Question

Gram staining is used to distinguish and classify bacterial species into two large groups. Discuss this statement with reference to the gram staining protocol. http://learn.chm.msu.edu/vibl/content/gramstain.html.                                                                                    write 1000/1500 words on this topic . please make sure in 1000 to 1500 words

Answer 1

• Gram Staining protocol is first discovered by bacteriologist Hans Christian Gram.
• Protocol :

1. ?Take a clean glass slide and take a loop of sample and make a thin smear.
2. Heat fix the organism.
3. Add Primary stain(Crystal violet) and keep for 1min.
4. Wash with distilled water
5. Add Mordant (Iodine) for 1min
6. Wash with distilled water and add decoloriser(70%Ethanol) for 15sec.
7. Wash it and add Secondary stain(Safranin) for 1min.
8. Wash and air dry.
9. See under oil immersion (100X).
10. Gram positive will give purple color and gram negative will give pink color.

• Gram staining helps to find two large group of micro organisms;
  1. Gram Positive
  2. Gram Negative

1. Gram Positive organism :

? Cellwall containing thick peptidoglycan layer, and thin lipopolysachharide layer. So, when primary stain(Crystal Violet) is added both the layer will take up the stain. But while treating with decolorizer(70%Ethanol) there will be leakage in lipopolysachharide layer and color will come out but peptidoglycan layer wil retain the primary stain. So after treating the cell with secondary stain(Safranin) there will be no uptake of safranin and the cell exhibit the primary stain and appear Purple or violet in color. Example: Staphaylococcus aureus, Bacilus subtilis etc.

2. Gram Negative Organism:

Cellwall contain thick lipopolysachharide layer and thin peptidoglycan layer. So it will easily take primary stain. But after treating with decoloriser (ethanol) the lipid layer gets ruptured and all the primary stains leak out. Later it take up the secondary stain(Safranin) and exhibit pink or reddish pink in color. Example: Helicobacter pylori, Escherecia coli etc.