Gram Stain Lab 2 - College of Al-Zahrawi University - PDF

Gram stain Second Stage- Pharmacy - College of Al-Zahrawi University Lab 2 7 October 2024 Assistant lecturer Muntadher Hameed INTRODUCTION Gram’s stain was originally devised by histologist Christian Gram (1884) as a method of staining bac...

Gram stain Second Stage- Pharmacy - College of Al-Zahrawi University Lab 2 7 October 2024 Assistant lecturer Muntadher Hameed INTRODUCTION Gram’s stain was originally devised by histologist Christian Gram (1884) as a method of staining bacteria in tissues. Gram positive bacteria stain purple, while Gram negative bacteria stain pink when subjected to Gram staining. They stain differently because of the fundamental differences in the structure of their cell walls. Approximately, 60%–90% of the Gram-positive bacterial cell wall is made up of peptidoglycan and interwoven teichoic acid, while only 10%–20% of Gram- negative bacterial cell wall is composed of peptidoglycan, forming 2–3 layers. The Gram-negative cell wall is also surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, lipoproteins and other proteins. Why we need to stain Bacteria??? Bacteria are transparent and colorless so they would be invisible to naked eye if observed under microscope thus bacteria should be stained with certain dyes in order to visualize bacterial cell or their internal structures using the light microscope. Figure 1-1 gram positive and negative structure.  Stains and Staining A stain is a substance that adheres to a cell, giving the cell color. The presence of color gives the cells significant contrast so they are much more visible. A stain is a dye consisting of a colored organic compound in the form of salt composed of positive and negative ion, one of these ions is responsible for color (a chromophore). Staining is a technique used in microscopy to enhance contrast in the microscopic image. Stains are frequently used in biology and medicine for viewing. Based on the charges: A stain is classified in to: - Basic stain/dyes – stain with +ve charge, example include crystal violate, methylene blue and safranin Acidic stain/dyes – stain with –ve charge, example nigrosin and India ink. Neutral stain/dyes – stain with both charges. Based on function of stain: 1. Simple staining – only one dye is used- differentiation among bacteria is impossible- Eg. Simple Staining. 2. Differential staining- more than one dye is used- Differentiation among bacteria is possible- Eg. Gram’s staining, Acid-fast staining. 3. Special staining – more than one dye used -Special structures are seen. Eg. Capsule staining, Spore staining  Principle of staining Basic stain (+ve charge) To stain -ve charged molecules of bacteria, mostly used because cell surface is –ve charge. Acidic Stain (-ve charge) To stain +ve charged molecules of bacteria, used to stain the bacterial capsules. How do you prepare a smear? From liquid media: Sterilize the loop by Bunsen flame then let it cool, shake the specimen container (broth culture) then withdraw one or more if needed loopful from the specimen and spread it on the center of a clean slide to form a thin film of 1- 2 cm in diameter, then sterilize the loop. Allow the smear to dry by air. The smear fixed by passing it (3- 4) times through the Bunsen flame then allow the slide to cool before staining. From solid media (slant or plate) Sterile the loop on Bunsen flame and let it cool. Place a loopful of clean water on the center of a clean slide. Re sterilize the loop, transfer a small portion of the growth, mix it with water thoroughly and spread the mixture evenly on the slide to form a thin film of 1- 2 cm in diameter. Dry and fix (as mentioned above)  Figure 1-2: preparation of bacteria smear Type of staining a. Simple Staining: This procedure uses only one basic dye e.g. Crystal violet or Methylene blue or Safranin to stain bacteria. The bacteria will simply take the color of the dye. Figure 1-3: simple staining B: Differential Staining: These involve more than one dye solution. The dyes may be added in several steps according to the procedure. Gram Stain Gram stain is used to stain bacteria. Bacteria stain either Gram- positive or Gram negative on the basis of the differences in their cell wall composition. What is the different between Gram positive and Gram-negative bacteria? Gram positive species have a thick peptidoglycan layer and large amount of teichoic acid and are therefore unaffected by alcohol decolorization and retain the initial stain (crystal volet) giving the organism violet appearance. Gram negative cell wall have a single peptidoglycan layer. The outer membrane is damaged by alcohol decolorizer allowing crystal violet- iodine complex to take out and be replaced by the counter stain (safranine) giving the organism a pink/ red appearance.