Gram Staining

• Gram Staining


  The most commonly used stain in the laboratory, used to differentiate two large groups of bacteria based on their different cell wall constituents

  
• Gram Staining developed by Danish physician Hans Christian Gram


  1884
• Gram-positive bacteria


  Bacteria that take up the basic dye
• Gram-negative bacteria


  Bacteria that allow crystal violet (basic dye) to be washed out with decolorizer
• Purpose of Gram Staining

  • Classifies bacteria on their forms, sizes and morphology
  • Classifies bacteria on the basis of gram reaction
  • Rapid, presumptive diagnosis of infectious agents
  • Assessing the quality of specimen

  
• Gram-positive cell walls


  • Contain thick peptidoglycan with numerous teichoic acid cross-linkages
• Gram-negative cell walls


  • Consist of a thinner layer of peptidoglycan and an outer lipid bilayer that is dehydrated during decolorization
• Theories of Gram Stain Reaction


  • Magnesium Ribonucleic Acid Theory
  • Benian Theory
  • Stearn and Stearn Theory
  • Lipid Content Theory
  • Teichoic Acid Content Theory
• Gram Positive


  With Teichoic Acid, Thicker Peptidoglycan
• Gram Negative


  No Teichoic Acid, Thinner Peptidoglycan Layer, outer membrane, Lipopolysaccharide and Periplasmic Space
• Gram Stain Reagents


  • Crystal Violet (Primary stain/Initial Stain)
  • Gram's Iodine (Mordant)
  • Acetone Alcohol (Decolorizer)
  • Safranin Red (Counterstain/Secondary Stain)
• Cell Wall


  Also known as PEPTIDOGLYCAN LAYER or MUREIN LAYER, Main Component is PEPTIDOGLYCAN, Basis of Gram Staining

  
• Most Critical Step in Gram Staining is DECOLORIZATION
• Hucker's Modification


  Gram Stain Technique for Fungi (Crystal Violet + Ammonium Oxalate)
• Carbol Fuchsin


  Can be used as COUNTERSTAIN/Secondary Stain in place of SAFRANIN to improve staining of some gram-negative organisms
• Higher forms of organism

  • Actinomyces
  • Nocardia
  • Streptomyces
  • Yeast
  • Molds
• Higher forms of organism like Actinomyces, Nocardia, Streptomyces, yeast and molds are GRAM POSITIVE
• ALL SPIRAL ORGANISMS are reported as GRAM NEGATIVE
• Mycoplasma and Ureaplasma


  Cell Wall LESS
• Gram Staining Procedure


  1. Cover smear with Crystal Violet for 1 minute, rinse with water

  2. Cover smear with Gram's Iodine for 1 minute, rinse with water

  3. Cover smear with 95% Ethanol or Acetone/Acid Alcohol for 30 seconds, rinse with water

  4. Cover smear with Safranin for 1 minute, rinse with water and air dry
• Errors in Gram Staining


  • Gram-positive becomes Gram-negative
  • Gram-negative becomes Gram-positive
• All COCCI are GRAM POSITIVE (+) except: NEISSERIA, BRANHAMELLA AND VEILONELLA
• Gram Positive Bacilli


  • Propionibacterium
  • Actinomyces
  • Rothia
  • Kurthia
  • Mycobacterium
  • Bacillus
  • Clostridium
  • Mubiluncus
  • Bifidobacterium
  • Corynebacterium
  • Erysipelothrix
  • Listeria
  • Lactobacillus
  • Eubacterium
  • Nocardia (weakly staining)
• All BACILLI are GRAM NEGATIVE (-) except: PARK My Big Car, My Big Car ELLEN
• gram staining uses what kind of stains

  differential stains
• what kind of microscopy is used in gram staining


  direct microscopy where the inoculum is from the primary soecimen
• Gram Staining


  The most commonly used stain in the laboratory, used to differentiate two large groups of bacteria based on their different cell wall constituents

  
• Gram Staining


  • Differential staining that can distinguish between types of bacteria

  
• Gram Staining developed by Danish physician Hans Christian Gram


  1884
• Gram-positive bacteria


  Bacteria that take up the basic dye
• Gram-negative bacteria


  Bacteria that allow crystal violet (basic dye) to be washed out with decolorizer

  
• Uses of Gram Staining


  • Classifies bacteria on their forms, sizes and morphology
  • Classifies bacteria on the basis of gram reaction
  • Rapid, presumptive diagnosis of infectious agents
  • Assessing the quality of specimen

  
• Theories of Gram Stain Reaction


  • Magnesium Ribonucleic acid Theory
  • Benian Theory
  • Stearn and Stearn Theory
  • Lipid Content Theory
  • Teichoic Acid Content Theory
• Crystal Violet


  Primary stain, stains all bacteria purple to blue
• Gram's Iodine


  Mordant, enhances reactions between cell wall and primary stain
• Acetone alcohol or Ethyl Alcohol


  Decolorizer, gram positive bacteria retain the primary stain because of the thick peptidoglycan and teichoic acid cross-links, gram negative bacteria lose the primary stain because of the large amount of lipopolysaccharide in the cell wall
• Safranin O


  Counterstain, no effect on gram positive bacteria
• Gram Positive Bacteria


  • Blue to purple
• Gram Negative Bacteria


  • Pink to red
• Most critical step in Gram Staining is the decolorization