• Gram staining is a method used to differentiate two groups of bacteria.
• Based on the cell wall.
• Developed by Hans Christian Gram in the 19^th
• This method is used to identify gram +^ve gram –^ve
• After performing staining the Gram-positive bacterium stain violet and Gram-negative bacterium stain pink.
• This technique of staining can’t define bacteria, used to general identification such as morphology.

 

How does Gram staining work?

• Gram staining is a three-step process:

Staining with crystal violet dye, decolorization, counterstaining

• Due to the difference of (thick and thin layer) cell wall, the cells are stained violet and red.
• The cell wall of the bacterium is made up of peptidoglycan.

Steps of Gram staining:

• Stain the cell using crystal violet dye.
• Add Gram’s iodine. (to form a complex between dye and iodine CV-I complex)
• Add decolorizer (ethyl alcohol/acetone).
• 
• Adding decolorizer dehydrates the cell.
• After adding decolorizer, the CV-I complex trapped between gram-positive cell walls because it is very thick, and the CV-I complex in the case of gram-negative is dissolved with water.
• Add counterstain (safranin)
• Adding safranin doesn’t disturb the gram-positive cell it only affects gram-negative cells and stain red.

 

Gram-positive bacteria:

• Contain a single membrane (monoderm) which is surrounded by a thick layer of peptidoglycan.
• Stain purple after gram staining.
• Teichoic acids are present on the peptidoglycan.

Gram-negative bacteria:

• A thin layer of peptidoglycan is present.
• LPS present (lipopolysaccharide)1

 

Difference between Gram Positive and Gram Negative bacteria:

Property	Gram-positive	Gram-negative
Cell wall thickness	Around 40-80nmnm	Thinner than gram-positive,30nm
Gram staining	Stain blue(violet/purple) color in gram staining	Stain pink in gram staining
LPS (Lipopolysaccharide)	Absent	present
Teichoic acid	Cell wall contains teichoic acid (-ve charged)	Teichoic acids are absent
Periplasmic space	Small and single	Two periplasmic space are present
Lipid content	Low	High (because of LPS)
PG (peptidoglycan layer)	Thick and multilayered	Thin and single-layered
Porins	Absent	present
Flagellar structure	2 rings	4 rings
Toxins	Exotoxin	Endo or exotoxin
Pathogenicity	Some belong to the pathogenic group	Most of them are pathogens
Outer membrane	Absent	Present
Mesosome	More prominent	Less prominent
Morphology	Cocci or rods (spore-forming)	Rods (non-spore forming)
Drug resistance	More susceptible	More resistant to drugs
Examples	Staphylococcus, streptococcus…	Escherichia, salmonella…
Resistant to drying	High	Sensitive/low

 

Peptidoglycan:

• Peptidoglycan is also known as
• PG is a polymer of sugars and amino acids.
• PG forms a mesh-like structure outside the plasma membrane.
• The sugar molecules (of Murein) of alternating residues NAM and NAG linked by β-1,4 (glycosidic bond)
• NAM (N-acetylglucosamine), NAG (N-acetylmuramic acid).
• NAM is a peptide chain of 3-5 AA (amino acids).
• In the peptidoglycan of gram-positive L-Lys is present and in gram-negative in place of L-Lys DAP (diaminopimelic acid) is present.

Question:

Can we heat fix the cell in this method of staining? Explain why?