Gram Stain
Staining your organisms to differeniate between positive- and negative-bacteria!
Step-by-Step: A beginner's guide to Gram
Materials Needed
- Ethyl alcohol (95% denatured)
- Iodine solution
- Crystal violet dye (1%)
- Safranin O dye (1%)
- Additional - your basic, everyday tools:
- Microscope slides
- Inoculating loops (or something similar)
- Distilled/Deionized water
- Immersion Oil
- Bunsen Burner/Flame (optional)
- Your microscope (ideally with 100x objective)
- Recommend: paper towels, wash bottle for water, and a timer/watch with seconds hand
Step 1: Fixing the Bacteria to the Slide
- Place a drop of water in the center of the slide.
- Use inoculating loop to collect bacteria. Then smear that bacteria into the water drop, creating a thin layer on the slide.
- Allow it to air dry, or pass it over a bunsen burner flame to evaporate the water.
- Note: Do not keep slide over the flame, do not put the slide directing in the flame. Simply pass it over a few times, let it cool, and repeat until water is gone/it's dry.
Step 2: Apply Crystal Violet Dye
- Recommended: Place slide on either paper towels or the like - dye will stain if it drips!
- Put several drops of Cystal Violet in center of slide completly cover the smeared cell area.
- Leave for 1 minute (60 seconds).
- Rinse off excess dye with water by tilting slide at an angle (do not directly hit stained cells with water - it can wash them off!).
Step 3: Apply Iodine & Ethyl Alcohol
- Put several drops of iodine in the center of the slide to completly cover the stained cell area.
- Leave for 1 minute (60 seconds).
- Rinse off excess dye with water by tilting slide at an angle (same as before).
- Add a few drops of ethyl alcohol to the tilted slide until it runs clear after passing over the stained cell area.
- Rinse off excess dye & alcohol with water again.
Step 4: Apply Safrinin O Dye
- Put just enough drops of safrinin in the center of the slide to completly cover the stained cell area.
- Leave for 1 minute (60 seconds).
- Rinse off excess dye with water by tilting slide at an angle (same as before).
- Use paper towel to blot off excess water along the edges.
- Allow to air dry completly.
Step 5: View on microscope!
- Once dry, place on micrscope. View as normal! If using 100x objective, remember to add immersion oil!
- Pink stained cells = Gram-Negative bacteria
- Purple stained cells = Gram-Positive bacteria