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Gram Stain Technique




  1. To differentiate between the two major categories of bacteria: Gram positive and Gram negative.
  2. To understand how the Gram stain reaction affects Gram positive and Gram negative bacteria based on the biochemical and structural differences of  their cell walls.

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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 most widely used  staining procedure  in microbiology  is the Gram stain, discovered by  the Danish scientist and physician Hans Christian Joachim Gram in 1884. 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.



Fig: Hans Christian Joachim Gram


Importance of a Gram Stain:


The Gram stain is a very important preliminary step in the initial characterization  and classification of bacteria.  It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope.  The bacteria present in an unstained smear are invisible when viewed using a light microscope. Once stained, the morphology and arrangement of the bacteria may be observed as well. Furthermore, it is also an important step in the screening of  infectious agents in clinical specimens such as direct smears from a patient.


The Gram stain procedure enables bacteria to retain color of the stains, based on the differences in the chemical and physical properties of the cell wall.


     1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet in the cell wall.

                           Example: Staphylococcus aureus


        Fig: Gram positive bacteria


         2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called Safranin.

                              Example: Escherichia coli



          Fig: Gram negative bacteria


 Bacterial Morphology:


Bacteria are very small unicellular microorganisms ubiquitous in nature. They are micrometers (1µm = 10-6 m) in size. They have cell walls composed of peptidoglycan and reproduce by binary fission. Bacteria  vary in their morphological features. 


 The most common morphologies are:


  Coccus (pleural: Cocci):


 Spherical bacteria; may occur in pairs (diplococci), in groups of four (tetracocci), in grape-like clusters (Staphylococci), in chains               (Streptococci) or in cubical arrangements of eight or more (sarcinae).

For example: Staphylococcus aureus, Streptococcus pyogenes


  Bacillus (pleural: Bacilli):


   Rod-shaped bacteria; generally occur singly, but may occasionally be found in pairs (diplo-bacilli) or chains (streptobacilli).

   For example: Bacillus cereus, Clostridium tetani


  Spirillum (pleural: Spirilla):


     Spiral-shaped bacteria 

For example: Spirillum, Vibrio, Spirochete species.


Some bacteria have other shapes such as:


Coccobacilli: Elongated spherical or ovoid form.

Filamentous: Bacilli that occur in long chains or threads.

Fusiform: Bacilli with tapered ends.





                                 Fig: Different bacterial morphology


Gram Stain Mechanism:


Gram Positive Cell Wall:


Gram-positive bacteria have a thick mesh-like cell wall which is made up of peptidoglycan (50-90% of cell wall), which stains purple.  Peptidoglycan is mainly a polysaccharide composed of two subunits called N-acetyl glucosamine and N-acetyl muramic acid. As adjacent layers of peptidoglycan are formed, they are cross linked by short chains of peptides by means of a transpeptidase enzyme, resulting in the shape and rigidity of the cell wall. The thick peptidoglycan layer of Gram-positive organisms allows these organisms to retain the crystal violet-iodine complex and stains the cells as  purple.

Lipoteichoic acid (LTA) is another major constituent of the cell wall of Gram-positive bacteria which is embedded in the peptidoglycan layer.   It consists of teichoic acids which are long chains of ribitol phosphate anchored to the lipid bilayer via a glyceride. It acts as regulator of autolytic wall enzymes (muramidases: Bacterial enzymes located in the cell wall that cause disintegration of the cell following injury or death.)




Medical Relevance of Gram Positive Cell Wall:


LTA also has antigenic properties that stimulate specific immune responses when it is released from the cell wall after cell death. Cell death is mailnly due to lysis induced by lysozymal activities, cationic peptides from leucocytes, or beta-lactam antibiotics.


Gram Negative Cell Wall:


Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the counter stain Safranin, thus appearing reddish or pink. They also have an additional outer membrane which contains lipids,  which is separated from the cell wall by means of  periplasmic space.



Medical Relevance of Gram Negative Cell Wall:


 The cell wall of Gram-negative bacteria is often a virulence factor that enables pathogenic bacteria to cause disease. The virulence of Gram-negative bacteria is often associated with certain components of the cell wall, in particular, the lipopolysaccharide ( otherwise known as LPS or endotoxin). In humans, LPS  elicits  an innate immune response characterized by cytokine production and  activation of immune system. Inflammation occurs as a  result of cytokine production, which can also produce host toxicity.


Stain Reaction:  


 The four basic steps of the Gram Stain are:



1) Application of the primary stain Crystal Violet (CV) to a heat-fixed smear of bacterial culture.

 CV dissociates in aqueous solutions into CV+ and  Cl – ions. These two ions then penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ions later interacts with negatively charged bacterial components and stains the bacterial cells purple.



2) Addition of Gram’s Iodine.

 Iodine (I – or I3 –) acts as a mordant and as a trapping agent. A mordant is a substance that increases the affinity of the cell wall for a stain by  binding to the primary stain, thus forming an insoluble complex which gets trapped in the cell wall.  In the Gram stain reaction, the crystal    violet and iodine form an insoluble complex (CV-I) which serves to turn the smear a dark purple color. At this stage, all cells will turn purple.


3) Decolorization with 95% ethyl alcohol.

 Alcohol or acetone dissolves the lipid outer membrane of Gram negative bacteria, thus leaving the peptidoglycan layer exposed and increases the porosity of the cell wall. The CV-I complex is then washed away from the thin peptidoglycan layer, leaving Gram negative bacteria colorless.


On the other hand, alcohol has a dehydrating effect on the cell walls of Gram positive bacteria which causes the pores of the cell wall   to shrink. The CV-I complex gets tightly bound into the multi-layered, highly cross-linked Gram positive cell wall thus staining the cells purple.


The decolorization step must be performed carefully, otherwise over-decolorization may occur. This step is critical and must be timed correctly otherwise the crystal violet stain will be removed from the Gram-positive cells. If the decolorizing agent is applied on the cell for  too long time , the Gram-positive organisms to appear Gram-negative. Under-decolorization occurs when the alcohol is not left on long enough to wash out the CV-I complex from the Gram-negative cells, resulting in Gram-negative bacteria to appear Gram-positive.


4) Counterstain with Safranin 

The decolorized Gram negative cells can  be rendered visible with a suitable counterstain, which is usually positively charged safranin, which stains them pink. Pink colour which adheres to the Gram positive bacteria is masked by the purple of the crystal violet (Basic fuschin is sometimes used instead of safranin in rare situations).



Fig: Colour changes that occur at each step in the staining process




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