Overview
This lecture explains the step-by-step procedure for performing the Gram staining technique to differentiate bacteria based on their cell wall composition.
Preparing the Slide and Smear
- Obtain a fresh pure bacterial culture with isolated colonies.
- Mark the area on a glass slide where the smear will be prepared.
- Sterilize the bacteriological loop by burning it and let it cool before use.
- Place a drop of water on the marked area of the slide.
- Re-sterilize the loop, pick a colony from the culture plate, and emulsify it in the water drop.
- Spread the bacterial suspension evenly within the marked area.
- Allow the smear to air dry so bacterial cells adhere to the slide.
- Fix the smear by passing the slide three times through a flame.
Gram Staining Procedure
- Cover the smear with crystal violet dye for 1 minute (primary stain).
- Rinse the slide with water to remove excess dye.
- Apply iodine solution (mordant) for 30 seconds to form a complex with crystal violet.
- Rinse with water and remove excess.
- Add alcohol-acetone mixture to decolorize the smear, affecting bacteria differently based on cell wall type.
- Rinse thoroughly with water after decolorization.
- Apply safranin dye as a counterstain for 1 minute to provide contrast.
- Rinse with water and dry the slide.
Visualization
- Once dry, add a drop of immersion oil to the slide.
- Observe the stained sample under a clear field microscope with the immersion lens.
Key Terms & Definitions
- Gram Staining — A differential staining technique to distinguish between Gram-positive and Gram-negative bacteria.
- Smear — A thin layer of bacteria spread on a microscope slide.
- Crystal Violet — Primary stain in Gram staining, colors all bacteria purple initially.
- Iodine Solution (Mordant) — Reagent that forms a complex with crystal violet in bacterial cells.
- Decolorizer (Alcohol-Acetone Mix) — Removes primary stain from some bacteria based on cell wall properties.
- Safranin — Counterstain providing a pink color to Gram-negative bacteria.
- Immersion Oil — Used to enhance image clarity at high magnification under a microscope.
Action Items / Next Steps
- Prepare your own Gram staining slides following the outlined steps.
- Review differences between Gram-positive and Gram-negative results for exam preparation.