Staining is a biochemical technique of coloring specimens.
Dyes are the chemical substances which commonly used to stain specimen.
There are three types of staining protocol or procedures:
Gram staining a differential staining method.
It is used to identify and differentiate bacteria into two groups i.e. Gram positive and Gram negative bacteria.
This classification is based on the physical properties of the bacterial cell wall.
Gram staining procedure is developed in 1884 by Hans Christian gram.
In this staining bacterial smear is of four different reagents:
Crystal Violet it is also known as primary stain.
Iodine it is referred as mordant because it increases the affinity of dyes or a stain for smear.
Alcohol or ethanol it is referred as de-colorizing agent that remove stain from the specimen.
Safranin it is referred as counterstain.
Procedures of Gram staining
Prepare a smear of bacteria culture and heat fix the smear.
Smear are first stained with Crystal Violet which is a basic dye it imparts purple color to all cells.
Washed the slide with distilled water for seconds.
Now, smear is treated with iodine (mordant), this permit the stain to retained by forming an insoluble CV-I complex (Crystal Violet-iodine complex).
Wash the slide with ethyl alcohol (95%) this is a differential step. At this step some bacteria retain the primary stain i.e. Crystal Violet and appears purple color while some bacteria not retain stain and loose purple color and appears colorless.
Bacteria that retain Crystal Violet and appears purple are classified as gram positive bacteria, while that not retain stain and loose the color after decolourisation are classified as Gram negative bacteria.
Wash the slide with distilled water and drain it. Now apply counter stain safranin (basic dye), and again wash the slide with distilled water and blot dry with absorbent.
Since Gram Positive Bacteria already retain purple color, they are not affected by counterstain but, Gram negative bacteria are colorless so, they directly stained by the safranin. Thus, Gram positive appear purple, and Gram negative appears red or pink.
The Gram Positive Bacteria cell walls are thick and chemically simple, in contrast Gram negative bacteria cell wall are thin and complex multi-layered structure.
When treated with alcohol gram-positive bacteria are not allowing the loss of CV-I complex and cells remains purple, while Gram negative bacteria have complex structure and have lipopolysaccharide layer which is absent in Gram Positive Bacteria due to which CV-I complex is leaked and resulting in decolorization which later take the counterstain and appears red.