Gram staining miceobiology student

microbiology student

1. 2 Gram Stain: It is the most important differential stain used in bacteriology because it classified bacteria into two major groups: Gram positive: Appears violet after Gram’s stain b) Gram negative: Appears red after Gram’s stain

2. 3 Types of staining techniques Simple staining (use of a single stain ) Differential staining (use of two contrasting stains separated by a decolorizing agent) For visualization of morphological shape & arrangement. Identification Visualization of structure Gram stain Acid fast stain Spore stain Capsule stain

3. Procedure A). Smear Preparation: Take a clean, dry and grease free slide. Transfer a loopful of culture by the sterile nichrome loop and make a smear on slide. Allow the smear to dry air. Fix the dry smear by passsing the slide 3-4 times through the flame quickly.

4. Contd … B). Grams staining: Place the slide on the staining glass rods. Cover the smear with crystal voilet or gentian voilet and leave for 1 minute. Wash with D/W. Flood the smear with Grams iodine solution and wait for 1 minute. Wash with D/W.

5. Contd … Decolourize with an alcohol or acetone for 10-30 seconds. Wash with D/W. Counterstain the smear with safranin and left for 1 minute. Allow the stained smear to dry in air . Observe under microscope.

6. 7 Gram +ve S.aureus Gram –ve E.coli Step 1: Crystal Violet Step 2: Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4: Safranin Red

7. 8 Results: Shape: Cocci Arrangment: irregular clusters Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Staphylococci

8. 9 Results: Shape: Rods Arrangment: Single Colour: red Gram’s reaction: Gram’s –ve Name of microorganism: Gram negative bacilli

9. Principle and mechanism

10. 11 Gram-positive bacteria Have a thick peptidoglycan layer surrounds the cell. The stain gets trapped into this layer and the bacteria turned purple. Retain the color of the primary stain (crystal violet) after decolorization with alcohol Gram-negative bacteria have a thin peptidoglycan layer that does not retain crystal violet stain. Instead, it has a thick lipid layer which dissolved easily upon decoulorization with Aceton-Alcohol. Therefore, cells will be counterstained with safranin and turned red .

11. Contd.. Protoplasmic PH : . The hydrogen ion concentration of the protoplasm of Gram positive bacteria (ph 2-3) is higher than that of Gram negative bacteria (ph 4-5). . The iodine treatment makes the cytoplasm further more acidic and serves as a mordant, i.e iodine combines with the dye and then fixes the dye in bacterial cell.