Gram staining

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<ul><li> 1. byDr. Khalid Yousuf MemonLecturer Dept: of Pathology</li></ul> <p> 2. Grams Staining OBJECT: To study the identification &amp; morphological features of micro- organisms by Grams staining 3. Grams Staining REQUIREMENTS: Glass slide, specimen, wire loop, tripod stand, crystal violet dye, grams iodine, spirit, 1/10 % safranin, water, cedar wood oil &amp; 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. Grams +ve and Grams vemicro-organisms. 5. Grams Staining Crystal violet is a basic dye, it forms acomplex with iodine &amp; get fixedpermanently in Grams +ve organisms. Thedye iodine complex is not washed out fromthese organisms ever. They are notdecolorized by spirit use. 6. Grams Staining This is because these organisms containthick and multilayered peptidoglycan,while Gram ve organisms have singlelayered peptidoglycan, therefore the dye isnot fixed permanently in them &amp; isremoved by spirit. 7. Grams 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. Grams Staining On the basis of Grams staining micro-organisms can be divided into four groups;according to their color &amp; shape Grams +ve cocci: Streptococci, Staphylococci. Grams +ve bacilli: Corny bacterium Diphtheria,Clostridia, Bacillus Anthrax. Grams ve Cocci: Neisseria. Grams ve Baccilli: E.Coli, Salmonella, Shigella,Proteus, Pseudomonas 9. Procedure 1. Take specimen on the wire loop &amp; spread iton the slide. 2. Fix the smear by passing it over the flame 2-3times. 3. Cover the smear with crystal violet for 1-2minutes. 4. Wash it &amp; than cover it with Grams stainingfor 01 minute. 5. Wash the smear with spirit till the blue colourof the slide stops to appear. 10. Procedure6. Than wash the slide with water &amp; cover it with 1/10 % safranin red for about 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 &amp; -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 &amp; G-ve Bacilli</p>

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