Gram staining
Principle
When the
bacteria is stained with primary stain Crystal Violet and fixed by the mordant,
some of the bacteria are able to retain the primary stain and some are
decolorized by alcohol. The cell walls of gram positive bacteria have a thick
layer of protein-sugar complexes called peptidoglycan and lipid content is low.
Decolorizing the cell causes this thick cell wall to dehydrate and shrink,
which closes the pores in the cell wall and prevents the stain from exiting the
cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is
bound to the thick layer of peptidoglycan of gram positive bacteria and appears
blue or purple in colour.
In case of gram
negative bacteria, cell wall also takes up the CV-Iodine complex but due to the
thin layer of peptidoglycan and thick outer layer which is formed of lipids,
CV-Iodine complex gets washed off. When they are exposed to alcohol,
decolourizer dissolves the lipids in the cell walls, which allows the crystal
violet-iodine complex to leach out of the cells. Then when again stained with
safranin, they take the stain and appear red in colour.
Procedure
- Take a clean, grease free
slide.
- Prepare the smear of
buttermilk or curd 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 wash 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.
Result
Violet
coloured rod shaped gram positive
bacteria observed.
