Gram Staining Procedure PDF

# Gram Staining The image depicts the steps involved in gram staining, a technique commonly used to differentiate between bacteria based on their cell wall composition. ## Steps of Gram Staining: - **Step 1:** Prepare a thin smear of bacteria on a clean slide - **Step 2:** Cover the smear with fe...

# Gram Staining The image depicts the steps involved in gram staining, a technique commonly used to differentiate between bacteria based on their cell wall composition. ## Steps of Gram Staining: - **Step 1:** Prepare a thin smear of bacteria on a clean slide - **Step 2:** Cover the smear with few drops of crystal violet for 1 min, this is called the primary stain. - **Step 3:** Wash excess stain with water. - **Step 4:** Add few drops of iodine solution on the smear for 1 min, this is called the mordant. - **Step 5:** Rinse in water. - **Step 6:** Wash with 95% ethanol for a minute, this is the decolorization step. - **Step 7:** Rinse with water. - **Step 8:** Stain with 1% safranin for 1 minute, this is the counterstain. - **Step 9:** Wash stain in excess water, this is the final step making the results visible under the microscope. ## Explanation of what happens in each step: 1. **The primary stain** colors all bacteria with crystal violet. 2. **The mordant** (iodine solution) forms a complex with the crystal violet which is able to bind to the thick peptidoglycan layers of gram-positive bacteria. 3. The decolorizing step uses ethanol to remove the primary stain (crystal violet-iodine complex) from gram-negative bacteria but not from gram-positive bacteria. 4. **The counterstain** (safranin) is used to stain the decolorized gram-negative bacteria, counterstaining them pink/red. ## Gram +ve vs Gram -ve - **Gram-positive bacteria** have a thicker peptidoglycan layer in their cell walls, and retain the crystal violet after the decolorization step. They will appear **purple** under the microscope. - **Gram-negative bacteria** have a thinner peptidoglycan layer and an outer membrane. The decolorizing step removes the crystal violet, and they become stained with the red counterstain (safranin). They will appear **pink/red** under the microscope. ## Visual Diagram of Gram Staining: - **Step 1:** Crystal violet is applied to the specimen. All bacteria are stained purple. - **Step 2:** Iodine is added, which forms a complex with the crystal violet and makes the stain more soluble. Some bacteria will have a stronger bond due to the thickness of the peptidoglycan layer. - **Step 3:** Alcohol is added, which washes away the crystal violet from bacteria with a thinner peptidoglycan layer. The bacteria with thicker peptidoglycan layers maintain the stain. - **Step 4:** Safranin is added, which stains the decolorized bacteria pink/red. Those with thicker peptidoglycan layers will remain purple. ## Summary Gram staining is a valuable tool for bacterial identification and classification, based on the difference in the cell wall composition of Gram-positive and Gram-negative bacteria. It plays a significant role in medical diagnostics, allowing us to diagnose infections, guide antibiotic treatment, and better understand the nature of bacteria.