Gram stain Protocol

Grams Iodine - Crystal violet
Safranin - Neutral red
Gram’s fuchsin - Gram’s decolourise
Gram’s Stain distinguishes between the two major classes of bacteria due to the differences in cell wall structure ;
Gram-positive bacteria, remain coloured after the staining procedure, and gram-negative bacteria, which do not retain dye.

In the staining technique, cells on a microscope slide are heat-fixed and stained with a basic dye, Crystal violet, which stains all bacterial cells blue. Iodide solution is then added that allows the iodine to enter the cells and form a water-insoluble complex with the crystal violet dye. The preparation is then treated with a decolourise solvent, in which the iodine-crystal violet complex is soluble.

Following solvent treatment, only gram-positive cells remain stained, possibly because of their thick cell wall, which is not permeable to solvent. After the staining procedure, cells are treated with a Counterstain which may be safranin, Gram fuchsin or Neutral Red. Counterstained gram-negative cells appear red, and gram-positive cells remain blue.

Although the cell walls of gram-negative and gram-positive bacteria are similar in chemical composition, the cell wall of gram-negative bacteria is a thin layer sandwiched between an outer lipid-containing cell envelope and the inner cell membrane, whereas the gram-positive cell wall is much thicker, lacks the cell envelope, and contains additional substances, such as teichoic acids, polymers composed of glycerol or ribitol.

The difference in reactivity between gram-positive and gram-negative bacteria is linked with differences in physiological properties of the two groups. Gram-positive bacteria are generally more sensitive to growth inhibition by dyes, halogens, many antibiotics, and to attack by phagocytosis and are more resistant to digestion by the enzymes pepsin and trypsin and enzymes in animal sera.

Gram Stain Pack
Contents
A Crystal Violet
B Grams Iodine Diluent
C Grams Iodine Concentrate
D Grams Decolouriser
E Counterstain: choice of (Safranin Neutral Red, or Gram Fuchsin)

Note
The Grams Iodine Concentrate (C) should be added to the Diluent (B) and mixed well before use. All solutions are now ready to use in dropper bottles.

Lugol’s Iodine
The addition of iodine to a stool concentrate highlights the
internal inclusions of cysts; e.g. the nuclei and glycogen mass, thus aiding their identification.

For example, the addition of iodine enhances refraction of the nuclei of Endolimax nana, stains the peripheral chromatin of the nuclei of Entamoeba species and demonstrates the well-defined glycogen mass which is a feature of pre-cysts or immature cysts if E. coli and cysts of Iodamoeba butschlii.

Iodine does not stain the body of Entamoeba species.

Crystal Violet
Primary gram stain Crystal Violet Solution is a violet stain used to differentiate bacteria.

Safranin
Safranin is a red stain used to differentiate bacteria, used in the Gram?s technique.

Grams Fuchsin
Used in the Gram?s technique

Lugol’s Iodine
Used in the Gram?s technique

Grams Decolouriser
Used in the Gram?s technique

Grams Carbon Fuchsin
Used in the Gram?s technique

Carbol Fuchsin for standard z/n stain
For use in acid fast staining.