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About Gram Positive and Gram Negative Bacteria

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I will like to know how one can clearly differentiate Gram Negative and Gram Positive bacteria under the microscope and also using reagents.


Oct 21st, 2017

Gram positive and gram negative refers to how a bacteria react to a gram stain. If it takes the initial stain, it will be purple and be considered gram positive. If it doesn't take the initial stain, it will be pink and gram negative. The difference is the outer casing of the bacteria. A gram positive bacteria will have a thick layer of peptidoglycan (a sugar-protein shell) that the stain can penetrate. A gram negative bacteria has an outer membrane covering a thin layer of peptidoglycan on the outside. The outer membrane prevents the initial stain from penetrating.

In more details 

Gram-positive bacteria are classified as bacteria that retain a crystal violet dye during the Gram stain process. Gram-positive bacteria will appear blue or violet under a microscope, whereas Gram-negative bacteria will appear red or pink. The difference in classification is largely based on a difference in the bacteria's cell wall structure. 

The following characteristics are generally present in a Gram-positive bacteria: 

1. A very thick cell wall (peptidoglycan) 
2. If a flagellum is present, it contains two rings for support (an additional two rings are not necessary, unlike in Gram-negative bacteria, because the thick cell wall is supportive enough) 
3. Teichoic acids are present, which serve to act as chelating agents, and also for certain types of adherence 

Gram-negative bacteria are those that do not retain crystal violet dye in the Gram staining protocol. Gram-positive bacteria will retain the dark blue dye after an alcohol wash, whereas Gram-negative bacteria do not. In a Gram stain test, a counterstain is added after the crystal violet, which colors all Gram-negative bacteria a red or pink color. The test itself is useful in classifying two distinctly different types of bacteria based on structural differences in their cell walls. 

Many species of Gram-negative bacteria are pathogenic, meaning they can cause disease in a host organism. This pathogenic capability is usually associated with certain components of Gram-negative cell walls, in particular the lipopolysaccharide (also known as LPS or endotoxin) layer. 

The following characteristics are displayed by Gram-negative bacteria: 

1. Cell walls only contain a few layers of peptidoglycan (which is present in much higher levels in Gram-positive bacteria) 
2. Cells are surrounded by an outer membrane of lipopolysaccharide outside the peptidoglycan layer 
3. Porins exist in the outer membrane, which act like pores for particular molecules 
4. There is a space between the layers of peptidoglycan and the secondary cell membrane called the periplasmic space 
5. The S-layer is directly attached to the outer membrane, rather than the peptidoglycan 
6. If present, flagella have four supporting rings instead of two 
7. No teichoic acids are present 
8. Lipoproteins are attached to the polysaccharide backbone whereas in Gram-positive bacteria no lipoproteins are present

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