Gram staining

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  • 1. byDr. Khalid Yousuf MemonLecturer Dept: of Pathology
  • 2. Gram’s Staining OBJECT: To study the identification & morphological features of micro- organisms by Grams’ staining
  • 3. Gram’s Staining REQUIREMENTS: Glass slide, specimen, wire loop, tripod stand, crystal violet dye, gram’s iodine, spirit, 1/10 % safranin, water, cedar wood oil & Microscope
  • 4.  THEORY:The Gram stain procedure was originallydeveloped by the Danish Physician HansChristian Gram to differentiatePneumococci from Klebsiella Pneumonia. Itis also known as “ Differential staining”, asit divides micro-organisms into two maingroups i.e. Gram’s +ve and Gram’s –vemicro-organisms.
  • 5. Gram’s Staining Crystal violet is a basic dye, it forms a complex with iodine & get fixed permanently in Gram’s +ve organisms. The dye iodine complex is not washed out from these organisms ever. They are not decolorized by spirit use.
  • 6. Gram’s Staining This is because these organisms contain thick and multilayered peptidoglycan, while Gram –ve organisms have single layered peptidoglycan, therefore the dye is not fixed permanently in them & is removed by spirit.
  • 7. Gram’s Staining Gram –ve organisms loose the dye and become colorless while the Gram + ve organisms retain the colour of crystal violet so when stained with counter stain i.e. 1/10 % Safranin , Gram –ve organisms appear pink in colour while Gram +ve organisms appear violet in colour by retaining the violet colour.
  • 8. Gram’s Staining On the basis of Gram’s staining micro- organisms can be divided into four groups; according to their color & shape  Gram’s +ve cocci: Streptococci, Staphylococci.  Gram’s +ve bacilli: Corny bacterium Diphtheria, Clostridia, Bacillus Anthrax.  Gram’s –ve Cocci: Neisseria.  Gram’s –ve Baccilli: E.Coli, Salmonella, Shigella, Proteus, Pseudomonas
  • 9. Procedure 1. Take specimen on the wire loop & spread it on the slide. 2. Fix the smear by passing it over the flame 2-3 times. 3. Cover the smear with crystal violet for 1-2 minutes. 4. Wash it & than cover it with Gram’s staining for 01 minute. 5. Wash the smear with spirit till the blue colour of the slide stops to appear.
  • 10. Procedure6. Than wash the slide with water & cover it with 1/10 % safranin red for about ½ minute. Than wash it with water.7. Allow the slide to dry in air. Put a drop of cedar wood oil.8. Than see the slide under oil immersion lens.
  • 11. RESULT  Gram +ve organisms appear violet in colour, Gram –ve organisms appear pink in colour. The epithelial cells and pus cells are also pink in appearance.
  • 12. Gram +ve Bacilli
  • 13. Gram +ve & -ve organisms
  • 14. Gram +ve in chains(S. Pneumoniae)
  • 15. Gram +ve BacilliClostridium Species
  • 16. Gram +ve cocciStaph: aureus
  • 17. Gram +ve Diplococci( Pneumococci)
  • 18. Gram +ve rods( Diphtheroids)
  • 19. G+ve & G-ve Bacilli