Bacteria are one of the oldest forms of life on earth, thought to have originated 3.22 billion years ago (Homann et al., 2018). There is an estimated one trillion species in existence, although most of these have not yet been discovered. They usually exist as single cell organisms (prokaryotic), however they form groups called colonies which are visible to the human eye.




Bacterial cells do not contain a nucleus- they contain loose genetic material housed in a cell membrane and cell wall. There are variations within the composition of these outer cell structures, therefore classifying bacteria into two groups: Gram-positive and Gram-negative (see Figure 1).


Gram-positive bacteria have a thin inner plasma membrane (composed of lipids) and a thick peptidoglycan layer (composed of carbohydrates and proteins).


Gram-negative bacteria have a thin inner plasma membrane, a thin peptidoglycan layer, and then another thin outer plasma membrane (Silhavy et al. 2010).


Gram-positive, Gram-negative and broad-spectrum antibiotics


Some antibiotics are target specific to Gram-positive or Gram-negative. For example, penicillin targets proteins within the peptidoglycan layer. This makes penicillin effective against gram-positive bacteria, because they contain a thick peptidoglycan layer which is also the outer most layer. Penicillin however is not very effective against gram-negative bacteria, because of the outer membrane protecting the peptidoglycan layer. Therefore, Gram-negative bacteria are already resistant to penicillin due to their outer structure.


When antibiotics target both Gram-positive and Gram-negative bacteria, they are known as broad-spectrum antibiotics.

Figure 1. Gram positive and gram negative cell wall structures.

Gram staining

Bacteria are identified as Gram-positive or Gram-negative by carrying out a Gram stain test, named after Hans Christian Gram (1853-1938) who invented the test (Colco, 2005):


  1. The bacteria sample is dried onto a slide, and the cells are flooded with crystal violet, a purple dye which binds to peptidoglycan.

  2. The cells are then flooded with iodine, a mordant which fixes the crystal violet dye to the peptidoglycan.

  3. The cells are then washed with alcohol, which decolourises the cells that are not stained.

  4. The cells are flooded with safranin, a counterstain which stains the remaining cells red.


Because the Gram-positive bacteria have an accessible peptidoglycan layer, they are stained purple by the crystal violet. The Gram-negative bacteria do not have an accessible peptidoglycan layer, and therefore do not retain the purple dye, and it is washed from the stain with alcohol, and instead retains the red counterstain safranin (see Figure 2).

Figure 2. Gram staining.



Colco R (2005) Gram Staining. Current Protocols in Microbiology. 00(1): Available at: Accessed August 2020.


Homann M, Sansjofre P, Van Zuilen M, et al. (2018) Microbial life and biogeochemical cycling on land 3,220 million years ago. Nature Geosci 11: 665–671.

Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol. 2(5) Available at:

Accessed August 2020.

© 2020 ABX

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