Explain in detail principle and significance of Gram staining.
Answers
Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet
The Gram stain is the most important staining procedure in microbiology. It is used to differentiate between gram positive organisms and gram negative organisms. Hence, it is a differential stain.
I would like to give an elaborate explanation for the reagents being used in this gram staining and why it is used!
First of all, the gram staining was named after Dr. Christian Grams to differentiate between gram positive and negative bacteria.
→ Bacterial cells are divided into 2 major groups based on their cell wall.
→ those Bacteria that accepts and retains gram dye like crystal violet are called Gram positive bacteria
→those bacteria that loses grams dye are called Gram Negative Bacteria
The gram stain uses four different reagents and their mechanism of actions are:
Crystal violet(primary stain) is a cationic stain attracted by anionic cell wall and stains all the cell purple
Gram's iodine(mordant) forms an insoluble complex by binding to the primary stain. The resultant CV-1 complexes wil intensity the colour of stain and all the cell's wil appear purple black. Only in positive cells, this cV-1 complexes binds to the magnesium-ribonucleic acid component of the cell wall. The resultant Mg-RNA-CV-1 complex is more difficult to remove than the smaller CV-1 complex.
Ethyl alcohol 95% serves as protein dehydrating agent. Its action is determined by the lipid concentration of the microbial cell wall. The low lipid concentration is important for the retention of Mg-RNA-CV-1 complex.
Therefore small amount of lipid content is readily dissolved by the action of alcohol, causing formation of minute cell wall pores. These are then closed by alcohol's dehydrating effect.
As a result. The tightly bound primary stain in difficult to remove and the cell's remain purple.
In Gram Negative cells, the high lipid concentration found In the outer layer of cell walls is dissolved by alcohol,creating large pores in the cell wall that do not closely appreciable on hydration of cell wall proteins.
This facilitates release of the unbound CV-1 complexes, leaving these cells colourless or stained.
4. Safranin, is the final stain (counter stain), which stains decolourized cells only. Gram Negative cells appear pink in colour and gram positive cells remain purple in colour.