How do you Gram stain?
Answer
Gram staining is a crucial microbiology technique that differentiates bacteria into two major groups: Gram-positive and Gram-negative, based on differences in their cell wall composition. This distinction is vital for identifying bacteria and guiding antibiotic treatment. There are four major steps in preparing a Gram stain.
1. Put on gloves and tie back long hair to prevent contaminating the bacteria sample you'll be testing.
2. Sterilize a glass microscope slide. Disinfect it with ethanol, dry it, and then flame the slide over a Bunsen burner.
3. If taking bacteria from a petri dish, sterilize and inoculation loop in a Bunsen burner flame until it glows, then let it cool. Use the loop to place a drop of sterile water on the slide, then sterilize and cool the loop again before transferring a tiny sample of bacteria and gently stirring them into the water.
4. Heat fix the smear. Quickly pass the slide two to three times through a Bunsen burner flame. Position the slide on a staining tray.
5. Using a pipette, flood the heat-fixed bacterial smear with crystal violet on a microscope slide. Both Gram-positive and Gram-negative cells will absorb this purple stain. appearing purple.
6. Rinse off the crystal violet gently with distilled water.
7. Next, flood the smear with iodine, then rinse. Both Gram-positive and Gram-negative cells still appear purple.
8. Decolorization with alcohol or acetone is applied. Gram-positive bacteria have a thick peptidoglycan layer in their cell wall, which becomes dehydrated and shrinks in the presence of alcohol. This traps the crystal violet-iodine complex inside the cell so Gram-positive bacteria retain the purple color.
Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane rich in lipids. The alcohol dissolves this outer membrane and the thin peptidoglycan layer cannot retain the crystal violet-iodine complex, leading to decolorization. Gram-negative bacteria become colorless at this stage.
9. Counterstain with Safranin--a pink to red dye. Gram-positive bacteria remain purple because the crystal violet is much stronger than the safranin and isn't displaced.
Gram-negative bacteria, which were decolorized, now pick up the safranin stain and appear pink or red.
10. The slide is rinsed, dried, and observed under a microscope, usually with an oil immersion lens.