Gram Staining Procedure and its Details

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Introduction

  • Gram staining procedure was discovered by Han’s Christian Gram in 1884.
  • Gram staining is a universal staining technique used for identification and classification of organisms.
  • In this staining, method bacteria are classified into two groups that are-

                                                                        1. Gram-positive bacteria

                                                                        2. Gram-negative bacteria

  • This classification of bacteria depends upon the property of a cell to retain or lose the primary stain after the treatment of decolourising agent. 
  • Gram staining is a basic and widely used technique.
  • This technique was modified by many scientists but the best result was obtained by Hucker and Conn’s modification.

Requirements

  1. A clean grease free slide.
  2. Bacterial cell suspension.
  3. Nichrome Wire loop.
  4. Primary stain – Crystal violet.
  5. Mordant- Gram’s Iodine.
  6. Decolourising agent- 95% alcohol ( 95% Ethanol).
  7. Counterstain- Basic fuschin or Safranin.

Procedure

  1. Take a clean grease free slide.
  2. Prepare a smear from a bacterial cell suspension on a slide by using nichrome wire loop.
  3. Air dry and heat fix the smear.
  4. Flood the smear with a primary stain that is Crystal violet and allow it to react for 1-2 minutes.
  5. After Crystal violet treatment water wash treatment is given to the slide.
  6. Further, the smear is treated with the mordant that is Gram’s Iodine for 1-2 minutes.
  7. Excess Gram’s Iodine is removed and the and the slide is further treated with a decolourising agent that is 95 % Ethanol.
  8. After Ethanol treatment the smear is water washed and flooded with counterstain that is Basic fuchsin or Safranin for 1-2 minutes.
  9. Finally, the slide is washed with water, air dried and observed under oil immersion.

Flow chart of Gram staining procedure

Flow chart of Gram staining procedure

Gram Staining Procedure

Gram Staining Procedure

  1. Cells stained with crystal violet appear violet colour are Gram-positive cells.
  2. Cells stained with counterstain i.e Basic fuschin or safranin appear pink in colour are Gram-negative cells.

Functions of agents used in Gram staining

  1. Crystal violet – It is a primary stain and a basic dye it stains all micro-organisms.
  2. Gram’s Iodine – Gram’s Iodine acts as a mordant and it forms a complex with crystal violet that is CV-I complex.This complex increases affinity between cell and stain.
  3. 95%  Alcohol (95% Ethanol) – It is a decolourising agent as well as a lipid solvent.It tries to decolourise the cell by removing the CV-I complex from the cell.
  4. Basic fuschin or Safranin – It acts as a counterstain. It stains the cells that are decolourised by alcohol.Only Gram-negative bacteria get decolourise and this counterstain gives pink colour to these cells.

Mechanism

  1. When a smear is stained with crystal violet it stains all cells to violet colour.
  2. After application of Gram’s Iodine, its molecules acts as a mordant and form a crystal violet – Gram’s Iodine complex that is CV-I complex.
  3. After CV – I complex formation this smear is subjected to decolourising treatment by using 95% Ethanol for 30 seconds.
  4. The gram-positive cell has some special features due to which CV – I complex is unable to come outside the cell they are-
  • The gram-positive cell has 1 to 4 % of lipid content due to low lipid content the cell get dehydrated by alcohol treatment and its pore size decreases so CV – I complex is trapped inside the cell.
  • Peptidoglycan layer account about 40 to 90 5 of the dry weight of Gram-positive cell so due to extremely dense cross-linkage  CV – I complex is trapped inside the cell.
  • The gram-positive cell contains Magnesium ribonucleate so this compound  Magnesium ribonuclease molecule forms a covalent bond with CV – I complex and thus it doesn’t allow CV – I complex to come outside the cell.
  • The gram-positive cell has 1 to 4 % of lipid content due to low lipid content the cell get dehydrated by alcohol treatment and its pore size decreases so CV – I complex is trapped inside the cell.
  • Peptidoglycan layer account about 40 to 90 5 of the dry weight of Gram-positive cell so due to extremely dense cross-linkage  CV – I complex is trapped inside the cell.
  • The gram-positive cell contains Magnesium ribonucleate so this compound  Magnesium ribonuclease molecule forms a covalent bond with CV – I complex and thus it doesn’t allow CV – I complex to come outside the cell.
  • The gram-negative cell contains 11 to 20 % of lipid content when Gram-negative cells are suspended in alcohol it dissolves the lipid and thus CV – I complex comes out.
  • Peptidoglycan content in Gram-negative cell wall is 5 to 10 % so due to less amount of cross-linkage  CV – I complex comes out easily.
  • Gram-negative cell lacks Magnesium ribonucleate molecules so CV – I complex is extracted easily from the cell.

7.  The cells which get decolourised by alcohol take the counterstain and appear pink in colour these cells are Gram-negative cells.

6.   After decolourisation treatment, the smear is treated with counterstain i.e Basic fuschin and Safranin.

Application

  1. Gram staining is a basic technique used for identification and classification of the cell.
  2. It is a useful technique in the diagnosis of the causative agent of a clinical infection.
  3. It is also helpful in studying morphological characters of cells.

Examples

  1. Gram positive bacteria – Bacillus, Staphylococcus, Streptococcus, Micrococcus etc.
  2. Gram negative bacteria – Pseudomonas, E.coli, Salmonella, Shigella, Proteus, Xanthomonas etc

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