In order to understand the action of the gram stain in differentiating the bacterium, we need to have a look at the cell wall composition of the bacteria .In them, the cell wall has a little difference .
Cell Wall in Gram positive bacteria –
- Has relatively thick layer of peptidoglycan ,
- Techoic acid (polymer of glycerol or ribotol connecterd with phosphate groups,
- The various layers of peptidoglycan are linked by covalent linking of techoic acid to muramic acid to form a mesh
Consequence— The crystal violet color is not allowed to decolorize , so the cells appear blue black
Cell Wall in Gram negative bacteria
- In gram negative bacteria, the cell wall is much thinner with low peptidoglycan concentration but high lipid content.
- Hence the crystal violet gets decolorized when washed due to restraining activity of the mesh like peptidoglycan in gram positive cells.
- Besides, in gram negative bacteria, the outer part of cell wall is high in lipid, polysaccharides and proteins, but there is a space called periplasmic space outside the central membrane external to which is the peptidoglycan followed by the final external membrane
- Thus another stain (counter stain ) becomes very important .
Well it is easy to use the different stains like H&E , Nile blue sulphate, or Neutral red or Leishman to stain different cells and observe them. But does it apply the same for bacteria? l forgot to mention that bacteria are the largest numbers of life forms on this planet, existing form within your intestine to the deep underwater vents of hot gases under the ocean or the Tundra! It is also interesting to note that all bacteria can be basically divided into two groups on basis of their staining properties by a stain called Gram stain, discovered by Hans Christian Gram in 1882–1884.
- Gram stain is based on the physical and chemical differences of cell wall in bacteria
- Bacteria can be categorized into two types-Gram positive and Gram negative.
- Heat fixed sections are placed in distilled water
- The section is flooded with Crystal violet or the Primary stain, as it colorizes all types of cells .
- Crystal violet is washed off after 1 minute with distilled water.
- The section is flooded Grams iodine solution , which is called a mordant or a helper.
- Iodine is washed off after approximately a minute.
- The slide is washed with 95% alcohol or alcohol -acetone solution to remove excess purple from some bacteria, it is decolorization agent.
- The slide must be kept at an angle and alcohol has to be poured till there is no purple tint.
- Slide is washed immediately with tap water.
- Slide is counter stained with safranin for 1 min.
- It is washed with distilled water, air dried and mounted for observation.
- Blue or Black- Indicates gram positive organisms
- Blue- Indicates fungal filaments
- Red- Indicates gram negative microorganism