Gram Positive Vs Gram Negative Bacteria

A differential staining method called Gram staining divides the bacteria into the Gram Positive and Gram Negative. Gram Staining was introduced by Danish Bacteriologist Hans Christian Gram in 1884.


 Reagents Used in Gram Staining

• Crystal Violet, the primary stain
• Iodine, the mordant
• A decolorizer made of acetone and alcohol (95%) 
• Safranin, the counterstain

Principle of Gram Staining:
The Gram Positive bacterium takes the colour of Crystal voilet and does not decolourize on alcohol treatment and appears purple in colour. However, the Gram negative decolourizes on alcohol treatment and takes the colour of safranine and appears as red in colour. The color difference in the Gram stain arises because of differences in the cell wall structure of gram-positive and gram-negative cells. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan as well as secondary polymers. 

Figure 1. Cell wall of Gram Positive Bacteria (Picture Credit-Biorender.com)

Gram-negatives have a thin peptidoglycan layer and in addition an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization ( Beveridge TJ,2001). The peptidoglycan layer makes up about 60-90% of gram positive cell wall which is just 10-20% in case of gram negative cell wall. Decolourizing the cell causes this thick cell wall to dehydrate and shrink causing the pores to close in cell wall and thus as the end of procedure the gram positive cells will be stained a purplish blue colour. Besides peptidoglycan layer, gram negative cells have an additional outer layer made up of lipid, polysachharides and proteins.The alcohol treatment will dissolve the lipid in the cell wall. As a result, the crystal-violet iodine complex will be leached out from the cells and thus stained with the colour of safranine as reddish-pink colour. 

The differentiation of bacteria into Gram Positive and Gram Negative Bacteria is preliminary step in the classification and differentiation of microorganisms.

Table 1 Procedure of Gram Staining 

Shape	Gram-Positive Bacteria		Gram-Negative Bacteria
Rods	Aerobe or Facultative anaerobe	Anaerobe	Aerobe	Facultative anaerobe	Anaerobe	Microaerophile
	Bacillus Corynebacterium Listeria Lactobacillus Nocardia Mycobacterium	Actinomyces Clostridium	Pseudomonas	Salmonella Shigella Klebsiella Proteus Escherichia Yersinia Bordetella Haemophilus Brucella Pasteurella Vibrio	Bacteroides Fusobacterium Prevotella	Campylobacter
Cocci	Aerobe or Facultative anaerobe	Anaerobe	Aerobe
	Staphylococcus Streptococcus Enterococcus	Peptostreptococcus	Neisseria
Spirochaetes				Aerobe	Anaerobe
				Leptospira	Borrelia Treponema

Some of the Gram Staining Pictures of Medically Important bacteria:

Gram Positive Cocci in Clusters

References: 

• Chesbrough M. (1984), Medical laboratory manual for tropical countries / Vol.II, Microbiology.
• Waites M.J and et al. (2001),  Industrial Microbiology: An Introduction, Blackwell Science.
• Beveridge TJ,(2001), Use of the gram stain in microbiology, 76(3):111-8.
• http://microbeonline.com/gram-staining-principle-procedure-results/
• http://austincc.edu/microbugz/gram_stain.php