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Presentation on Gram Staining.pptx
- 1. Dr.Yogesh Bhagrava Sir ByAdil Ali Msc Microbiology 1st Semester Submitted To Gram Staining
- 2. Content History Introduction Principle Requirements Reagent Functions of Reagent The Gram stain procedure Result Precautions Reference
- 3. Gram staining developedby Hans Christian Gram,a Danish Doctor working in Berlin While examining lungs tissue from a patients who had died of pneumonia, he discovered that certain stains were preferentially taken up and retained by bacterial cells. Gram devised his technique to enable bacteria to be seen more readily in stained section of lung tissue. He published his method in 1884, and included in his short report the observation that the typhus bacillus did not retain the stain. Hans Christian Gram 1853-1938 History
- 4. Introduction Gram Staining isthe common, important and most used differential staining techniques in microbiology. This test differentiate the bacteria into Gram Positive and Gram negative Bacteria, which helps in the classification and differentiations of microorganisms. Gram staining involves four process staining with a water-soluble dye called Crystal Violet, Gram Iodine, Decolorization and counterstaing usually with safranin.
- 5. Principle When the bacteriais stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in colour.
- 6. In case ofgram negative bacteria, cell wall also takes up the CV- Iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin, they take the stain and appears red in color.
- 7. Requirements A cleangrease free slide Bacterial cell suspension Tissue Paper Nichrome wire loop Spirit lamp/Bunsen burner Oil immersion Microscope
- 8. Reagents Primary Stain(Crystal Voilet) Mordant (Gram’s Iodine) Decolorizing (Alcohol 95%) Counterstain (Fuschin or Safranin)
- 9. Functions of Reagents Crystal Violet - It is a primary stain and a basic dye it stains all Microorganisms. Gram’s Iodine – Gram’s Iodine acts as a mordant and it forms a complex with crystal violet that is CV-I complex. This complex increase affinity between cell and stain. Alcohol – It is a decolorizing agent as well as a lipid solvent. It tries to decolorize the cell by removing the CV-I complex from the cell. Fuschin or Safranin – It acts as a counterstain. It stains the cells that are decolorized by alcohol. Only gram negative bacteria get decolorize and this counterstain gives pink to these cells.
- 10. The Gram StainProcedure Take a clean grease free slide. Let the smear should be air dried. Heat fixation is mostly used to a fix the bacteria to the slide so that they don’t rise out during the staining procedure. Prepare the smear with the help of nichrome wire on the clean glass slide.
- 11. Primary Staining:The smear is covered with crystal violet, for one minute and wash with water.
- 12. Mordant: Itis then covered with Gram’s iodine, kept for 1 minute, and washed with water. Crystal Violet – Iodine Complex (CV-I complex)
- 13. Decolourization: The smearis covered with alcohol and is washed with water immediately with in 5second.
- 14. Counter staining: Thesmear is then covered with safranin, kept for 30 seconds and wash with water. • Let it dry the smear • Mount the smear • Observe under oil immersion microscope
- 17. Result GRAM POSITIVE: Blue/PurpleColor GRAM NEGATIVE: Red/Pink Color
- 18. Examples G+Ve G-Ve Streptococcus pneumoniaeSalmonella Typhi Mycobacterium tuberculosis E.Coli Difference between Gram positive and Negative bacteria Gram positive bacteria (G+) have cell walls that contain thick layers of peptidoglycan (90% of cell wall). These stain purple Gram negative bacteria (G-) have walls with thin layers of peptidolyacn (10% of cell wall), and high lipid content.
- 20. Precautions Gram stainmaterials will stain skin and other materials. Wear chemical splash goggles and chemical-resistant gloves. Wash hands thoroughly with soap and water before leaving the laboratory. Follow all laboratory safety guidelines. Please review current Material Safety Data Sheets for additional safety, handling and disposal information. Check the reagent for the expire date before the using. Care should be taken when sterilizing the inoculating loop and when flame-fixing slides.
- 21. Reference Infection,Immunity andInflammation – University of Mosul Bio Practicals – Nagaraj Balasubramanian Stains & Staining – Dr. Rakesh Sharda Microbiology – Pelczar Jotscroll.com https://www.jotscroll.com/gram-stain-procedure-gram-staining- steps-and-results Microbiology info.com https://microbiologyinfo.com/gram-staining-principle-procedure- interpretation-examples-and-animation/ Slideshow FLINN SCIENTIFIC BIO FAX
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