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Gram Staining – Principle, Procedure, Purpose and Examples

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Gram stain is a technique to impart color to the bacterial cell to differentiate between gram-positive bacteria and gram-negative bacteria based on cell wall composition. Gram Staining is a laboratory procedure that consists of four reagents crystal violet (primary stain), iodine (mordant), decolorizer (ethyl alcohol), and safranin (counter stain) to stain the bacterial cell. Hans Christian Gram is a Danish bacteriologist who named this stain and developed this method in 1884.

The basic function of this technique is to differentiate between bacteria based on the chemical and physical properties of the cell walls. The difference in the cells can be identified by the cell wall as the gram-negative bacteria has a thin cell wall due to which the violet stain gets washed out with ethanol whereas the cell wall of gram-positive bacteria is thicker because of which violet stain stays out and give pink color to the bacteria.

What is Gram Stain?

Gram stain is a laboratory technique that differentiate bacterial cell on the basis of bacterial cell wall composition. It consists of four steps crystal violet (primary stain), Gram’s iodine (mordant), decolorizer (ethyl alcohol), and safranin (counter stain). These reagent impart color to the bacterial which differentiate between gram positive (purple) and gram negative (red).

Gram-Negative Bacteria

If the bacteria are colored pink or red that shows Gram-negative bacteria. Gram-negative bacteria lose the strain because the cell wall is not present in -ve bacteria.

Gram-Negative Bacteria

Gram negative bacteria

Gram-Positive Bacteria

Gram positive bacteria are those which keep primary stain i.e. crystal violet due to their thick peptidoglycan layer in cell wall and appear purple in light microscope.

Gram-Positive Bacteria

Gram positive bacteria

Gram Staining Principle

The basic principle behind this technique is that some bacteria have the ability to retain the dye while the other bacteria don’t have the ability to retain the dye after staining with crystal violet dye. This differentiation is possible due to the difference in the cell wall of bacteria.

  1. Gram positive bacteria have high peptidoglycan content and two types of teichoic acids, wall teichoic acid and lipoteichoic acid. Wall teichoic acid keeps the cell wall intact and lipoteichoic acid have affinity toward anionic crystal violet dye. When we pour crystal violet (first step), it gets attached to lipoteichoic acid and reaches to the cell cytoplasm where it bind to magnesium ion and forms a CV-Mg-RNA complex, while in gram negative bacteria there is no teichoic acid and have less content of peptidoglycan. So the dye did not penetrate so well and forms a weaker complex.
  2. In Second step, iodine is poured which act as a mordant i.e., it intensify the color and form a stronger complex in gram positive bacteria which is known as CV-Mg-RNA-I complex.
  3. In third step, ethanol is poured that has a dual property (1) lipid solvent and (2) dehrydation. So lipid gram positive has low lipid content, which get easily dissolved and form small pores which easily get shrink also that prevent washing off of complex in gram positive bacteria and appear purple at this time and in Gram-negative bacteria have a high content of lipid this results in formation of large pores which does not get shrink so easily. This causes to flood off the complex from gram negative bacteria and appear colorless this time.
  4. In fourth step, safranin is poured which stains the gram negative bacteria red in color.

Gram Staining Requirements

  1. Crystal violet (primary stain)
  2. Iodine (mordant)
  3. 95% Ethanol (decolorizer)
  4. Safranin
  5. Glass slide
  6. Inoculating loop
  7. Burner

Gram Staining Procedure

There are different steps in this procedure which is needed to be followed step by side to successfully complete this technique. Those steps are given below:

  1. Firstly, get a free slide which should be clean and free from grease.
  2. With the loop pour the sample and make a smear of it.
  3. Dry the slide with air and then provide heat to the slide. This heat is given to the sample to fix the sample on the slide so that it doesn’t get washed off too easily. Moreover, through the heat, some of the bacteria get killed but it gets fixed properly.
  4. Now after the heat finally the dye (crystal violet) is poured onto the slide and minimum for 30 seconds and maximum for 1 minute it is kept on the slide and then it is washed with water. This time is given so that the bond is formed between the dye and the cell membrane of the bacteria. Moreover, through the heat, some of the bacteria get killed but it gets fixed properly. 
  5. The mordant or it can be said the iodine solution is applied and it is also kept for a maximum 1 minute and then the slide is washed with normal water. It gets trapped in the cell after making a bond with the crystal dye.
  6. Now it’s the turn for acetone or alcohol to be used on the slide and then after 10-20 seconds, the slide is washed again with water. These two agents work as decolorizers.
  7. The last step is the application of safranin for 1 minute and then again it is rinsed with water.
  8. The slide undergoes blot and air dry and now it is ready for observation under the microscope.

Gram-Staining-Procedure

Purpose of Gram Staining

There are many purposes and uses of this technique that are explained briefly

  1. The main purpose of this technique is the identification of the different types of bacteria i.e., the difference between gram-positive and gram-negative bacteria.
  2. It is a laboratory technique that can only be performed in labs. The main thing which is responsible to give color to the bacteria is their cell wall composition and thickness. There are a lot of bacterial families and species which can not get differentiated by this technique. For e.g., archaea, mainly archaebacteria.
  3. Some of the bacteria are gram-variable which means they can stain anyone either positive or negative while some are doesn’t stain any dye, they neither come under positive nor under negative. Such bacteria which doesn’t come under either the negative or positive category such bacteria are not identified by this technique.
  4. There are other techniques that are used for such bacteria. For such bacteria genetic sequencing, molecular techniques are used for their identification in detail. In medical research, this technique is very helpful.
  5. When the infection is there inpatient then staining is done on the fluid of his body. This technique is fast result giving so it is used more often than culturing in the case of infection. It is also used in the treatment of the patient.
  6. It is used in some special cases of infection. For e.g., in septic arthritis synovial fluid is used for gram staining, and in meningitis cerebrospinal fluid is used for diagnosis. In case of infection in the throat, lungs, skin wounds, or genitals this technique is used. It is also used to check infection in some body fluids such as urine and blood.

Examples Gram Staining

  1. Gram negative bacteria: E. coli, Pseudomonas aeruoginosa
  2. Gram Positive bacteria: Staphylococcus aureus, Streptococcus sp.

FAQ’s – Gram Staining

Q1. What is the Most Important and Crucial step in Staining?

As the main purpose of this staining is to differentiate between positive and negative bacteria. Decolorization is very important step in this technique because it rinses off the violet crystal dye and then help in differentiating between the bacteria.

2. What is the Purpose of the Gram Stain Procedure?

The Gram stain is a method to classify bacteria as Gram-positive or Gram-negative based on their cell wall characteristics, involving a series of staining and decolorization steps.

3. What is Gram staining and explain its Procedure?

The Gram stain categorizes bacteria into Gram-positive or Gram-negative groups based on cell wall properties. This involves specific staining and decolorization steps, providing a comprehensive bacterial classification.

4. In a Cell what are the things that can be known by Gram Staining.

The structure of its cell wall and cell shape can be determined by this technique.

5. What is the Purpose of the Reagents in the Gram Stain?

The primary stain imparts color to all cells, while the mordant intensifies the primary stain’s color. The decolorizing agent is then used to establish a color contrast and safranin stains gram negative bacteria.



Last Updated : 13 Jan, 2024
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