Staining is an auxiliary technique used in microscopic
techniques used to enhance the clarity of the microscopic image. Stains
and dyes are widely used in the scientific field to highlight the
structure of the biological specimens, cells, tissues etc.
The Gram staining method was first described in 1844 by the Danish bacteriologist Hans Christian Gram, after whom the test was named.
The Gram staining test for bacteria is one of the most important tests in microbiology and is often one of the first tests performed in the identification of bacteria.
Gram staining is a differential staining technique that differentiates bacteria into two groups: gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary stain, purple. Gram-positive bacteria are not decolorized by alcohol and will remain as purple. After decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative organisms.
The mechanics of the Gram staining method is that the bacteria cell walls retain the crystal violet and subsequently added iodine, which complexes with the crystal violet, preventing the easy removal of the dyes. This step is known as the
“fixing the dye” step. During the subsequent addition of a decolorizer, a mixture of acetone and ethanol solvents, Gram-positive cell walls dehydrate, closing the pores in the cell wall, resulting in the retention of the crystal violet: iodine
complexes. In contrast, the decolorizer dissolves the higher lipid content of Gram-negative bacteria and the primary stain is able to leach into the solvent, essentially washing away the dye, leaving the Gram-negative bacteria unstained.
The length of the decolorization stage is critical as prolonged decolorizing will remove the primary stain from the Gram-positive cells and this will lead to false negatives during characterization of the microorganisms.
Finally, in order to visualize the unstained Gram-negative bacteria, a counter stain is added. Safranin, a basic stain that stains bacteria red. Some bacteria stain weakly with Safranin and the alternative counter stain Fuchsin is used.
Procedure for Gram's Staining
After the smear has been dried, heat-fixed, and cooled off, proceed as follows:
The Gram staining method was first described in 1844 by the Danish bacteriologist Hans Christian Gram, after whom the test was named.
The Gram staining test for bacteria is one of the most important tests in microbiology and is often one of the first tests performed in the identification of bacteria.
Gram staining is a differential staining technique that differentiates bacteria into two groups: gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary stain, purple. Gram-positive bacteria are not decolorized by alcohol and will remain as purple. After decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative organisms.
The mechanics of the Gram staining method is that the bacteria cell walls retain the crystal violet and subsequently added iodine, which complexes with the crystal violet, preventing the easy removal of the dyes. This step is known as the
“fixing the dye” step. During the subsequent addition of a decolorizer, a mixture of acetone and ethanol solvents, Gram-positive cell walls dehydrate, closing the pores in the cell wall, resulting in the retention of the crystal violet: iodine
complexes. In contrast, the decolorizer dissolves the higher lipid content of Gram-negative bacteria and the primary stain is able to leach into the solvent, essentially washing away the dye, leaving the Gram-negative bacteria unstained.
The length of the decolorization stage is critical as prolonged decolorizing will remove the primary stain from the Gram-positive cells and this will lead to false negatives during characterization of the microorganisms.
Finally, in order to visualize the unstained Gram-negative bacteria, a counter stain is added. Safranin, a basic stain that stains bacteria red. Some bacteria stain weakly with Safranin and the alternative counter stain Fuchsin is used.
Procedure for Gram's Staining
After the smear has been dried, heat-fixed, and cooled off, proceed as follows:
- Place slide on staining rack and cover specimen with crystal violet. Let stand for 1 minute.
- Wash briefly in tap water and shake off excess.
- Cover specimen with iodine solution and let stand for 1 minute.
- Wash with water and shake off excess.
- Tilt slide at 45° angle and decolorize with the acetone-alcohol solution until the purple color stops running. Wash immediately with water and shake off excess.
- Cover specimen with safranine and let stand for 1 minute.
- Wash with water, shake off excess, and gently blot dry. The smear is now ready to be read. (Use oil immersion lens.)
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