Gram staining is a widely used technique in microbiology to distinguish between Gram-positive and Gram-negative bacteria. By applying a series of stains and washes, students observe differences in bacterial cell walls that cause some cells to appear purple (Gram-positive) and others pink (Gram-negative) under the microscope.

1. Use aseptic technique to prepare a bacterial smear on a clean microscope slide.
2. Heat-fix the smear by quickly passing the slide through a flame to kill bacteria and attach them to the slide.
3. Flood the slide with crystal violet stain for about one minute, then rinse gently with water.
4. Apply Gram’s iodine solution for one minute to form a crystal violet–iodine complex, then rinse.
5. Decolorize briefly with alcohol or acetone until runoff is clear; rinse immediately with water.
6. Counterstain with safranin for about one minute, then rinse and blot dry with bibulous paper.
7. Observe under the compound microscope with oil immersion (100x objective). Gram-positive bacteria will appear purple; Gram-negative bacteria will appear pink/red.

How to Perform a Gram Stain - Centers for Disease Control and Prevention (CDC):

Gram Staining - Bio-Rad Laboratories:

• Compare Gram staining results for different bacterial species (e.g., *E. coli* vs. *Staphylococcus aureus*).
• Use environmental samples (soil, water, or swabs) to test for mixed populations of Gram-positive and Gram-negative organisms.
• Compare with alternative staining methods (acid-fast, spore staining).
• Practice the procedure with prepared slides before attempting live cultures.

• Always use aseptic technique when handling bacterial cultures.
• Work in a clean, designated lab space; disinfect benches before and after use.
• Wear gloves, lab coat, and safety glasses at all times.
• Use heat carefully when heat-fixing slides; avoid burns.
• Dispose of bacterial cultures, slides, and staining reagents in accordance with biosafety guidelines.
• Only use non-pathogenic bacterial strains in student laboratories.

• Why do Gram-positive bacteria retain the crystal violet stain while Gram-negative bacteria do not? (Gram-positive bacteria have a thick peptidoglycan layer that retains the stain, whereas Gram-negative bacteria have a thinner wall and an outer membrane that allows decolorization.)
• What would happen if you forgot the decolorization step? (All bacteria would appear purple, making it impossible to distinguish between Gram-positive and Gram-negative.)
• Why is it important to heat-fix the bacterial smear before staining? (To kill the bacteria and adhere them to the slide so they don’t wash away.)
• How can Gram staining help in medical microbiology? (It provides rapid preliminary identification of bacteria, guiding treatment decisions such as antibiotic choice.)
• What are the limitations of the Gram stain? (Some bacteria do not stain well, and results may vary with culture age or technique.)