Endospore and Capsule Staining PDF

Endospore Staining (Schaeffer-Fulton Method): 1. Prepare the Smear: Prepare a bacterial smear on a slide and allow it to air dry. Heat-fix the smear by passing it through a flame 2-3 times. 2. Apply Malachite Green: Place the slide over a steaming water bath....

Endospore Staining (Schaeffer-Fulton Method): 1. Prepare the Smear: Prepare a bacterial smear on a slide and allow it to air dry. Heat-fix the smear by passing it through a flame 2-3 times. 2. Apply Malachite Green: Place the slide over a steaming water bath. Cover the smear with a small piece of paper towel Flood the smear with a malachite green stain and let it sit for 5-10 minutes, ensuring the stain does not dry out. Add more stain if needed. 3. Rinse with Water: Remove the slide from the steam and let it cool. Gently rinse the slide with water to remove excess malachite green. 4. Counterstain with Safranin: Flood the smear with safranin and let it sit for about 30 seconds to 1 minute. 5. Rinse and Dry: Rinse the slide again with water to remove excess safranin. Gently blot the slide dry with absorbent paper. 6. Examine Under a Microscope: View the slide under the microscope using the oil immersion lens. Endospores will appear green, and the vegetative cells will appear red. Capsule Staining (Maneval’s Method): 1. Prepare the Smear: Place a drop of India ink or Congo red (negative stain) on one end of a clean slide. Mix a small amount of bacterial culture into the drop without spreading too much. Using another clean slide, spread the drop by dragging the edge of the second slide across the first one, creating a thin smear. Allow the smear to air dry (do not heat-fix). 2. Stain with Maneval’s Solution: Once dry, flood the smear with Maneval’s stain (a basic stain) and let it sit for 1-2 minutes. 3. Rinse Gently: Gently rinse the slide with water to remove excess Maneval’s stain, being careful not to wash away the smear. 4. Air Dry: Allow the slide to air dry completely (do not blot or heat). 5. Examine Under a Microscope: Observe the slide under the microscope using an oil immersion lens. Capsules will appear as clear halos around the bacterial cells, while the background will be dark (from the India ink or Congo red) and the bacterial cells will be stained pink/red (from Maneval’s stain).