staining is a procedure established by Christian Gram in the late 19th century. It classifies bacteria that remain purple (violet) after the multi-step staining using the crystal violet, iodine and safranin solutions as Gram +, and those that appear pink as Gram – [1].

Gram-staining of bacteria: Gram+ (purple) and Gram- (pink)

Why are some cells stained pink whereas others are violet? To understand, we have to look at the composition of their cell walls. The cell wall is composed of layers of peptidoglycan, a complex molecule composed of smaller sugar and aminoacid molecules linked alternatively in a chain. Aminoacids make up peptides (and peptides form proteins), simple sugars scientifically termed monosaccharides form glycans (polysaccharides in other words) and together they make up a peptidoglycan. Cell walls of Gram+ bacteria have thicker peptidoglycan layers in their cell walls than of Gram- bacteria. So, what happens during Gram-staining is that initially, crystal violet solution stains all cells purple, however, it gets washed off from those bacteria having cell walls with thin peptidoglycan layers. Safranin solution re-stains those cells that lost their purple color in pink while bacteria with thick peptidoglycan layers in their cell walls remain purple. The loss of color during staining attributes the minus sign (-) and we name these bacterial cells Gram – . For preserving their purple color since the beginning, we give a thumbs up and denominate the other cells Gram + [1] !

differences in the cell walls of Gram- and Gram+ bacteria

An outer membrane supports the thin peptidoglycan layer in Gram- bacteria. This outer membrane is like the cytoplasmic membrane where proteins that enable selective permeability, porins, and attachment, adhesins, are dispersed in a lipid bilayer. In Gram- bacteria, lipopolysaccharide (LPS) molecules are found attached to the outer membrane and if released to the bloodstream, they may give rise to endotoxic shock in humans [2].

The outer membrane confers several advantages to Gram- bacteria: it protects the peptidoglycan layer from cleavage by the lysozyme enzyme and impedes the intake of antibiotics (a chemical that inhibits the growth of microorganisms or kills them). An enzyme is a protein that accelerates the rate of biochemical reactions that take place in a cell. Lysozyme is a naturally occurring enzyme found in our bodily secretions such as tears, saliva, and mothers’ milk. It can be considered as an antimicrobial agent because it cleaves the bonds between sugar and peptide chains in the peptidoglycans of bacterial cell walls, killing bacteria. Gram+ bacteria are more sensitive to the antimicrobial actions of penicillin, acids, iodine, detergents and lysozyme [2, 3, 4].


  1. Coico, R. (2006). Gram Staining. Current Protocols in Microbiology, 00: A.3C.1-A.3C.2
  2. Madigan, Martinko, and Parker (2003). Brock Biology of Microorganisms, 10th edn. Prentice Hall/Pearson Education Upper Saddle River, NJ, USA
  3. May and Grabowicz (2018). The bacterial outer membrane is an evolving antibiotic barrier. Proceedings of the National Academy of Sciences, 115(36): 8852-8854
  4. Oliver and Wells (2015). Lysozyme as an alternative to growth promoting antibiotics in swine production. Journal of Animal Science and Biotechnology, 6:35