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Gram staining

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Nagaraju Yalavarthi
Nagaraju Yalavarthi

Christian Gram developed the gram staining during the year 1884, it is preliminarly used to classify the microorganisms

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Gram staining
Aim of this lecture  To incorporate the basic knowledge on stain, staining and applications of staining After this lecture you should know  What is staining  Difference between the dye and stain  Components of stain
What is Staining?  It is the artificial coloration of a substance to facilitate examination of tissue, microorganism or other cells under microscope
What is the difference between stain and dye? Stain  Used to stain biological specimens  Pure form  Large molecular structure  It contains pigment, binder and carrier material  Solubility is less Dye • Used as coloring agent for general purposes • Crude form • Small molecular structure • Pigment and carrier • More soluble
Components of stain
History  Hans Christian Joachim Gram, Danish bacteriologist and physician  Developed Gram staining technique in 1883 and published his findings in 1884 in Friedlander’s Journal  Was working with respiratory disease in lung tissue from cadaver at Municipal Hospital Berlin and his accidental spillage of lugol’s iodine over lung tissue sections led to the development of Gram Staining.  While examining the lung tissue from the patients who had died of pneumonia, he observed that certain stains were preferentially taken up and retained by bacterial cell.
 His initial work with this staining process was performed on Klebsiella pneumoniae and Streptococcus pneumoniae.  He did not use a counter stain in his procedure. It was a few years later, that the German pathologist Carl Weigert (1845-1904) from Frankfurt, added a final step of staining with safranin.
Procedure  I. Primary stain- Crystal violet  II. Mordant- Gram’s Iodine  III. Decolorizer- Acetone or alcohol or mixture of acetone alcohol  IV. Counter stain- Neutral red or Safranine
Result
Difference between gram positive and gram negative
Gram staining
Principle of gram staining  Three hypothesis a. Cell wall related b. PH related c. Magnesium ribonuclease related
a) Cell wall permeability hypothesis  Gram positive cell wall has thicker peptidoglycan layer as compared to Gram negative cell wall .  Gram negative cell wall contains a thick layer of lipopolysaccharide in their cell wall which is absent in Gram positive bacteria .  During Gram staining a dye iodine complex (CVI complex) or lake is formed within the cell wall after staining with crystal violet and on subsequent treatment with iodine .This complex is insoluble in water but soluble in alcohol.  Now when we add decolourizer (Acetone alcohol ), the dye iodine complex is trapped inside the thick peptidoglycan layer of Gram positive bacteria so they are not decolorized.  But outer lipopolysacharide layer of Gram negative cell wall is easily dissolved by the decolourizer due to which dye iodine complex is easily washed away from Gram negative cell.
b) Presence of magnesium ribonucleate  Magnesium ribonucleate present in Gram positive bacteria has affinity for basic dyes. As a result Gram positive bacteria take the color of crystal violet .  It has also been proved that Gram positive bacteria become Gram negative upon removing this material.
c) pH hypothesis  Cytoplasmic pH of Gram positive bacteria is more acidic (2-3) as compared to Gram negative bacteria(4-5) and since the primary stain used (crystal violet ) is basic in nature it develops more affinity with the Gram positive cytoplasm .  The difference in pH is mostly due to presence of Teichoic acid in the cytoplasm of gram positive cell, which is absent in Gram negative cell.
Utility  Gram staining is a method of differentiating bacterial species into two large groups (Gram- positive and Gram-negative)  The Gram staining is almost always the first step in the identification of bacteria  It is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to Gram- variable and Gram indeterminate groups as well
Importance of gram staining  Important test for the rapid presumptive diagnosis of infectious agent  To classify the bacteria as a Gram positive or Gram negative  To study the morphology of bacteria  To study the arrangement of bacteria  To find out the evidence of capsule  To find out the evidence of spore  To find out the evidence of pus cells  To find out the evidence of epithelial cells  To find out the evidence of Yeast cells
Summary of this lecture  Gram staining was invented by Christian Gram during 1884  Gram staining contains four components crystal violet iodine alcohol saffranin  Gram staining is generally used to classify the microorganisms
Thank you so much for your attention

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Gram staining

  • 2. Aim of this lecture  To incorporate the basic knowledge on stain, staining and applications of staining After this lecture you should know  What is staining  Difference between the dye and stain  Components of stain
  • 3. What is Staining?  It is the artificial coloration of a substance to facilitate examination of tissue, microorganism or other cells under microscope
  • 4. What is the difference between stain and dye? Stain  Used to stain biological specimens  Pure form  Large molecular structure  It contains pigment, binder and carrier material  Solubility is less Dye • Used as coloring agent for general purposes • Crude form • Small molecular structure • Pigment and carrier • More soluble
  • 6. History  Hans Christian Joachim Gram, Danish bacteriologist and physician  Developed Gram staining technique in 1883 and published his findings in 1884 in Friedlander’s Journal  Was working with respiratory disease in lung tissue from cadaver at Municipal Hospital Berlin and his accidental spillage of lugol’s iodine over lung tissue sections led to the development of Gram Staining.  While examining the lung tissue from the patients who had died of pneumonia, he observed that certain stains were preferentially taken up and retained by bacterial cell.
  • 7.  His initial work with this staining process was performed on Klebsiella pneumoniae and Streptococcus pneumoniae.  He did not use a counter stain in his procedure. It was a few years later, that the German pathologist Carl Weigert (1845-1904) from Frankfurt, added a final step of staining with safranin.
  • 8. Procedure  I. Primary stain- Crystal violet  II. Mordant- Gram’s Iodine  III. Decolorizer- Acetone or alcohol or mixture of acetone alcohol  IV. Counter stain- Neutral red or Safranine
  • 10. Difference between gram positive and gram negative
  • 12. Principle of gram staining  Three hypothesis a. Cell wall related b. PH related c. Magnesium ribonuclease related
  • 13. a) Cell wall permeability hypothesis  Gram positive cell wall has thicker peptidoglycan layer as compared to Gram negative cell wall .  Gram negative cell wall contains a thick layer of lipopolysaccharide in their cell wall which is absent in Gram positive bacteria .  During Gram staining a dye iodine complex (CVI complex) or lake is formed within the cell wall after staining with crystal violet and on subsequent treatment with iodine .This complex is insoluble in water but soluble in alcohol.  Now when we add decolourizer (Acetone alcohol ), the dye iodine complex is trapped inside the thick peptidoglycan layer of Gram positive bacteria so they are not decolorized.  But outer lipopolysacharide layer of Gram negative cell wall is easily dissolved by the decolourizer due to which dye iodine complex is easily washed away from Gram negative cell.
  • 14. b) Presence of magnesium ribonucleate  Magnesium ribonucleate present in Gram positive bacteria has affinity for basic dyes. As a result Gram positive bacteria take the color of crystal violet .  It has also been proved that Gram positive bacteria become Gram negative upon removing this material.
  • 15. c) pH hypothesis  Cytoplasmic pH of Gram positive bacteria is more acidic (2-3) as compared to Gram negative bacteria(4-5) and since the primary stain used (crystal violet ) is basic in nature it develops more affinity with the Gram positive cytoplasm .  The difference in pH is mostly due to presence of Teichoic acid in the cytoplasm of gram positive cell, which is absent in Gram negative cell.
  • 16. Utility  Gram staining is a method of differentiating bacterial species into two large groups (Gram- positive and Gram-negative)  The Gram staining is almost always the first step in the identification of bacteria  It is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to Gram- variable and Gram indeterminate groups as well
  • 17. Importance of gram staining  Important test for the rapid presumptive diagnosis of infectious agent  To classify the bacteria as a Gram positive or Gram negative  To study the morphology of bacteria  To study the arrangement of bacteria  To find out the evidence of capsule  To find out the evidence of spore  To find out the evidence of pus cells  To find out the evidence of epithelial cells  To find out the evidence of Yeast cells
  • 18. Summary of this lecture  Gram staining was invented by Christian Gram during 1884  Gram staining contains four components crystal violet iodine alcohol saffranin  Gram staining is generally used to classify the microorganisms
  • 19. Thank you so much for your attention