Gram's Staining - Microbiology Notes
GRAM'S STAINING
INTRODUCTION
The Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram.
It is a very important differential staining because it separates bacteria into two broad categories namely
gram-positive and gram-negative.
This differential staining requires the use of at least four reagents that are applied sequentially. The first
reagent is called the primary stain-crystal violet (because it is applied first), which will impart its color to all
cells in conjunction with the second reagent iodine, the mordant. In order to establish color contrast the third
reagent used is the decolorizing agent. It may or may not remove the primary stain from the cell, depending
upon its cellular composition. Finally, to stain the decolorized cells, another secondary stain of different color
is applied. The cells which are not decolorized (i.e. those which have retained primary stain), will not be
affected by the counter stain.
STAINING METHOD
Principle
Several theories have been proposed to explain the mechanism of Gram's staining, however, the one based
on physicochemical nature of cell wall of bacteria is widely accepted. Cell walls of gram-negative bacteria
possess higher percentage of lipids in their cell wall as compared to gram-positive bacteria. During staining
the primary stain crystal violet forms complex with mordant iodine (CV-I) in the cell wall. When gram-positive
bacteria are decolorized with ethanol, the alcohol is thought to shrink the pores of the thick peptidoglycan.
Thus, the dye iodine complex is retained during the short decolorization step and the bacteria remain violet.
In contrast, gram-negative peptidoglycan is thinner and more cross-linked and contains larger pores. Alcohol
treatment also may extract enough lipid from the gram-negative wall to increase its porosity further. For these
reasons, alcohol more readily removes the crystal violet-iodine complex from gram-negative bacteria. These
cells subsequently take on the color of counterstains- the safranin.
Requirements
1. Young cultures of Escherichia coli, Bacillus subtilis & Staphylococcus aureus
2. Crystal violet stain, Gram's iodine, 95% ethanol and safranin stain.
GRAM'S STAINING
INTRODUCTION
The Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram.
It is a very important differential staining because it separates bacteria into two broad categories namely
gram-positive and gram-negative.
This differential staining requires the use of at least four reagents that are applied sequentially. The first
reagent is called the primary stain-crystal violet (because it is applied first), which will impart its color to all
cells in conjunction with the second reagent iodine, the mordant. In order to establish color contrast the third
reagent used is the decolorizing agent. It may or may not remove the primary stain from the cell, depending
upon its cellular composition. Finally, to stain the decolorized cells, another secondary stain of different color
is applied. The cells which are not decolorized (i.e. those which have retained primary stain), will not be
affected by the counter stain.
STAINING METHOD
Principle
Several theories have been proposed to explain the mechanism of Gram's staining, however, the one based
on physicochemical nature of cell wall of bacteria is widely accepted. Cell walls of gram-negative bacteria
possess higher percentage of lipids in their cell wall as compared to gram-positive bacteria. During staining
the primary stain crystal violet forms complex with mordant iodine (CV-I) in the cell wall. When gram-positive
bacteria are decolorized with ethanol, the alcohol is thought to shrink the pores of the thick peptidoglycan.
Thus, the dye iodine complex is retained during the short decolorization step and the bacteria remain violet.
In contrast, gram-negative peptidoglycan is thinner and more cross-linked and contains larger pores. Alcohol
treatment also may extract enough lipid from the gram-negative wall to increase its porosity further. For these
reasons, alcohol more readily removes the crystal violet-iodine complex from gram-negative bacteria. These
cells subsequently take on the color of counterstains- the safranin.
Requirements
1. Young cultures of Escherichia coli, Bacillus subtilis & Staphylococcus aureus
2. Crystal violet stain, Gram's iodine, 95% ethanol and safranin stain.
