Gram staining is a simple, differential staining method which helps to group bacteria according to their Gram character (Gram positive or Gram negative). It is one of the initial steps carried out in pathological laboratories for the identification of etiological agent and hence is one of the most important staining techniques in medical microbiology. The Gram reaction depends on the growth phase of the organism, young and growing bacteria gives most consistent reaction.
Principle - Gram staining depends on the structure and composition of the cell wall of bacteria. Gram positive bacteria have thicker peptidoglycan layer than gram negative bacteria. First, primary stain, crystal violet stains all the cells purple. Addition of iodine (mordant) forms crystal violet iodine complex within the cell wall. The complex formed is larger than crystal violet so it cannot be easily washed out from the intact peptidoglycan layer. Application of alcohol (decolorizer) decolorizes the stain. Since gram negative organism have thin peptidoglycan layer and have additional lipopolysaccharide layer which gets dissolved due to the addition of alcohol, so gram negative organism fails to retain the complex and gets decolorized . On other hand gram positive continues to retain the complex and remain purple. To observe the decolorized cells secondary stains like Basic fuchsin or Safranin is added which stains the gram negative organisms pink.
Procedure
1. Prepare the smear and heat fix it.
2. Add crystal violet (primary stain) - 1minute.
3. Wash and add Gram’s iodine (mordant) - 1 min.
4. Drain off iodine, add alcohol (decolorizer) -15 seconds
5. Wash and add Basic fuchsin or Safranin (secondary stain)- 1 min.
6. Wash, dry and observe under oil immersion lens.
Observation
Purple cells - Gram positive
Pink cells – Gram negative
Gram staining also helps in studying the morphological features and cellular arrangements of the organisms.
Gram character of some organisms.
Gram negative rods – Escherichia coli, Pseudomonas aeruginosa.
Gram negative cocci – Neisseria sps., Morexella sps.
Gram positive rods – Clostridium botulinum, Bacillus cereus.
Gram positive cocci – Staphylococcus aureus, Streptococcus pyogenes.
Principle - Gram staining depends on the structure and composition of the cell wall of bacteria. Gram positive bacteria have thicker peptidoglycan layer than gram negative bacteria. First, primary stain, crystal violet stains all the cells purple. Addition of iodine (mordant) forms crystal violet iodine complex within the cell wall. The complex formed is larger than crystal violet so it cannot be easily washed out from the intact peptidoglycan layer. Application of alcohol (decolorizer) decolorizes the stain. Since gram negative organism have thin peptidoglycan layer and have additional lipopolysaccharide layer which gets dissolved due to the addition of alcohol, so gram negative organism fails to retain the complex and gets decolorized . On other hand gram positive continues to retain the complex and remain purple. To observe the decolorized cells secondary stains like Basic fuchsin or Safranin is added which stains the gram negative organisms pink.
Procedure
1. Prepare the smear and heat fix it.
2. Add crystal violet (primary stain) - 1minute.
3. Wash and add Gram’s iodine (mordant) - 1 min.
4. Drain off iodine, add alcohol (decolorizer) -15 seconds
5. Wash and add Basic fuchsin or Safranin (secondary stain)- 1 min.
6. Wash, dry and observe under oil immersion lens.
Observation
Purple cells - Gram positive
Pink cells – Gram negative
Gram staining also helps in studying the morphological features and cellular arrangements of the organisms.
Gram character of some organisms.
Gram negative rods – Escherichia coli, Pseudomonas aeruginosa.
Gram negative cocci – Neisseria sps., Morexella sps.
Gram positive rods – Clostridium botulinum, Bacillus cereus.
Gram positive cocci – Staphylococcus aureus, Streptococcus pyogenes.
