* The Gram staining is fundamental to the phenotypic characterization of bacteria.
* It is a method of staining used to distinguish and classify bacterial species into two large groups : gram - positive bacteria and gram - negative bacteria.
* The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.
* This gram stain technique continues to be a standard procedure in medical microbiology.
Principle :
"The Gram staining procedure differentiates organisms of the domain Bacteria according or cell wall structure. Gram- positive cells have a thick peptidoglycan layer and stain blue to purple. Gram - negative cells have a thin peptidoglycan layer and stain red to pink".
Steps :
Gram - positive cell wall :
* Gram - Positive bacteria have a thick mesh - like cell wall which is made up of peptidoglycan (50 - 90% of cell wall), which stains purple.
* Peptidoglycan is mainly a polysaccharide composed of two subunits called N- acetyl glucosamine and N- acetyl muramic acid.
* As adjacent layers of peptidoglycan are formed, they are cross linked by short chains of peptids by means of a transpeptidase enzyme, resulting in the shape and rigidity of cell wall.
* The thick peptidoglycan layer of Gram - positive organisms allows these organisms to retain the crystal violet - iodine complex and stains the cells as purple.
Gram - Negative Cell wall :
* Gram - negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall).
* Therefore, they lose the crystal violet - iodine complex during decolorization with the alcohol, rinse, but retain the counter stain Safranin.
* As a result, gram negative cells appear reddish or pink.
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| Gram positive VS Gram negetive cell wall |
Procedure :
* Take a clean, grease free slide.
* Prepare the smear of suspension on the clean slide with a loopful of sample.
* Air dry and heat fix.
* Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water.
* Flood the gram's iodine for 1 minute and wish with water.
* Then, wash with 95% alcohol or acetone for about 10-20 seconds and rinse with water.
* Add Safranin for about 1 minute and wash with water.
* Air dry, Blot dry and observe under microscope.
Stain Reaction :
The four basic steps of the Gram Stain are :
1) Application of the primary stain crystal violet (CV) to a heat fixed smear of bacterial culture.
* CV dissociates in aqueous solutions into CV+ and Cl- ions.
* These two ions then penetrate through the cell wall and cell membrane of both Gram - positive and Gram - negative cells.
* The CV+ ions later interacts with negatively charged bacterial components and stains the bacterial cells purple.
2) Addition of Gram's Iodine
* This step is known as "fixing the dye".
* Iodine (I - or - I3 -) acts as a mordant and as a trapping agent.
* A mordant is a substance that increases the affinity of the cell wall for stain by binding to the primary stain, thus forming an insoluble complex which gets trapped in cell wall.
* In the Gram stain reaction, the crystal violet and iodine form an insoluble (CV - I) which serves to turn the smear a dark purple color.
* At this stage, all cells will turn purple.
3) Decolorizations with 95% ethyl alcohol
* This step is known as "Solvent treatment".
* Alcohol/ acetone dissolves the lipid outer membrane of Gram negative bacteria, thus leaving the peptidoglycan layer exposed and increases the porosity of the cell wall.
* The CV - I complex is then washed away from the thin peptidoglycan layer, leaving Gram negative bacteria colorless.
* On the other hand, alcohol has a dehydrating effect on the cell walls of gram positive bacteria which causes the pores of the cell wall to shrink.
* The CV - I complex gets tightly bound into the multi-layered Gram positive cell wall thus staining the cells purple.
4) Counterstain with safranin
* The decolorized Gram negative cells can be rendered visible with a suitable counterstain, which is usually positively charged safranin.
* Safranin stains them red.
* Red colour which adheres to the Gram positive bacteria is masked by the purple of the crystal violet.
* Basic fuschin is sometimes used instead of Safranin in rare situations.
Observation :
Summary
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| Gram's staining Summary |
